> San . Ne Dts - ae : RSE Se on: Sab Sree waa Se a ent Sn ee ne Se ee aE YR NA A RT RN A A A ENR SN on = Se Pa Dihy ow ety: ee a eet a ———— Sa eee : Serene JOURNAL OF MORPHOLOGY. EDITED BY C. O. WHITMAN, With the Co-operation of EDWARD) PHELPS” ALLIS; MILWAUKEE. Vou. XLV. BOSTON, U.S.A.: GINN & COMPANY 1898 j is) | 7 hy : Ve tks arg Fis i yi whe Oy i T CONTENTSOr WoL. XIV: No. 1. — June, 1897. PAGES I. James F. Porter. io. New Gregaripida si, .) eae I-20 TE. F.C. Case. On the Osteology and Relationships of PR BLOSEC Ee 5 a. So oe, 8 ee eas 21-60 III. Atsro D. Morritt. The Innervation of the Auditory Epithelium of Maustelus Canis, De Kaye. 61-82 IV. J. PrayraiR McMurricu. The Epithelium of the so-called Midgut of the Lervrestrial Isopods 3 - .. 2 . 83=104 No. 2. — June, 1808. I, EstHer F. Byrnes. Experimental Studies on the Development of Limb-Musclesin Amphibia. . . . . 105-140 II. Howarp S. Brobe. A Contribution to the Morphology of Dero LE as ai NA AEM A STORE 141-180 he A. Ds MEAD: The Origin and Behavior of the Centrosomes tthe ANNE BER. os Na oe EBIR=2TS iv TV. Mi VIL. LB IV. CONTENTS. Acnes Mary CLAYPOLE. The Embryology and Odgenesis of Anurida Maritima (Guér.) ‘ : CLARA LANGENBECK. Formation of the Germ Layers in the Am- phipod Microdeutopus Gryllotalpa Costa . SIMON FLEXNER. The Regeneration of the Nervous System of Planaria Torva and the Anatomy of the Nervous System of Double-Headed Forms FRANKLIN P. MALL. Development of the Ventral Abdominal Walls in Man No. 3. — September, 1808. GEORGE LEFEVRE. Budding in Perophora . Epwarp Puevrs ALLIS, JR. On the Morphology of Certain of the Bones of the Cheek and Snout of Amia Calva . ALBERT C. EYCLESHYMER. The Location of the Basis of the Amphib- zan Embryo . KATHARINE Foot. The Cocoons and Eggs of Allolobophora FGCU GA. esi an On aes PAGES 219-300 301-336 337-346 347-366 367-424 425-466 467-480 481-506 Volume XIV. June, 1897. Number I. JOURNAL OF MORE TIOLOG ¥. TWO NEW GREGARINIDA|! By JAMES F. PORTER. I. A Gregarine from Clymenella. (Monocystis clymenellae 7. sf.) THERE is found in the body cavity of Clymenella torquata a gregarine which I have been unable to identify with any described species. It resembles, however, in many ways Monocystis magna from the earthworm, recently described by Wolters,? a species to which it is probably closely allied, though parasitic in a salt-water annelid. On cutting open a parasitized Clymenella one finds in the body cavity, but generally in the posterior half only, numerous white, opaque, oval bodies a little larger than starfish eggs. Microscopic examination shows that these are gregarines in the encysted condition. The cysts are often found scattered singly through the body wall; but in much-infested worms they are more frequently met with in clusters of as many as eight, ten, or even more, and are surrounded by loose connective tissue. 1 Contributions from the Zodlogical Laboratory of the Museum of Comparative Zoology at Harvard College, under the direction of E. L. Mark, No. LXXXII. 2 Arch.fiir mikr. Anatomie, Bd. XX XVII, 1890, pp. 99-134. 2 PORTER. [Vou. XIV. A fair idea of the number and relative position of the cysts in the body cavity of the host can be obtained from PI. I, Fig. 1, which was drawn from a cross section of a greatly infested worm, and shows sections of eighteen gregarine cysts. Ordinarily the cysts appear white and opaque (PI. I, Fig. 2); mature specimens, however, when illuminated from beneath, are quite translucent and are seen to be filled with sporocysts. I have not been able to throw much light on the question, whether or not there is a conjugation or a division preceding the beginning of spore formation. However, the few cases observed which have any bearing on this question seem to point to a conjugation. In the only case of the unencysted condition found (Pl. I, Fig. 3), a division or conjugation — unfor- tunately one cannot say positively which — is in the act of taking place. This case alone cannot be taken as satisfactory evidence of either process; but in a dozen or more recently encysted gregarines I have noticed a constriction of the protoplasm which strongly suggests a conjugation, the evidence of which has not yet been fully obliterated (Pl. I, Fig. 4). I infer this because, apparently, in this form no division of the protoplasm precedes spore formation (Pl. I, Figs. 5,9, 10,12). It therefore seems probable that the case represented in PI. I, Fig. 3, is a conjugation preparatory to encystment. The close proximity of the two nuclei, which might at first sight be taken as evi- dence of recent division, is a condition which is entirely in harmony with the ordinary method of conjugation in monocystic eregarines, where, as is well known, the corresponding (ante- rior) ends of the copulating individuals are the ones to come in contact and fuse. It is to be inferred that the time intervening between encyst- ment and the formation of the first sporogonia is very short, because the number of cysts found either filled with sporocysts or in the process of spore formation is very large, the number of those in other conditions being comparatively small. Before the development of spores actually begins, however, the nucleus breaks up (PI. I, Fig. 6) and eventually disappears entirely (Pl. I, Fig. 7). During this process the character of the protoplasm changes from a loose vacuolated state to a more No. 1.] TWO NEW GREGARINIDA. 3 compact and finely granular condition. Thus at f7’gl" (Pl. I, Fig. 6) the protoplasm is still extensively vacuolated, while at prpl' this, more properly speaking, frothy condition has given — place to fine granules, some of which are shown highly magni- fied in Pl. I, Fig. 8. The compact condition of the protoplasm at the close of this stage is shown by the numerous cracks run- ning through hardened specimens (Pl. I, Fig. 7). The cracks are due, of course, to the method of hardening. I believe that the disintegration of the nucleus continues until the chromatic matter no longer exists as such; at any rate, until it becomes divided into particles so fine as to be invisible even under the highest powers of the microscope. One of the reasons for believing that a complete dissolution of the nucleus takes place is that with its disappearance the chemical char- acter of the protoplasm seems to change. I believe that the protoplasm then develops slightly acid qualities, for I have found that it acts on the haematoxylin stain, turning it gradually to a reddish tint, while the tissue enveloping the cyst retains its original blue color. This is very noticeable in my old slides, where all the cysts in this stage of development stand out as red spots from their blue surroundings (PI. I, Fig. 7). Very frequently the first sporogonia are formed before the network state of the protoplasm has been completely replaced by the finely granular condition. The protoplasm shrinks away from the wall of the cyst and then sends out homogene- ous, translucent, amoeboid processes from the whole or part of its periphery (Pl. I, Figs. 9, 10). The granules of the central mass are carried out through these processes and, collecting together in clusters, help to form the sporogonia (PI. I, Fig. 11). Meanwhile numerous chromatic particles appear, which, by arranging themselves into rings (Pl. I, Figs. 11, 14, and Pl. I, Fig. 18), form the nuclei of the sporogonia. As more and more sporogonia are formed, the central mass of protoplasm becomes smaller and smaller, large vacuoles appear, and sporogonia are likewise seen in these (Pl. I, Fig. 12). A fine filamentous network can be distinguished in the cyst (ji/, ri/, Pl. I, Fig. 12); it is perhaps a useless rem- nant of the original protoplasmic mass. 4 PORTER. [Vor. XIV. The process of spore formation seems to be somewhat differ- ent in the later stages of sporulation; for, as the protoplasm becomes riper, sporogonia can be seen developing not only at its periphery but also within it (Pl. I, Fig. 13, sfo’go’). During this process the particles of chromatin appear at first to collect together in large masses (Pl. I, Fig. 13, chr) and then to be distributed to the forming sporogonia. At length the proto- plasm that still remains breaks up into small masses (PI. I, Fig. 15, 7/2) and finally becomes employed in the formation of additional sporogonia. One frequently finds, however, in mature cysts — that is, those containing only ripe sporocysts— a small mass of loose protoplasm left over when the formation of the sporogonia has ceased. The sporogonia that are formed first do not wait in their development for those that are formed later, but continue their growth and metamorphosis without interruption, so that one finds in the same cyst sporogonia in all stages, from such as are just formed to those that are mature (PI. I, Fig. 15, spo’go and spo’'go', and Pl. II, Fig. 16). In one case, I have noticed within the cyst at about this stage delicate transparent membranes (Pl. I, Fig. 15, 0). What their purpose is I cannot say, unless possibly they are connected with the formation of the sporocysts. The development of the sporogonia into sporocysts each con- taining eight spores, is as follows: The nucleus first, apparently without mitotic changes, divides into two (PI. II, Fig. 20), then each of these divides, making four (PI. II, Fig. 21), and finally each of the four divides, thus producing in all eight nuclei (PI. I, Figs. 15, sfo’go, and Pl. II, Fig. 17). The example shown in Pl. II, Fig. 19, seems to be an exception to this rule, for here there is a well-developed nucleus at one pole, and near the other a collection of chromatic particles not very compactly arranged and without nuclear membrane or precise boundary. The only explanation I can offer is that abnormally the division has resulted in nuclei of very unequal size. After their formation the sporogonia increase greatly in size. Pl. II, Fig. 17, shows five sporogonia, four before any division of the nuclear substance, and one after the three successive No. 1.] TWO NEW GREGARINIDA. 5 divisions which result in eight nuclei. After the first nuclear division the resulting nuclei migrate to opposite poles of the sporogonium (Pl. II, Fig. 20 8). When the second division has occurred, two of the nuclei occupy the two poles of the sporogonium; the remaining two lie on opposite sides of the chief axis, half-way between the poles (Pl. II, Fig. 21). I am unable to say what the positions of the eight nuclei are at the close of the final division. The chromatic substance fre- quently. has the form of eight rings (Pl. II, Fig. 17), and these are usually arranged near the periphery of the cyst. With the metamorphosis of the rings into more homogeneous and elon- gated nuclei, they take up positions in the periphery of the cyst at or near its equator. It is frequently seen in cross sec- tions (Pl. II, Fig. 24) that the nuclei lie very near the periphery, and closely approximated in two rows of four each, which occupy opposite sides of the cyst. It is possible that each of the clus- ters is descended from one of the two nuclei resulting from the first division of the nucleus of the sporogonium; but I have not satisfactory proof of this. In other cases (Pl. II, Fig. 25) the nuclei lie near the periphery, but are not thus evenly divided in their grouping. In still other cases there is no evi- dence of a particular grouping; but this condition may be due to disarrangement caused by mechanical influences in killing, hardening, etc. During the metamorphosis of the nuclear substance the sporogonia produce two enveloping membranes: an outer one, which I shall call the capsule, and an inner one, which I shall call the cyst (Pl. II, Figs. 22, 23). The outer envelope is a very transparent, unstainable struc- ture of an elongated cylindrical or spindle-shaped form. It apparently has two openings, one at either end (PI. II, Fig. 31). Around each opening the wall of the capsule is thickened (a, Beit Figs: 22,°23,.209; 31). ° This’ thickening constitutes a somewhat rigid ring around the opening of the capsule, which is thus perhaps prevented from being accidentally closed. The two ends of the capsule generally differ slightly from each other; usually one end has more of a neck-like prolongation than the other, and it is this end which generally presents the 6 PORTER. [Vou. XIV. more prominent ring. In some cases, however (Pl. II, Fig. 31), the ring at the other end is the thickest. It is not always pos- sible to make out an opening at the end opposite that which is prolonged into a neck (PI. II, Fig. 23); but when it does exist it may be larger than the opening at the neck end (PI. II, Figs. 29, 31). A certain amount of shriveling is sometimes to be observed on the part of the capsule, but this is less likely to affect the neck end than the opposite end (PI. II, Figs. 22, 29, 30). The orifice at the neck end of the capsule varies in diameter from about that of a spore to two or three times that size. The inner envelope, which does not fill the capsule com- pletely, although of a more or less spindle-shaped form, is much shorter than the capsule, but of nearly the same diameter, so that the space left between the capsule and the cyst is princi- pally at the ends. The cyst is really flask-shaped, having but a single orifice, which terminates in a more (PI. II, Fig. 22) or less (Pl. II, Figs. 23, 30) neck-like prolongation of the end cor- responding to that end of the capsule which I have called the neck end. The opposite end of the cyst is rounded or slightly conical. The orifice sometimes exhibits a thickened rim; it varies a little in diameter, being usually somewhat smaller than that of the capsule, and of a size sufficient to allow the passage of only one spore at a time (PI. II, Figs. 22, 29, 32). In the early stages of spore formation the cyst is completely filled by its protoplasmic contents. In later stages nearly or quite all of the contents is concentrated into the spores, which then by no means fill the cyst completely. Very frequently the whole cyst and contents slip out of the capsule (Pl. II, Figs. 26, 32). Not knowing this, I was at first greatly perplexed at being unable, as I supposed, to stain the spores. I prepared my slides by first breaking open the living cysts by slight pressure on the cover glass, and then killing, fixing, and staining on the slide, as one would in preparing bacteria. After such treatment I almost always found, much to my astonishment, that while thousands of sporocysts remained fixed to the slide, nothing was stained. I attributed this to the inadequacy of the stain, and not until after trying No. I.] TWO NEW GREGARINIDA. 7 many experiments with a dozen or more different stains did I discover that there was nothing left to stain, that the cysts containing the spores had escaped from the capsules and had been washed away. I have not been able to determine how either of the envelopes is formed, but they are both undoubtedly secreted by the proto- plasm of the sporogonium, the capsule first, the cyst afterwards. The protoplasm in the cyst gradually collects into cord- or band-like structures apparently in connection with the elon- gated nuclei, of which they appear as though tail-like appen- dages (Pl. II, Figs. 22, 23). The bands at first are only feebly stainable; later they stain more deeply. Large globules of a colorless substance appear in the cyst at the same time; finally, the protoplasm becomes entirely consumed in the formation of the tails. At the time of the first appearance of the tail-like proto- plasmic portions of the spores, the nuclei are elongated, cylin- drical rods, usually constricted slightly in several (3 to 5) places, so as to present a rather evenly monilate form. With the com- pletion of the protoplasmic portion of the spore, the chromatic mass becomes shorter and somewhat thicker, and stains more deeply than formerly. It finally assumes an appearance as though made up of only two oval bodies of equal size united end to end. Each portion is about twice as long as thick, and the union of the two is often so intricate that there is only a trace or even no evidence of a constriction, and the nucleus then is a single oval body some three or four times as long as thick. The tail portion of the immature spores is apt to contain vacuoles, and to shrink somewhat in the process of hardening (PE Il, Fig. 27). The spores represented in Pl. IJ, Fig: 28; are abnormal, or else have been subjected to too great pressure. In many of the sporocysts I have found the spores arranged in a regular manner, with their head ends, that is, their nucleated ends, in or near the equatorial plane, four of their tails being turned in one direction and four in the opposite. In fully as many cases, however, the spores had apparently no definite arrangement in the cyst (PI. II, Fig. 32). 8 PORTER. [VoL. XIV. The fate of the spores after their escape is still unknown. They are so minute that it would be almost impossible to iden- tify them in the tissues of the host. I have found, however, imbedded in the wall of the intestine of Clymenella, on the body-cavity side, numerous large amoeboid cells, evidently foreign organisms (Pl. II, Figs. 34-36); these, I think, may possibly be the early amoeba-like stage of this gregarine devel- oped directly from spores, but I have seen no intermediate stages. After trying many methods of killing and many kinds of stains, I found that the best results were invariably obtained by killing in a concentrated aqueous solution of corrosive sublimate, and staining in Kleinenberg’s haematoxylin. The aqueous stains were, as a rule, unsatisfactory, although I suc- ceeded very well with Mayer’s aqueous haemalum. Notwithstanding the greatest care in hardening the specimens and in passing them gradually into chloroform or into oil of thyme, I could not prevent the sporocysts from shrinking more or less. Pl. II, Fig. 33, represents a portion of a section through a nearly mature gregarine cyst; it shows sections through sporocysts in various directions and the great amount of shrinkage that has taken place. The best way that I have found for preparing slides of sporo- cysts consists in staining the gregarines zz zofo and breaking open the cysts with needles after they have been transferred to balsam ready for mounting. About one-fourth of all the worms I have examined were infested. I have collected Clymenella as late as the last of November and as early as the middle of April, and have never failed to find infested worms. My material was collected at Woods Holl, and at the mouth of the Saugus River, near Lynn, Massachusetts. II. A Gregarine from Rhyncobolus. While collecting Clymenella I frequently dug up fine speci- mens of Rhyncobolus Americanus, Verrill. Curiosity led me to search the intestines of a few of these, where, to my sur- No. I.] TWO NEW GREGARINIDA. 9 prise, I discovered a very large gregarine belonging to the order Polycystidea. After this I naturally examined every specimen of Rhyncobolus that came in my way; but I found that the parasitized worms were rare,—perhaps 10% of those examined, —and even these contained only a few gregarines, seven or eight at most. This gregarine is generally found with its anterior, that is, the larger, end (PI. III, Fig. 37) buried in the wall of the host's intestine, and its posterior end projecting out into the intestinal canal. It is easily distinguished from its surroundings, appear- ing as a white opaque body about 0.7 mm. in length. The body of the parasite is composed of an exceedingly large deutomerite (Pl. III, Fig. 37, dew’mer), containing the nucleus in its anterior portion, and a small protomerite (f7’mer), which bears an epimerite (e’mer). Its surface is covered with fine longitudinal striations (PI. III, Figs. 37-39) due to longitudinal folds in the cortical layer of the body (Pl. III, Figs. 41, 42). On focusing just below the surface, cross striations can occasionally be seen (PI. III, Fig. 37); they are due to circular muscular fibres. In optical longitudinal sec- tions of the parasite they are seen along its edges as black dots (ETL, Pizs..37,.38)- The outer cuticular wall appears very thick in longitudinal sections (Pl. III, Fig. 40); but this appearance is misleading, as cross sections of the parasite (Pl. III, Figs. 41, 42) clearly show. It is produced by the peculiar relation of the circular muscle fi- bres to the relatively thin cuticular covering and to the cortical portion of the parenchyma, which lies immediately below the cuticula. At short and tolerably regular intervals the circular contractile fibres come nearer to the deep surface of the cuti- cula and apparently have here a closer connection with that structure than over the intervening regions. The result is that the subcuticular layer of the parenchyma is thrown into longitudinal ridges, separated by sharp, deep furrows. The height of the ridges and the narrowness of their bases are to a certain extent dependent on the degree of contraction of the muscular fibres. The more the muscles shorten, the thinner and higher are the ridges. These ridges are so numerous and 10 PORTER [VoL. XIV. close-set, and of so nearly uniform height, that in longitudinal sections, optical or actual, they have the effect, especially with low powers, of a very thick cuticular covering, as thick as the ridges are high. But the real thickness of the cuticula is only a very small fractional part of the height of the ridges, and is therefore many times less than the apparent thickness of the cuticula. That this relation of cuticula and contractwe fibres is not an accidental or variable one is shown by the fact that the cuticula is not of uniform thickness, but presents at regular intervals, strictly correlated with its relation to the muscle fibres, longitudinal thickenings. These thickenings correspond with the crests of the ridges, and the middle of each thickening is raised into a sharp ridge (PI. III, cw, Figs. 41, 42). On focus- ing with a high power a little above the level of the bottom of the furrows, the surface of the gregarine appears striated longi- tudinally (Pl. III, Fig. 43), granular stripes alternating with nar- rower clear ones. The clear stripes are caused by the depressions between the ridges; the darker granular stripes are optical longitudinal sections of the subcuticular substance of the ridges. At a slightly deeper focusing are seen fine, parallel, transverse markings much nearer together than the longitudinal stripes; these are due to the very fine, highly refractive circu- lar muscle fibres (Pl, III, wu, Fig. 43). In cross sections these circular fibres (Pl. III, Figs. 41, 42, mz) can be seen running entirely around the parasite, stretching in succession from the cuticula at the bottom of one furrow to that at the bottom of the next. When the muscles are relaxed the ridges become low, rounded, and blunt (Pl. III, Fig. 41); but upon the contrac- . tion of the muscles the furrows become narrower and deeper, and the ridges higher and more pointed (Pl. III, Fig. 42). The protomerite of this gregarine has the form of a biconvex lens set in a corresponding depression of the anterior end of the deutomerite (Pl. III, Figs. 44, 46, pr’mer). It is composed of a very compact and finely granular protoplasm. The anterior portion is slightly denser and more deeply stained than the posterior half. From the centre of its anterior surface the epimerite (PI. III, Figs. 44, 46, e’mer) arises as a conical structure tapering off at its anterior end into a long filament (//). NOT. ‘ TWO NEW GREGARINIDA. Vi On removing the gregarine from the host the epimerite is generally torn off, remaining behind fastened to the intestine. The filament being, so far as discovered, a simple structure unprovided with hooks, probably maintains its hold by a more or less sinuous course in the intestinal cells of the host. When the epimerite is not torn off, it invariably carries with it an irregular mass of protoplasm, part of the intestinal cell of the host in which it had anchored itself (Pl. III, pr’p/, Figs. 44, 46). The conical base of the epimerite is apparently continuous with the contents of the anterior portion of the protomerite, for there is an orifice through the cuticular wall of the proto- merite, and the base of the epimerite is composed of protoplasm very similar to that of the protomerite. The orifice is made very distinct by a considerable thickening of the cuticula at its margin (Pl. III, Figs. 44, 46, fas). In the cases where the epimerite has been torn off, the cuticula immediately surround- ing the orifice appears pulled out a little by the strain of the rupture, thus leaving a cup-like depression at the anterior end of the protomerite (Pl. III, Fig. 48). Longitudinal sections through this cup show that the cuticular thickening just alluded to bends over its rim and extends about half-way down into the bowl (PI. III, Fig. 45, fas). Cross sections through this region in the case of individuals retaining the epimerite show a row of teeth-like processes (de) extending inwards from the margin of the cup (Pl. III, Fig. 47). These project radially toward the axis of the epimerite, — I think from the lowermost portion of the cuticular band which bounds the orifice, — and perhaps serve to hold the epimerite more securely to the protomerite. The parallel longitudinal ridges of the body wall of the gregarine, on reaching the protomerite, converge towards the cuticular ring surrounding the cup (PI. III, Fig. 47); they become narrower and lower as they approach the ring, until at length they are so minute that in sections cutting across them (Pl. III, Fig. 49, cvs) they resemble short cilia. The cuticular crests run up almost to the ring, where they terminate in a slight thickening (Pl. III, Fig. 48, cz/). I2 PORTER: [VoL. XIV. The deutomerite is unusually large, as we have seen. It is composed of a very loose and highly vacuolated protoplasmic network, which does not stain. The large nucleus (Pl. III, Fig. 46, 77) is usually situated, as I have said, in the anterior portion of the deutomerite; in living specimens, however, I have seen it move half the length of the animal. It generally contains several nucleoli, and in some of these, in addition to two or three vacuoles, I have noticed one or more very dark bodies. In one case I discovered a second nucleus in the posterior half of the animal (PI. III, Fig. 38). Here the deutomerite was slightly constricted just in front of the posterior nucleus; this certainly suggests preparation for division, and together with the condition shown in PI. III, Fig. 39, which looks like a recently divided individual, makes it seem probable that division is one of the normal modes of reproduction with this gregarine. I have found that this gregarine will live for seven or eight hours in sea water after being forcibly removed from the host. In watching the living specimens my attention was attracted by the very interesting movements made by the animal, which reveal a surprising amount of strength and a most remarkable manner of locomotion. Most of the movements can be readily accounted for by contractions of the cortical portion of the parenchyma or the circular muscular fibres. Thus the movement in the direction indicated by the arrow in Pl. III, Fig. 50, or from the condition of a to condition of in Pl. III, Fig. 51, is undoubtedly due to the contraction of the cortex on the convex side of the body. The creases in the cortex (Pl. III, Fig. 50, ~wg) are due to the con- traction of some of the circular muscle fibres; I believe they divide off successive areas of longitudinal contraction. This gregarine is able to obtain a firm hold on smooth sur- faces by using its anterior end as a sucker. On keeping a few of them for a time in a watch glass some have fastened them- selves in this manner so tightly to the bottom that they could not be removed without tearing them to pieces. PI. III, Fig. 53 shows the appearance of the anterior end when fastened in this way to a cover glass. No. 1.] TWO NEW GREGARINIDA. 13 The strength of the gregarine was well shown in one case where, after having fastened its head to the slide, it raised the middle portion of its body and turned it through an arc of 180°, like the bail of a bucket, its head and the tip of its tail alone resting on the slide during the process (PI. III, Fig. 52). From the fact that the general shape of the animal did not change during the operation, it is probable that the entire movement was caused by the contraction of a very small portion of the body wall. The area of contraction must have been limited to the region of a (PI. III, Fig. 52), since the head alone was fixed, the tail being free and changing its position slightly. The most interesting movement observed, however, is that of locomotion. It is a “very slow movement of translation in a straight line’ without any apparent contraction of the walls of the body. It is probably caused by a very slight undulatory motion of the under surface of the animal. My material, obtained at Woods Holl, was all collected in the months of July and August. Perhaps this is the reason why I have not found the encysted stage of this gregarine. This work was begun in the summer of 1891 at the sugges- tion and under the direction of Dr. C. O. Whitman; it was completed during the past winter under the very kind and care- ful supervision of Dr. E. L. Mark in the zodlogical laboratory of Harvard College. I wish to express a deep feeling of indebtedness to my instructors for whatever there may be of value in this paper. CAMBRIDGE, Mass., May 21, 1895. 14 PORTER. EXPLANATION OF PLATES. All the figures were outlined with an Abbé camera lucida. The very high magnifications were obtained with a Zeiss 1.5 mm. apochromatic homogeneous immersion objective and compensating oculars nos. 4, 6, and 12. ABBREVIATIONS. cau, cavity. gran, granules. chr, chromatin. mb, membrane. cps, capsule. mu, muscle. cp. sng, blood corpuscles. nl, nucleus. crs, crest of ridge. prmer, protomerite. cta, cuticula. ; pr pl, protoplasm. cul, ridge. rtl, reticulum. cys, cyst. TUL, crease. de, teeth. Spo, spores. dep, depression. SpO'L0, deu’mer, deutomerite. spo'go’", ¢ Sporogonia. e’mer, epimerite. SpOg0,” Sas, band. sul, furrow. jil, filament. vac, vacuole. 16 PORTER. EXPLANATION OF PLATE I. Fic. 1. Cross section of Clymenella torquata, showing sections of eighteen gregarine cysts. X 40. cys, cysts before the beginning of spore formation. Fic. 2. Mature living cyst illuminated from above. X 136. Fic. 3. Conjugating gregarines drawn from a living specimen. X 136. Fic. 4. Gregarine just after encystment, drawn from living specimen. X 136. Fic. 5. Early stage in spore formation, drawn from parasite. X 136. Fic. 6. Section of a gregarine showing the breaking up of the nucleus before encystment. X 350. cav, cavity caused by the shrinking of the protoplasm away from the sur- rounding tissue of the host. prpl, granular protoplasm. pr’pl,” frothy or vacuolated protoplasm. Fic. 7. Section of gregarine, imbedded in tissue of host, after disappearance of nucleus and before the beginning of sporulation. X 350. a, 8, y, 5, spaces occupied by other cysts. Fic. 8. A portion of the central mass of protoplasm shown in Fig. 9 highly magnified. X 3100. -— Fic. 9. Section of a cyst showing the formation of sporogonia and the con- dition of the central mass of protoplasm. X 350. Fic. 10. Another section of the same cyst. X 350. Fic. 11. Portion of the same cyst more highly magnified, showing structure of recently formed sporogonia, their manner of formation, and the condition of the protoplasm. X 1540. rtl, protoplasmic network. Fic. 12. Section of cyst in a later stage of the formation of sporogonia. X 230. cys, wall of cyst. a, former position of wall of cyst, determined by surrounding tissue of host. vil, fine filamentous network. Fic. 13. Part of central mass of protoplasm, shown in Fig. 12, more highly magnified. X I100. spo’go, recently formed sporogonia. spo’go,, sporogonia not yet fully formed. chr, mass of chromatin granules. Fic. 14. Sporogonia from same cyst. X 3100. Fic. 15. Section of cyst when spore formation is almost completed. X 400. pr pl, protoplasm not yet converted into sporogonia. spo’go, sporogonia with eight nuclei. spo’go,’ a sporogonium after the first division of the nucleus. mb, exceedingly thin transparent membrane. SEL del. 2 Tih dash cWorner 8Woxter, Prank Ml, Pim one ae Le vie pir ns wn rae ner: hy " vty ; TP) au ie - ae y ve a ai oi AN Seyi Vi ’ ie Maree a) Pia a nee lie Ea | ts fi “4 a) es jails i hy ; tani “i! (O42 ye eed i Wi ig i yy) wm cate Pay hh 7 18 PORTER. EXPLANATION OF PLATE II. Fic. 16. Section of a cyst containing sporogonia in several stages of develop- ment. X 400. cys, the shrivelled cyst. 8, former position of the wall of the cyst as determined by the surrounding tissue. a, see explanation of Fig. 17. Fic. 17. Group of sporogonia, seen at a in Fig. 16, more highly magnified. X 1540. Fics. 18, 19. Recently formed sporogonia killed with Perenyi’s fluid stained with borax carmine. X 3100. ‘ Fic. 20. Sporogonia after the first division of the nucleus; treatment of the preparation the same as that of Fig. 18. X 3100. a, immediately after the division of the nucleus has taken place. 8, a little later stage than that shown at a. Fic. 21. Sporogonium after the second division of the nucleus; otherwise same as FIG. 20. X 3100. Fic. 22. Sporocyst showing early stage in the development of the spores. X 3100. Two sac-like envelopes already formed ; the outer one (a) the capsule ; the inner one (8) the cyst. a, thickening of the cuticula at the mouth of the capsule. 8, narrow neck of inner cyst. Fic. 23. Sporocyst flattened somewhat by pressure of cover glass. Spores in a little more advanced stage than in Fig. 22. X 3100. Fic. 24. Cross section through the middle of sporocyst and contents in the condition represented in Fig. 23. X 3100. Fic. 25. Cross section of another sporocyst of the same age as Fig. 24. Fic. 26. Inner cyst of sporocyst ; spores not yet fully matured; four of them have been squeezed out. X 3100. pr pl, thread-like portions of contents of cyst not employed in formation of spores. Fic. 27. Two spores forced out of cyst. X 3100. a, clear area due to shrinkage of nucleus. Fic. 28. Two abnormal spores. X 3100. Fic. 29. Mature sporocyst containing seven spores, one in process of emerg- ing: the eighth spore had already escaped. a, thickening of cuticula around the opening at upper end of capsule. Fic. 30. Mature sporocyst with seven spores, three of them leaving the cyst. Probably one spore had already entirely escaped. X 3100. Fic. 31. Mature sporocyst showing a frequent arrangement of spores in cyst. Stained with Bismarck brown. X 3100. Fic. 32. Inner cyst showing irregular arrangement of the eight spores, some of which are considerably curved. Stained in Bismarck brown. X 3100. Fic. 33. Portion of a section through a gregarine cyst showing sections through sporocysts in various directions. X I100. Fic. 34. Cross section of intestine of Clymenella torquata showing large amoeboid cells, possibly early amoeba-like stage of this gregarine. X 77. Fic. 35. Amoeboid cell in wall of intestine near its outer body-cavity surface, more highly magnified. X 350. Fic. 36. Another amoeboid cell of larger size and more deeply imbedded in the wall of the intestine. X 350. ui ry Vier Waist, \) . \ ye ly We Lite ; iad as taut lanl Nahe’ he ee iy ¢ mar Al 20 PORTER. EXPLANATION OF PLATE III. - Gregarine parasitic in Rhyncobolus Americanus. Fic. 37. Normal appearance of a detached gregarine. X 77. Fic. 38. Individual with two nuclei, perhaps about to divide. X 77. Fic. 39. Gregarine, possibly just after division has taken place. X 77. Fic. 40. Longitudinal section showing -apparently a thick cuticular wall. X 187. Fic. 41. Cross section through the middle of the parasite showing the actual thickness of the cuticula and the longitudinal folds into which it is thrown. X 1100. mu, circular muscle fibres, in this case somewhat relaxed. Fic. 42. Cross section, the circular muscular fibres (mz) contracted. X 1100. Fic. 43. Surface view, focusing at the level of the bottom of the furrows (sz/), and showing the circular muscle fibres (wz). X 1100. Fic. 44. Section through the anterior end of the gregarine. X 1100. The protomerite (f7’mer) is seen to be filled with finely granular compact protoplasm. The conical epimerite (e’#er), terminates in a long slender filament (//), and is enveloped in a loose mass of protoplasmic substance (f7’f/), probably derived from the intestinal epithelium of the host. fas, thickened cuticular band forming a rim around the cup-shaped depression. This is left behind whenever the epimerite is torn away from the protomerite. Fic. 45. Section through the cup-shaped depression at the anterior end of the gregarine. X II0oo. Fic. 46. Section through anterior end of gregarine. X goo. cp. sng, blood corpuscles scattered in the protoplasm from host. Fic. 47. Surface view of anterior tip of gregarine. The most of the epimerite has been cut away. Tooth-like processes (de) can be seen projecting radially into the base of the epimerite, which remains in the cup-shaped depression. X II0o. Fic. 48. Side view of the cup-shaped depression and the anterior tip of the gregarine. X II00o. Fic. 49. Section through the protomerite. In the middle of the figure the radial ridges (crs) are cut crosswise. X I100. Fics. 50-52. Different forms and positions assumed by living gregarines. Fic. 53. Outline of the head of a living gregarine when pressed against the cover glass and used as a sucker. Pl. MSc emer pipe. dew'mer pA MU. pe ‘<< ON THE OSTEOLOGY AND RELATIONSHIPS ‘OF PROTOSTEGA. E. €. CASE. INTRODUCTION. THE systematic arrangement of the sea turtles, or Pzzxata, has long been a mooted point among zodlogists. Prior to the year 1870 there was practical unanimity in placing Dermochelys near the members of the Chelonzidae. In the year 1871 Cope (1) separated these forms, and placed Dermochelys in a distinct group, Athecae, opposed to and of equal rank with the Cryptodira and Pleurodiva. One year later, in discussing the genus Protostega (2) he placed it ‘near the Sphargidae in the sub- order Athecae, and in some points to be approximated to the Cheloniidae.”’ In 1875 he established the family Protostegidae (3), a name he had used two years earlier (4). The group Athecae was apparently accepted by Gervais in his description of Dermochelys (Sphargis) (5), and the separa- tion of the group was recognized by Seeley (6), who in 1880 placed Dermochelys in a group Dermatochelyidae, of equal rank and value with two opposing groups, Peltochelytdae and Aspidochelyidae. Doderlein (7) accepted Cope’s classification with the addition of the group 7vionychotdea, and this group was subsequently adopted by Cope (11). Bottger (29) in 1895 recognized the Athecae. LDollo (8) in 1886 published a paper in which he raised the value of Cope’s group Azkecae by placing it in oppo- sition to all the remaining Zestudines grouped under the name Thecophora. This idea he subsequently defended in two papers which appeared in 1887 (9) and 1888 (10). Dollo was supported by Smith-Woodward (12), Bernard (18), and by Boulenger, both alone (13) and in collaboration with Giinther (14). These later authors substituted, as did Lydekker, (15, 16) the name Zestudinata for Dollo’s Thecophora. 22 CASE. [VoL. XIV. In 1873 Riitimeyer (17) disregarded Cope’s classification of two years previous, and placed Dermochelys among the Pznnata. In 1886, the same year as Dollo’s first paper, Baur published anote in the Zoologischer Anzeiger (19), in which he claimed that the separation of Dermochelys from the Chelonitdae was abso- lutely artificial. He maintained his position in papers appearing at intervals from 1888 to 1893 (20-26). Zittel (27) in his text-book, and later Dames (28), disregarded the group Azheeae, the former considering the Dermochelyidae as a family of the Cryptodzra. There are then at present three views as to the position of Dermochelys. (1) It is closely related to the Cheloniidae, being merely a specialized form. (2) It is the sole representative of a group equal in rank to all the remaining Testudines. (3) It is the representative of a group of equal rank with the Z7zony- choidea, Cryptodiva, and Pleurodira+ Paleontology alone can decide which of these theories is correct, and, fortunately, a turtle from the middle cretaceous of Kansas, Protostega Cope, is known, which from its intermediate form affords most valuable evidence in completing the phylogeny of the existing sea turtles. This paper is concerned in describ- ing additional remains of this animal, and discussing its relationships to allied forms. Description of Protostega and Comparison with Related Forms. The material used in the following descriptions of Protostega consists of two specimens, both from the Niobrara cretaceous of Kansas. The first and larger specimen comprises the 1 It may be of interest to give here the synonymy of Dermochelys. 1816: Dermochelys, Blainville, Bull. des Sciences par la Société philomatique de Paris, année 1816, p. 119 (wrongly printed 111). (See Baur’s discussion of the names Dermochelys, Dermatochelys, and Sphargis, Zool. Anzeiger, no. 270, 1888.) 1820: Sphargis, Merrem, Versuch eines Systems der Amphibien, p. 19. 1822: Coriudo, Fleming, Philosophy of Zoology, vol. ii, p. 271. 1828: Scytina, Wagler, Oken Isis, 1828, part 2, p. 861. 1829: Dermatochelys, Wagler, Nat. Syst. Amphib., S. 133. 1832: Chelyra, Rafinesque, Atlantic Journal and Friend of Knowledge, vol. i, no. 2, Philadelphia, Summer of 1832, p. 62. No. I.] RELATIONSHIPS OF PROTOSTEGA. 228 greater part of the plastron and limbs of a very large individual. The bones are in excellent condition, and the sutures very distinct. There are present the hyoplastra of both sides, the hypoplastron of the left side, and weathered fragments of that of the right, the xiphiplastron of the left side, and the distal end of that of the right, the nuchal and eight peripherals determinable as belonging in series on the left side, and fragments of others, the humeri, scapulae, and coracoids of both sides, and the proximal ends of the radius and ulna (?). The femur of the left side, the pubis and ilium of the same side with the distal end of the ischium, the ischium of the right side with the distal ends of the ecto- and ento-pubis. Several incomplete ribs, Pl. IV. The second and smaller specimen preserves the humerus, scapula, and coracoid of the left side, a singularly complete pelvis, and some incomplete ribs. The greater part of the skull is in fairly good condition, showing the basi-, supra-, and ex-occipitals, the paroccipital, quadrates, petrosals, quadrato- jugal, and squamosal, the basisphenoid, pterygoids, and pala- tines, and the almost perfect lower jaw. Skull. — The supraoccipital was greatly flattened from side to side in the process of fossilization. The ridge forming the upper edge of the bone slants downward and backward, its dis- tal part is incomplete, though apparently only a small part has been lost. The superior-anterior portion bears a narrow face which slants downward and forward for a considerable distance. These regions are almost identical with the same regions in the Chelonitdae, and are widely different from Dermochelys, where the upper edge of the supraoccipital is almost horizontal, and is broad and rounded. The face on the anterior aspect is broad, horseshoe-shaped, and almost vertical. The region bearing the articular faces for the exoccipitals, petrosals, and paroccipitals is moderately expanded and is quite solid, showing the absence of any great amount of intercalated cartilage, such as occurs in Dermochelys, where the articular faces are represented by rugose pits, and are not distinguish- able one from the other. The articular faces for the petrosal and paroccipital meet on the summit of a ridge running out- wardly from the external edge of the anterior semicircular canal. 24 CASE. [Vou. XIV. This canal is represented by a deep triangular pit, no part of which is covered by processes from the sides. This condition of the semicircular canal is exactly that of the Chelontidae, and differs widely from Dermochelys, in which it is roofed by three distinct processes meeting in the middle and leaving three foramina for communication with the other semi- circular canals (Pl. V, Fig. 1). The exoccipital of the left side is badly crushed, but is still in sutural union with the paroccipital of the same side, and the two are in connection with the supraoccipital. The exoccipital of the right side is separate and almost perfect. The ascending process for the supraoccipital is short and strong. The descend- ing process is short, and did not reach connection with the pterygoid. The articular face for the paroccipital is deeply excavated. The condylar foramen is near the condylar portion, which is well ossified and free from osseous connection with the same region of the basioccipital. This complete and sepa- rate ossification of the condylar region is a point of decided difference between the Chelontzdae and Dermochelys; in the latter the region is almost entirely cartilaginous, and the three bones are weakly anchylosed in old specimens (PI. V, Fig. 2). The Jdasioccipital is a comparatively broad and short bone with well-ossified condylar portion and strong lateral processes terminating in rugose extremities which extended between the pterygoids and the exoccipitals. The under surface is nearly smooth, and lies in the plane of the horizontal axis of the skull. The articulation for the basisphenoid is confined to its anterior end. In every particular but that of the ossified condylar portion the basioccipital of Protostega agrees with Dermochelys. In the Cheloniidae the lateral processes are small, and the pterygoid articulates with the exoccipital; this causes the basioccipital to lie largely between the exoccipitals, instead of below them as in Dermochelys. The inferior surface of the basioccipital in the Cheloniidae varies from being almost horizontal to being inclined steeply downward and forward, and the basisphenoid may cover it far back towards its middle (Pl. V, Fig. 3; a, from above; 4, from below). No:t.] RELATIONSHIPS OF PROTOSTEGA. 25 The petrosals are both present in a very perfect condition. They are roughly triangular, and have a strong ridge, partly due to pressure, on the external surface. The external face also shows a deep excavation corresponding to a similar excavation on the antero-interior portion of the quadrate. The union of the sides of these two excavations forms the foramen for the external carotid artery, and probably excluded the paroccipital from any part of the foramen. The external semicircular canal is represented by a deep pit bridged in its antero-superior region by a bony bar reaching from side to side, and leaving in front of it a foramen for communication with the anterior canal. The articular face for the basisphenoid is broad and strong. The formation of the carotid foramen, as well as the nature of the semicircular canal, is typically that of the Chelonizdae. In Dermochelys the paroccipital takes large part in the forma- tion of the foramen, and the pit in the petrosal is entirely free from any bony processes (Pl. V, Fig. 4; a, from within; 4, from without). The paroccipital of the right side is present in almost perfect condition. The bone is elongated and reaches connection with the squamosal, a character which never appears in Dermo- chelys. The posterior or external half of the posterior semi- circular canal is roofed by a bony process pierced by two foramina which communicate with the other canals. This character of the posterior semicircular canal appears in the Chelontidae, and is very different from Dermochelys, where there is a single bony process which does not reach entirely across the canal (Pl..V;, Fig: 's): The basisphenoid is badly crushed, but retains somewhat its original character. It is almost round in outline, with a smooth under surface. The upper face is traversed in a longitudinal direction by two deep grooves. There is no trace of an ante- riorly extending rostrum on the thickened anterior end. The smooth under surface appearing largely on the base of the skull, with no trace of a ridge where it meets the basioccipital, is similar to the condition found in Dermochelys, though in that genus the basisphenoid takes much larger part in the 26 CASE. [Vou. XIV. formation of the base of the skull, and separates the pterygoids for a greater distance than it did in Protostega. The guadrates are present in excellent condition; they are still connected with the pterygoids, which are in turn united with the imperfect palatines. The articular face for the quadrato-jugal is strongly developed, and stands on the summit of a prominent ridge. The anterior edge is thin and rounded in outline; near its inferior portion there is developed a short, stout process, which fits into a deep groove on the external face of the pterygoid. This process gave attachment to the colu- mellar plates or the cartilage of its lower end. This strong process of the quadrate is present in Dermochelys; in the Cheloniidae it is very slender, and may even be absent as in Lepidochelys. Near the anterior inferior portion of the inner side lies the groove which in connection with the groove on the petrosal forms the foramen for the external carotid artery. The condylar face is divided into two parts; one, the pos- terior, looking slightly upward and backward; the other saddle- shaped, and looking almost directly downward. The upper posterior portion of the bone shows a strong face for attach- ment with the squamosal. The most distinctive feature of the bone, and one which is shared with none of the other sea turtles as far as observed, is the manner of attachment to the pterygoid; the posterior portion of that bone reaches almost to the condylar face, instead of being separated from it by a con- siderable space. The quadrate stood at almost a right angle to the pterygoid (Pl. V, Fig. 6). . The stapes was comparatively a very large bone. The distal end of one preserved in the stapedial notch of the right quad- rate is larger than the same bone in a skull of Dermochelys twice as large as the skull here described. The pterygotds of both sides are present in fairly good con- dition, the internal edges only being broken and crushed. They are long, slender bones with rounded external edges, decidedly concave external margins, and with no trace of an ectopterygoid process. The posterior portion articulates far down on the quadrate as described, and the posterior external face shows a No. 1.] RELATIONSHIPS OF PROTOSTEGA. 27 deep groove running forward and upward which receives the epipterygoid process from the quadrate. The posterior half is not perforated by a branch of the car- otid artery as in the Chelonitdae, nor does any foramen for this artery appear on the back of the skull as in that family. In these points of difference from the Cheloniidae, and in the fact previously mentioned that it is separated from the exoccipitals by the lateral processes of the basioccipital, the pterygoid agrees with Dermochelys (Pl. V, Fig. 6). The falatines are present in an imperfect condition. The anterior and interior portions are gone, and the whole bone is distorted by pressure; enough remains, however, to show that there were deep choanae located far forwards which were not roofed by the vomer and palatines. This condition of the internal nares is largely that of Dermochelys, in which the choanae are far forward, and are not roofed by the palatines and vomer. The articulation with the maxillaries was by a deep, elongated, triangular region, as in the typical Cheloniidae (EL V, Fig. 6). The vomer is present in a fragmentary condition. It did not have a process descending between the palatines, and help- ing to roof the choanae, as in the Cheloniidae. The guadratojugal of the right side is triangular in general outline. The posterior edge is concave, and the whole bone is convex from above downwards. The superior edge is narrow, and there is no prolongation of the antero-inferior portion, as in Dermochelys (Pl. V, Fig. 7). The sguamosal of the right side shows a broad concave sur- face for the upper end of the quadrate. The posterior inferior portion shows no groove as in the Cheloniidae. The anterior portion is thin and expanded. The mandible is present in a singularly perfect condition, the only parts injured being the posterior portions of the com- plementaries. It is figured in Pl. V, Fig. 8. The whole jaw resembles very much that of the Cheloniidae. The dentary is broad from above downwards, with the upper surface slightly concave in the region of the symphysis, and marked by deep pits. The symphysis is broadly triangular, it 28 CASE. [Vou. XIV. extends farther back on the lower surface than on the upper, and its posterior part is marked by a deep pit. The dentary reaches very nearly to the posterior extremity of the man- dible, covering in large part the complementary and the sur- angular. The complementary is present and complete; it is largely covered externally by the dentary, which also overlaps the superior margin, and appears on thé supero-internal edge. The posterior end rises rather high, and terminates abruptly. Its postero-inferior angle articulates with the angular, and forms a bridge over the cavity in the ramus. The presplenial. Dr. Baur has drawn my attention to the fact that the element described by him (44) in the jaw of cer- tain pleurodiran turtles (the group Chelyotdea), in the Croco- dilia, and in the Lacertilia as the presplenial, also occurs in Protostega and Toxochelys. It is an elongated element articu- lating with the angular and splenial behind, the dentary below, and the coronary above, occupying the same position as in the Chelyoidea, but extending far forward, and covering the groove on the internal surface of the ramus except in its anterior extent. It is pierced by a foramen near its anterior edge. Its general form and relations are shown in the accompanying figure. weil ae. . Dr Dice Vo 94 89935 : wisn IY > B20, 920 2S The sflenial is distinct from the angular, and appears as a long, splint-like bone reaching forward towards the symphysis. It appears largely on the under surface of the mandible. The angular is short and broad; it sends a process upwards to meet the complementary. The posterior end shows a large face concave from above downwards, and looking almost directly backwards. It rises above the surangular, concealing it on the interior aspect. No. T.] RELATIONSHIPS OF PROTOSTEGA. 29 The surangular is broad and short, joining the complementary by its antero-superior portion. The posterior portion bears an articular face, concave from above downwards, and looking slightly inwards; the external margin of this face ends on a thin ridge. The articular is well ossified. Its articular face, slightly saddle-shaped, looks backward and upward. The postero-inferior part is rounded, and shows largely on the inferior surface of the mandible. There is no process on the infero-internal por- tion of the articular, as in the Cheloniidae, and this allows the condylar face of the angular to curve towards the articular at the bottom instead of bending toward the median line of the jaw. Plastron. — The hyoplastron is a large, heavy plate, thickened in the middle and becoming thin towards the edges, which are extended into long, slender, radiating processes. It is roughly triangular in shape. The antero-internal portion is bent slightly inward, and carried as a strong wing toward the median line, where the terminal processes meet those of the same of the opposite side. The antero-external edge is smooth, free from processes, and concave. The external edge is thickened, and the terminal processes are comparatively short and strong. The articulation with the hypoplastron was by small and numerous closely interlocking processes amounting almost to a sutural union. The connection occupied nearly half the posterior edge of the bone (Pl. IV). The ypoplastron is of almost equal size with the hyoplastron. Its general shape is more nearly round, it is furnished with processes on the edges, and those on the posterior internal edge meet those from the same bones of the opposite side, The posterior edge was furnished with two long processes diverging posteriorly, between which the xiphiplastron articulated. The appended measurements will give an idea of the size of these plates, though the loss of the distal ends of the processes makes exact measurements impossible (PI. IV). Greatest length hyoplastron . . . . . . .649 meters ss breadth a 6 tet wo gg = BIRO WIS. |G s length hypoplastron. . . . . . .578 S breadth se Pete: al eee ee 30 CASE. [VoL. XIV. The xiphiplastron is an elongated bone articulating by an interlocking joint between the posterior processes of the hypo- plastron. It differs from all sea turtles in the peculiar bending of the bone near its middle; originally directed inwards and backwards, it changes its course abruptly, and is directed inwardly at almost a right angle to its original course. It articulated strongly with the xiphiplastron of the opposite side (Pl. IV and Pl. V, Fig. 9). There is nothing preserved of the ento- or epi-plastron. The general shape of the plastron was broadly ovate with the posterior end truncate. The fontanelle was diamond-shaped, and bridged at its anterior and posterior extremities by the lateral processes from the different plates. The plastron stands midway between that of Dermochelys and that of the Chelonitdae. In Dermochelys the union of the hyo- and hypo-plastra is by the overlapping of the extremities of the slender bones which have lost their radial processes. The xiphiplastron is straight, and articulates by overlapping with the hypoplastron. In Protosphargis Cap., the plastral bones are more robust than in Dermochelys. The marginal processes are retained to some extent, and the hyo- and hypo-plastra articulate by the interlocking of a few digital processes. The xiphiplastron is straight, and articulates with the hypoplastron by overlapping. The Cheloniidae have a broad sutural union between the two plates of the plastron. The marginal processes are confined to the distal ends of the bones, leaving the edges near the suture round and smooth. An approximation to this state can be noticed in Protostega, where the marginal processes near the union of the plates are shorter than those on the ends. The nature of the processes varies among the members of the Che/- onttdae. In Lepidochelys kempt Garm. they are numerous and irregular, standing on a base that leaves the main body of the plate in a curve, thus forming an oval or rounded fontanelle. In Chelonia there are generally only three processes on the hyoplastron, two of which project from the body of the bone at right angles, and meet across the squarish fontanelle, while the other passes obliquely forward toward the epiplastron. No. 1.] RELATIONSHIPS OF PROTOSTEGA. 31 The series with Dermochelys at one end, and Chelonia at the other, is marked by a constant variation in the size of the plas- tral elements, the nature of union of the bones, and the presence and position of the marginal processes. Carapace. — The vzbs are present in fragmentary condition in both specimens. The head was well developed and separated from the costal plate, the proximal end of which was expanded and produced into slender digitations. Examination of a speci- men of Chelonia mydas shows that the distance from the point of union of the rib head with the costal plate to the vertebral articulation is greater than the distance from the same point to the neural edge of the costal plate. In Pvotostega, as these specimens show, the opposite is true even when as in this case the measurements are carried only to the broken ends of the digitations. This shows that there was proportionally less room between the proximal ends of the costal plates in Protostega than in the living sea turtles, and in all probability too little room to allow the presence of neurals. This supposition is further borne out by the digitated proximal ends of the ribs and the entire absence of anything that can be referred to neurals in the known specimens. The expansion of the ribs extends for about half their length (Pl. VI, Fig. 19). The nuchal plate is very peculiar in form, resembling most nearly the nuchal of the soft-shelled turtles. In the present specimen the plate lies directly on the hyoplastra, having been crushed down on them, and has preserved them in their cor- rect relative positions; it is thickened in the middle, becoming thin laterally, and expanded into broad wings. The distal ends of the wings are irregular in outline, and probably articulated weakly with the first peripheral. The upper surface shows two low rugose ridges which run from the center out into the wings, and there disappear. The anterior edge was concave, and beveled from above downward and backward. The posterior edge is continued into a long, slender process running back over the vertebral column. The postérior end of the process is broken off, but apparently only a small portion is missing. There is no process on the under side for articulation with the posterior cervicals (Pl. V, Fig. 10). 32 CASE. (Vor. XIV. Following are some measurements of the nuchal plate. Length from tip to tip of wings .. . $9 ->. . 599 meters Length from middle of anterior edge to ea y gceurtoy process .168 ‘« Thickness incenter. . . Moe, ee a eet 0 Breadth of left wing in Beets oe. ni SSG YON, We desc ghaed bien aiekey Canam The peripherals are represented by eight from the left side, which are determinable as belonging in series, and several detached bones whose position is doubtful. The series extends from the second (?) to the ninth. The anterior, which probably joined the nuchal, is unfortunately lost. The second (?) periph- eral is slender, concave on its outer edge, and bears no facet for arib. It articulates strongly with the third. The third is strong and broad, and bears a deep pit for a rib near its external margin. The thickened external margin is turned slightly downward and inwards. In the succeeding peripherals the length becomes greater than the breadth, and the external margin becomesacute. The turning in of the margin begun on the third becomes broader and broader, shoving the pit before it till in the posterior peripherals it occupies half the under surface of the bones, and its anterior edge underlies the centrally located pit. The inner edges of the peripherals are irregular, and extended into slender processes. Pl. V, Fig. 11, shows the upper surface of the fifth and sixth, and gives a good idea of the general shape of the peripherals and their strong articulation one with another. The following measurements are accurate in so far as the broken condition of the inner edge would permit. Length second (?) pone See ae reel O pILELETS Breadth ‘“ moran (84 fou sam Sesion » ie Length third x el et Soe deere zO2 Re Breadth ‘“ ue ROM nh rok avon Length fourth se MMC ar S ditetoe fe Breadth “ ey Siete s oo EA The condition of the carapace of Protostega as described above is heralded in the young of Chelone Benstedii Owen, where the costal plates taper from the proximal to the distal end, and in Al/opleuron Baur (Chelone Hoffmani Gray), where the ribs have become very slender and the costalia short and No: 1-] RELATIONSHIPS OF PROTOSTEGA. 33 broad. In Protosphargis and Dermochelys the rib head is not covered by an expansion of the upper surface of the rib. The loss of the neurals may find its initial step in the condi- tion of Eosphargis (Chelone gigas Owen). There are in that genus, as described by Lydekker (30), six or seven large plates overlying the ribs; these were considered by Owen in the original description as neurals, but are considered by Lydekker as dermal scutes. It is difficult to see, however, whence the “median dorsal row of large carinated scutes”’ may have taken their origin if they are not neural plates which have lost con- nection with the vertebrae, and become laterally expanded so as to cover the ribs in part. This loss of connection between the neurals and vertebrae may be observed in the recent Lepidochelys kempt Garm., where 1, 2, 3, and also 8 are freer (21). The strong peripherals of Protostega were possibly present in Losphargis (30), and peripherals have been observed in Protosphargis (31). They were very slender in Protosphargis, and were considered by the original describer as phalanges, but were later shown by Baur to be peripherals. Most of the Chelontzdae have the typical number, eleven, but Thalassochelys and Lepidochelys have more, the number being varied by the introduction of one or two extra plates between the 5 and. 3 (22). The nuchal plate of Protostega differs widely from that of the living sea turtle, but in no point more widely than in the complete absence of the process on the under side for articula- tion with the last cervical. In Osteopygis, a sea turtle from the cretaceous, there is no trace of this process, but in Lytoloma, a form from the upper cretaceous and eocene, the eocene forms show the beginning of the process in a small tubercle (22). The carapace of Pvrotostega is now seen to be intermediate between the Dermochelytidae and the Cheloniidae, with several primitive characters which are ancestral to both. The vertebrae are represented by only two, from the caudal region. These are deeply concave in front, with the arch ossi- fied with the centrum. The anterior zygapophyses extend well forward of the anterior edge of the centrum, and the top of the 34 CASE. [VoL. XIV. arch is broad and rugose. There is a triangular articular face at the base of the arch on the anterior portion of the centrum. The description of the vertebrae from Pvofostega given by Cope (32) is here quoted to show their general nature. He says of the vertebrae: “These have been recognized chiefly by their neural arches, which are separate. They are in form something like an X, the extremities of the limbs car- rying the zygapophysial surfaces. The only point of contact with the centrum is a wide process, which stands beneath the anterior zygapophyses, and spreads out foot-like obliquely for- ward and outward to beyond the line of the anterior margin. Its surface extends nowhere posterior to the surface of the zygapophysis above it, but a little farther inward. Its outer margin rises ridge-like to the under side of the neural arch, and each one, forming a semicircle, forms the boundary of the neural, and turning outward, forms the zzzer boundary of the posterior or down-looking zygapophysis. The space be- tween these apophyses is roofed over so as to produce a shallow zygantrum, which, however, only seems to roof over the deep emargination of the neural arch of the vertebra immediately following. The anterior zygapophyses are often broken away, so that the neurapophysial supports look like the missing pair, when the difficulty ensues that both pairs look downward. The top of the neural arch is, in two cases, broad and flat; in two others there is an obtuse keel. «The centra, apart from their arches, are puzzling bodies, especially since in the present case they are somewhat flattened by pressure. They differ materially in size, one of them being twice the size of the others. The smaller ones are of the ball- and-socket type, and have a deep longitudinal groove on each side. The thickened portion of the centrum forms the inferior boundary of the pit groove, while a thinner portion, possibly a diapophysis, limits it above. It is, however, thin, and has no great length. There is no sign of chevron bones and articula- tion, so that these vertebrae may have been cervical. Their bodies are, however, shorter and wider than in those vertebrae of any known tortoise. A groove on the upper surface repre- sents the neural canal, while a flat area on each side in front No. I.] RELATIONSHIPS OF PROTOSTEGA. 35 supports the neuropophyses. The large centrum exhibits the superior groove and antero-lateral platform for support of the neural arch. One end is cupped obliquely, while the other is nearly plane, with the same obliquity and a slightly raised margin. Its outline is subtriangular. The lower side of this centrum possesses a short keel posteriorly. The sides exhibit no pit, but have a thin edge, which is concave behind the middle and then turned outward. I can see no articulation for a rib.” These vertebrae are stated by Cope to be most closely related to Dermochelys. Unfortunately, the material is too limited to admit any positive conclusions to be drawn as to the relationship of Protostega ; but it is necessary here to note the close resemblance between the cervical vertebrae of Dermo- chelys and the Chelonitdae. Both have the strong articular process for the nuchal plate on the last cervical, and the articular faces between the 6 and 7 are plain. Limbs.— The humerus is very broad and strong. The area: for cartilaginous attachment on the mesial process is entirely separate from the area on the head, which is in turn separate from the radial process. In the smaller specimen the areas are all united. This is evidently a variation due to age, as the same thing is observable in large and small specimens of Chelonia mydas. The radial process lies near the center of the shaft, and is very prominent. It is simple, instead of having the U or V shape of the same process in existing sea turtles and in Psephophorus. The ento- and ecto-condyles and the entepicondylar and ectepicondylar processes are strong and prominent. The ectepicondylar foramen is quite large. The shaft of the bone was somewhat flattened and constricted beneath the head (Pl. VI, Fig. 12). MEASUREMENTS. Length from distal end to top of head. . . . . . . . .348 meters Greatest width at distal end . . . . AL es OGD (Se Length from exterior edge of head to end of mesial process .175 “ The humerus shows a somewhat close resemblance to that of Psephophorus and Dermochelys. The radial process is simple, stands higher on the shaft, and lacks the downward prolonga- 36 CASE. [VoL. XIV. tion shown in those forms. The higher position of the radial process is a primitive character, and is well shown in Lytoloma (33) and in Chelonia girundica (34), as figured by Delfortrie. The vadius and w/na are apparently represented by the proximal ends of two bones that from their size could not have belonged to the posterior extremity. They are so crushed as to afford no distinctive characters. The scapulae show a broad angle between the scapula proper and the proscapular process. Both parts are strongly com- pressed, but show on their ends large areas for cartilaginous attachment. The neck of the glenoid portion is short in com- parison with existing members of the Chelonitdae. The pro- scapular process is much the shorter. The glenoid articular portion shows two faces: one for the coracoid and the other forming part of the glenoid cavity. The whole bone is very strongly built (Pl. VI, Fig. 13). The coracoids are long, slender bones, greatly thickened proximally where they articulate with the scapulae. Distally the shaft becomes flattened and very thin. Upon the upper surface there is a strong ridge running from the proximal end out into the flattened part of the shaft, where it disappears. This ridge is present in both Chelonta and Dermochelys, but is absent in 7halassochelys. In the latter form the whole bone is proportionately shorter and stouter (Pl. VI, Fig. 14). MEASUREMENTS. Length of most nearly complete bone . . . .405 meters Breadth qistaliende 29 sat. x5 pic eel kot omens MOISES The pubis has a very large and distally expanded ectopubis. It is much larger than the entopubis, and joins it at almost a right angle; in these respects it differs from the living sea turtles, where the two processes meet at an angle. The great- est axis of the ectopubis is in almost a line with the axis of the whole pubis. In the Pzzxzata these two meet at an angle. The entopubis joins the main body of the bone at almost a right angle by a very short and very broad neck, the anterior edge of which nearly reaches the edge of the acetabular face. The symphysial faces of the entoischia were nearly straight, INO T.(] RELATIONSHIPS OF PROTOSTEGA. 37, so that they touched for a great part of their length. The process bearing the articular faces for the other bones of the pelvis is short and strong (Pl. VI, Fig. 15). MEASUREMENTS. Length from proximal end to end of ectopubis . . . . . . .243 meters se “« external edge of bone to ischial symphysis . . . .169 “ a “« point on shaft opposite center of entoischium to proximal end sea fs 1m oak eS ey OST The zschtwm is somewhat hourglass-shaped in profile, with the distal end the largest and the middle of the bone much con- tracted. The broader portion of the shaft is thin, and the anterior edge rounded and thickened. The symphysial edge is somewhat convex, the two bones meeting probably in the middle portion only. The pubo-ischiatic foramen was small in Dermochelys (Pl. VI, Fig. 16). The z/ium is a short, strong bone, concave on its lower sur- face, and angularly convex above from before backwards. The distal articular surface is confined to the end of the bone. The center of the upper side is rugose for cartilaginous attachment (Pl. VI, Fig. 17). The figure of the ilium is made from the smaller specimen, as it is much more perfect than the larger. The femur is much smaller and more slender than the humerus. The distal end is expanded. The shaft is contracted below the head, which was supported on a well-developed neck (El Way. Figs 18). MEASUREMENTS. Renpthortemur. 2) ek be is a |; . .295 meters Greatestibreadthadistalyendijy sian en ne eo ne Breadth center of shakes iii. a). io 4 +.) 2098) Y“ Protostega has, then, the following points in common with the Cheloniidae: the peripherals, the condition of the plastron (part.), the lack of such a large amount of intercalated cartilage in the articulations of the bones of the skull, the nature of the semicircular canals in the paroccipital, petrosal, and exoccipital, and the shape of these bones; the formation of the foramen for the external carotid by the petrosal and quadrate to the almost complete exclusion of the paroccipital, the form and position of 38 CASE. [VoL. XIV. the quadrate, the form of the squamosal and its close articula- tion with the quadrate, the articulation of the paroccipital with the squamosal, the well-ossified and separated condylar portions of the exoccipital and basioccipital, the manner of articulation of the palatines with the maxillaries, the posterior nares (part.), and the form of the mandible. With Dermochelys it agrees in, the broad basioccipital with its lateral processes preventing the articulation of the pterygoid and exoccipital, the broad basisphenoid separating the ptery- goids on the base of the skull (to a less extent than in Dermo- chelys), the nonappearance of the pterygoids on the posterior aspect of the skull and their not being perforated by a branch of the carotid artery, the large groove on the pterygoid for the epipterygoid process of the quadrate, the large epipterygoid process of the quadrate, the posterior nares (part.), and the vomer, the lack of a carapace, the large nuchal, the humerus (part.), and the plastron (part.). There should also be mentioned here the stapes, which is even larger than in Dermochelys. Points separating Pvotostega from both forms are the lack of dermal ossifications on the back, the manner of articulation of the pterygoid and quadrate, the presence of a presplenial bone in the jaw, the lack of any articular process on the under side of the nuchal, the simple radial process of the humerus, and the peculiar bent form of the xiphiplastra. Protostega is distinctly an intermediate form. In the paper containing the description of Pyvotostega (2) Cope attempted a restoration from the material at his command. He estimated the head as 243 inches long, and by assuming the proportions to be near those of Chelonza, the neck and cara- pace as 1382 inches, making a total length of 154% inches, or 12.83 feet. (He evidently deducted 8 inches from the neck as remaining within the carapace.) From the ribs and vertebrae he estimated the width of the carapace to be 36} inches, and the length 118 inches. The series of marginals did not justify this length, but he considered that they were not united, and that the intervening spaces would make up the deficiency. His final conclusion was that the carapace was more elongate and narrower than in any existing form of sea turtle. No. I.] RELATIONSHIPS OF PROTOSTEGA. 39 In a recent paper (43), Dr. O. P. Hay has described portions of the plastron of a large specimen of Protostega, and attempted a restoration. The materials on which his restoration was based were the almost complete hyo- and hypo-plastra of one side and a fragmentary nuchal. Regarding the plastron he says (p. 58): “In Zhalassochelys the anterior end of the epi- plastra extends in front of a line joining the bottoms of the excavations for the fore limbs a distance equal to that from the bottom of the excavations for the fore limbs to those of the hind limbs. This, in the Pvotostega plastron before me, amounts to 84 cm. The xiphiplastra of Zhalassochelys extends behind the excavations for the hinder limbs as far as do the epiplastra from the anterior excavations. If these proportions hold good for Protostega, the whole length of the plastron would amount to at least 2.4 meters’’; and further (p. 59): ‘had the breadth of the body of Pvotostega possessed the same ratio to the length that we find in Thalassochelys, the lower side of the animal would have been about 2.2 meters wide.”’ In regard to the fontanelle: “if we have placed the plastral bones aright, there is left between them a great fontanelle. Where the hypoplastra are widest, this is about 43 cm. in width, and oppo- site the union of the hyo- and hypo-plastron about 90cm. This is somewhat smaller, however, than the fontanelle found in Protosphargis, and much smaller than that of Dermochelys.” The head he estimates as 32 cm., from the snout to the end of the occipital condyle, and concludes as follows (p. 62): “The length of the carapace of Chelonia has a ratio to the plastron of about 31 to 24. Hence the length of the carapace of my specimen must have been close to 3.1 meters. The neck of our living marine turtles projects beyond the front of the cara- pace a distance equal to at least 4 of the length of the carapace. Hence we are safe in allowing 50 cm. for the neck outside of the shell. We have, therefore, for the length of this turtle the following figures: ead Mgnt ih ice Noni shee UILeLeTS Neck beyond carapace . . .50 “ Carapacev pment! tiie couse LON pace POtali Msc shi hol ia wise cnashy os Meee SOB eee) 40 CASE, [VoL. XIV. The specimens just described afford material for quite accu- rate measurements, which give results different from those obtained by Cope and Hay, the main discrepancy being in the relative length of the carapace to its breadth. The present specimen shows the peculiar bent condition of the xiphiplastra, which was not indicated in the specimens described by the authors mentioned. This would account for a considerable reduction of the length of the plastron, and a still further reduction is quite certainly to be found in the condition of the epiplastra. In none of the known specimens has any trace of epiplastra been discovered, and neither in the specimen here described nor in Dr. Hay’s specimen can I find any trace of attachment of the epiplastra. Moreover, the anterior ends of the hyoplastra meet over the anterior end of the fontanelle. In the plate of Protosphargis given by Capellini the restored epiplastra extend beyond the exterior end of the hyoplastra a distance of one-tenth the length of the plastron as restored. This restoration is open to doubt, however, as the close resem- blance of Protosphargis to Protostega makes it possible that the distal ends of the xiphiplastra were incurved as in Proftostega. Only the proximal ends of both epiplastra and xiphiplastra are known. It may be assumed, however, for the purposes of this restoration, that the epiplastra extended in front of the hyo- plastra a distance of one-tenth the length of the plastron. The distance from the posterior edge of the conjoined xiphiplastra to the anterior extent of the hyoplastra is 1.15 meters; adding to this one-tenth the length of the plastron, we have 1.27 meters, instead of 2.4 meters, as estimated by Hay. Fortunately, in the process of fossilization, the nuchal plate was pressed down upon the plates below, preserving them in their normal position, and rendering it possible to give exact measurements of the fontanelle. It was bridged in its anterior and posterior extent by the processes from the plastral plates, and at its widest part measured .§25 meters, instead of .go, as estimated by Hay. If we assume the ratio of the carapace to the plastron as 31 to 24, as in Chelonia, the carapace was 1.64 meters long. In a three-fourths grown specimen of Chelonia the ratio of the No. 1.] RELATIONSHIPS OF PROTOSTEGA. 41 breadth of the plastron to the breadth of the lower surface of the turtle is as 5 to 6. The distance across the plastron in this specimen of Pvotostega in its widest place is 1.029 meters; and this, according to the ratio stated, would make the lower surface of the turtle 1.235 meters wide. The widest part of the carapace in Chelonia does not correspond with the widest part of the plastron, but is broader somewhat behind it, so the general form of the carapace was not long and narrow, but almost round. As shown in Pl. V, Fig. 6, the quadrate, pterygoid, and pala- tine of the smaller specimen are all united and very slightly distorted by pressure, especially in a linear direction. The measurements of these bones, including length of quadrate, length of condylar face of quadrate, and length from posterior end of quadrate to anterior end of palatine, are almost exactly the same as that of a skull of Chelonia mydas, which measures .197 meters from snout to occipital condyle. The humerus of the smaller specimen is six-elevenths as large as the same bone in the larger specimen, both being in excellent condition. If it is assumed that the same ratio applies to the head, the larger specimen would have a skull measuring .363 meters from snout to occipital condyle. No material is at hand to give exact measurements of the neck, but assuming with Hay that the neck extended in front of the carapace a distance equal to one-sixth of the carapace, it would have a length of .278 meters. The exact figures are: Plastron, from xiphiplastra to anterior end, hyoplastra . . . 1.15 meters Breadth of fontanelle at suture between hyo- and hypo-plastra. .525 “ Breadth of plastron at widest part of hyoplastra. . . . . . 1.029 “ The estimated figures are: iengta of carapace = =’. . .' “1.640 meters enpthvotbeady) Siete te fh 6 gOgt Wength Gh mechs. Mies ve Weel a) Ue lp 27 Ou Ne Ota mee usar Cer Viele au ffl t21-p' 20977 3th ace Widthtof carapacelnn ts. <6 \ 1687. BOULENGER, G. A. Remarks on a Note by Dr. G. Baur on the Pleu- rodiran Chelonians. Ann. and Mag. Nat. Hist. Oct., 1888. BOULENGER AND GUNTHER. Article in Eucyclopaedia Britannica, vol. xxiii; and BOULENGER. Catalogue of Chelonians. 1889. LYDEKKER, R. JVature, vol. lx, no. 1, p. 6. 1889. LYDEKKER, R. Catalogue of the Fossil Reptilia and Amphibia of the British Museum, part iii. 1889. RUTIMEYER, L. Die Fossilen Schildkréten von Solothurn und der ibrigen Jura-formation. Neue Denkschriften der allgemeinen Schweizerischen Gesellschaft fiir die ges. Naturw., Bd. xxv. Ziirich, 1873. BERNARD, FELIx. Paléontologie. Paris, 1895. 31. 32. 33- 34. 35: 36. 37- 38. 39- 40. 4l. CASE. [VoL. XIV. Baur, G. Osteologische Notizen tiber Reptilien. Zoolog. Anzeig., no. 238. Nov. 22, 1886. Baur, G. Unusual Dermal Ossifications. Sczezce, xi, no. 268, p. 144. March 23, 1888. Baur, G. Osteolog. Not. Zoolog. Anzeig., no. 285. 1888. Baur, G. Osteolog. Not. Zoolog. Anzeig., no. 298. 1889. Baur, G. Die systematische Stellung von Dermochelys Blnv. Szolog. Centralblatt, Bd. ix, nos. 5,6. Mair und 15, 1889. Baur, G. Nachtragliche Bemerkungen uber die systematische Stellung von Dermochelys Blnv., Bd. ix,‘nos. 20, 21. Dec., 1889. Baur, G. On the Classification of the Testudinata. Am. /Vat,, p. 530. June, 1890. ; Baur, G. Notes on the Classification of the Cryptodira. Am. Vat. July, 1893. ZITTEL, CARL VON. Handbuch der Palaeontologie, p. 517. 1889. DAMES, W. Die Chelonier der Norddeutschen Tertiarformation. Palacontolog. Abhandlungen Herausgegeben von Dames und Kayser, Neue Folge, Bd. ii, Heft 4. BOTTGER. Zoolog. Centralblatt, erster Jahrg., nos. 21-25. Jan., 1895. LYDEKKER, R. On the Remains of Eocene and Mesozoic Chelonia and a Tooth of (?) Ornithopsis. Quart. Journ. Geol. Soc. May, 1889, p. 241. CAPELLINI, GIOVANNI. I] Chelonio Veronese (Protosphargis vero- nensis Cap.) scoperto nel 1852 nel cretaceo superiore presso Sant’ Anna di Alfaedo in Valpolicella. Reale Academia dei Lincet (Anno cclxxxi, 1883-1884). Roma, 1884. 36 p., 7 pl. Corr, E.D. Report of the U.S. G.S. of the Territories, Hayden, 1876. Vol. ii, Cretaceous Vertebrata. DoLLo, L. On the Humerus of Euclastes. Geol. Mag., vol. v, no. 6. Dec. 3, 1888. DELFORTRIE, M.E. Les Chéloniens du Miocéne Supérieur de la Gironde. Actes de la Société Linnéene de Bordeaux, tome xxvii, 4¢ livre. 1870. DeE BLAINVILLE. Bull. des Sciences par la Société philomatigque de Paris, année 1816, p. 119. MEYER, H. v. Neues Jahrbuch fiir Mineralogie, Geognosie, Geologie und Petrefactenkunde. K. C. Leonhard und H. G. Bronn, 1847, p. 579: Corr, E.D. Proc. Acad. Nat. Sct. Phil., p.147. 1868. CopE, E.D. TZvans. Am. Phil. Soc., vol. xiv, part i, p. 144. 1870. FITZINGER. Axnnal. Mus. Wien, vol. i, p. 121. 1835. WINKLER, T. C. Les Tortues fossiles conservées dans le Musée Teyler et dans quelques autres Musées. Harlem, 1869. UpaGus, C. La Machoire de la Chelonia Hoffmani Gray. Avznales Soc. de Belge, tome x, pp. 25-35, Pl. I. No. 1.] RELATIONSHIPS OF PROTOSTEGA. 55 42. UBAGHS, C. Le Crane de la Chelone Hoffmani. Bud. de la Société Belge de Géologie, de Paléontologie et d’Hydrologie, tome ii. Brux- elles, 1888. pp. 383-392, Pl. X-XII. 43. Hay, O. P. On Certain Portions of the Skeleton of Protostega gigas. Field Columbian Museum Publications, Zoélogical Series, vol. i, no. 2. Chicago, 1895. 44. Baur, G. Ueber die Morphologie des Unterkiefers der Reptilien. Anatomischer Anzeiger, Bd. xi, no. 13. 1895. EXPLANATION OF PLATE IV. | Photograph of plastron with nuchal plate and peripherals. LEA AR logy Vol. XW. Journal of Morpho Case dei ' = a ees x , oe a> ‘ >» Fe. 58 CASE. EXPLANATION OF PLATE V. Fic. 1. Supraoccipital 4. The badly crushed petrosal and paroccipital are not shown. Fic. 2. Exoccipital 4. Right side. Fic. 3. Basioccipital 4. a, from above ; 4, from below. Fic. 4. Petrosal 4. a, from within ; 4, from without. Fic. 5. Paroccipital 4. Right side. Fic. 6. Quadrate, pterygoid, and palatine 4. Right side. Fic. 7. Quadrate-jugal 4. Right side. Fic. 8. Lower jaw. Fic. 9. Xiphiplastron 1. Left side, showing attachment with hypoplastron and xiphiplastron of right side. Fic. 10. Nuchal plate a little over +. Fic. 11. Fifth and sixth peripherals. Left side. Journal of Morphology Vol.xwv: :: - 4 Si 60 Fic. Fic. Fie. FIG. FIG. FIG. Fic. Fic. 12: se 14. ES: 16. Ez. 18. 19. CASE. EXPLANATION OF PLATE VI. Humerus }. Right side. Scapula 4. Right side. Coracoid 3. Right side. Pubis 4. Right side. Ischium 4. Right side. Ilium 4. Right side (taken from smaller specimen). Femur 4. Left side. Rib head 4. (Taken from smaller specimen.) THE INNERVATION OF THE AUDITORY EPI- THELIUM OF MUSTELEUS CANTS,, De Kay: ALBRO D. MORRILL. CONTENTS. Pack VI EI 0 TT be ce ee Ey eee Bs te es see ise vss ube 3bs cds cdogeusnbenecmepestesbieppesoeuoeecete 57 ti Jrecent work on anditory epitheliwnn.oi:.b nes ceseecnsesseecepacestoramescne 57 Ze INeKVeleMCIN gS INMGROS?S) LOWE UC sas: -seeye ce soe een sano nescence eee) OG PLEO Bi et ope as oa cases ee SER Senn ose coca aee poiren eae dune oonent darstenanconackaae ty OF Ill. JLnnervation of Auditory Epithelium tn Musteluts...........ceeeececceccceeeesees 68 MN SLA PISACLE SESE EE AERC Ory ET ee er ee ee ee eee ee nutbeaeereceerass 71 I. Historical. 1. Recent work on auditory epithelium. — The application of the methods of Ehrlich and Golgi in their original and variously modified forms to the study of the auditory epithelium has greatly increased our definite knowledge of its innervation. Although there is considerable general uniformity in the results obtained by different observers, there are several dis- puted points, particularly in the interpretation of the results obtained. One of these which is of the greatest importance is the exact relation of the terminal branches of the nerve fibers to the auditory or hair cells. The numerous investigations carried out by Retzius by the old methods, as well as by the most recent, have led me to give a brief statement of the results of his earlier observations before considering more recent conclusions. In his great work, Retzius ('84) states that in general he was able to trace the nerves into the auditory epithelium and follow the naked nerve fibers between the basal layers of the support- 1 I wish to express my indebtedness to Dr. Howard Ayers, at whose suggestion the work was undertaken; also to the Director of the Marine Biological Laboratory at Woods Holl, Mass., for many privileges enjoyed during the progress of the work. 62 MORRILL. [Vou. XIV. ing cells in a nearly perpendicular direction until about half the height of the epithelium was reached. At this point branches were given off extending horizontally at the base of the hair cells, and each was applied quite firmly to from three to five hair cells at their proximal ends. The determination of the exact relation existing between the nerve fibers and the hair cells was not accomplished, owing to the inadequate methods then in use. In the ear of man he describes a shell-like expansion of the nerve at the base of the hair cell in which these cells were sup- ported. In the pigeon’s ear he saw fine varicose nerve fibers ascending between the hair cells, as had been already observed by several investigators. That the relation of the nerve fibers to the hair cells was uncertain is shown in Retzius’ account of the auditory epithe- lium of the rabbit: “Ihre definitive Endigung an oder in den Haarzellen ist deshalb gewissermassen noch ein ungeloste Frage.” Again, in the account of the cat’s ear: “Es scheint also als ob die feinen Fibrillen sich dem Protoplasma Haarzellen anlegten und sich an demselbern einigermassen befestigten ob sie aber in dasselbe eindringen ist sehr schwer zu entscheiden.” Kaiser (91), with the Golgi method, found the nerves of the auditory epithelium ending in cup-like structures, which were hyaline and contained numerous highly refractive granules. The hair cells fit into these structures, as he says, like eggs in egg cups. Kaiser considers these cup-like structures as made up of nervous material, which form a connection between the nerves and hair cells. On this account he considers them of the greatest importance. Ayers (92) states that in the cochlea the sensory or hair cells are directly continuous with the fibers of the cochlear nerve. In a paper (93) devoted wholly to a study of the rela- tions of the auditory nerve and hair cells the previous statement is confirmed. ‘From the center of the base of each hair cell issues a nerve fiber which in a favorable case admits of being traced through a ganglion cell of the cochlear ganglion into the collection of fibers which pass to the brain.”” These fibers were No. 1.] EPITHELIUM OF MUSTELUS CANIS. 63 found to exhibit many varicosities in their course which vary greatly in size. The largest varicosities were nearly as large as the ganglion cells. It was thought, from results still unpub- lished obtained with Ehrlich’s method, that these varicosities were of a cellular nature, probably sheath cells. The nerve fibers stained in this way were found to be uniform in size. Dr. Ayers considers the hair cell as a genuine nerve cell and the cell of origin of the auditory nerve fiber. He thinks that at an early stage of development the ganglion cells are produced by the division of the superficial hair cells, and as development advances a protoplasmic thread connects the two cells. The nerve fiber soon begins to develop from the proximal end of the bipolar ganglion cell and extends to the brain. The relations existing between the hair cells and nerve fibers in the maculae and cristae acusticae were found to be essentially the same as in the cochlea only simpler. In the summary we find these statements: “That there is no fundamental difference between the acoustic and olfactory elements yet made known.” “ That all fibers of the auditory nerve proceed out of hair cells alone so far as has yet been satisfactorily determined.”’ Niemack ('92), in his observations on the auditory epithelium of the frog and rabbit with Ehrlich’s method, found that the nerve after losing its sheath divided dichotomously many times. The final branches were of equal size, and, after rising to the level of the proximal ends of the hair cells, extended horizon- tally, forming a close network about their bases or a kind of sieve, in the meshes of which the hair cells rest. He found, as Kuhn had previously done in fish and amphibia, two kinds of nerve endings: one connected with the proximal ends of the hair cells, while in the other the nerve fibers pass in the inter- stices between the hair cells to end free at the surface in knob- like enlargements. Niemack found triangular swellings in the nerve fibers at their point of branching, as had been previously observed by Retzius. In the maculae he found a granular layer separating the nerve fibers from the base of the hair cells; a thin mantle of violet granules was also found covering the surface of these cells. 64 MORRILL. [VoL. XIV. When examined with high powers, the dark varicosities of the nerves could be distinguished. He concludes after careful examination that the granular mantle had no connection with the nerve fibers: ‘“ Mit Nervenfasern zeigten sie keinen zusammenhang.” Geberg ('92) found both kinds of nerve endings: one free in knob-like enlargements at the surface between the hair cells, and the other in nerve fibers attached to the surface of the cells, but not continuous with them. Retzius ('92) did not obtain satisfactory results with Ehrlich’s method, but with Cajal’s modification of Golgi’s method used on embryonic or very young chickens and mice he secured good results. In comparing auditory hair cells and olfactory nerve cells, he says: ‘ Die Haarzellen sind deshalb keine Nervenzellen sie sind den Riechzellen keinwegs gleichzustellen.” The bipolar ganglion cells of the acusticus are considered by him to be the true auditory nerve cells and correspond with the olfactory nerve cells. In numerous preparations he never saw a nerve fiber arising from a hair cell. The hair cells he classes as secondary sensory cells. Retzius, who had previously described a structure similar to the hyaline cup observed by Kaiser connecting the nerves and hair cells, does not accept the latter’s view in regard to its nature. In 1893 Retzius confirmed his earlier observations by the study of rat and trout embryos. In this publication he states that he does not always find Kaiser’s cup-like structure present. In the rat the hair cells were seldom stained, and then were of a clear chestnut-brown color, which did not interfere with the study of the relation of the nerve fibers and hair cells. Van Gehuchten’s results, obtained wholly independently of Retzius, were almost exactly the same in the main points. Lenhossék ('94) found in his study of the auditory epithelium of young mice that supporting cells were frequently stained a deep black with the Golgi method, while the hair cells when stained at all were of a clear brown color. As Retzius had previously observed, he saw the deep black nerve fibers clearly outlined on the surface of the hair cells. He did not find the NOs Te] EPITAELEACE ORVMUSTELUS: CANIS. 65 nerve fibers extending so near the surface as Retzius (93), Kaiser ('91), Niemack (92), and Geberg ('92), but thinks the difference between his results and those of Retzius may be due to the age of the embryos studied. At the point of division or separation of the nerve fibers he observed the characteristic tri- angular thickening, and says that the nerve fibers were of nearly equal size. He did not find the wide-meshed network of nerves described by Niemack as existing at the base of the auditory epithelium of the frog and rabbit. The horizontal nerve fibers at the base of the hair cells were generally toothed on their upper surface, mainly at points where branches arise. He did not find any free endings at the surface and questioned the value of Niemack’s sections, in which fibers were traced to the surface. Nothing was observed to support Kaiser’s view. The nerve fibers which did not end free terminated in thick- ened knobs in contact with the surface of the hair cells. No more than two or three branches were observed to end in con- tact with a single hair cell. The number of nerve fibers was so great that their relations could only be studied when isolated nerves and their branches were stained. Lenhossék calls attention to the fact that it is easy for an inexperienced observer to be deceived when many nerves are stained, and to be led to think that anastomoses exist. In thick sections the tangle of fine varicose horizontal fibers and their cut branches at the base of the hair cells may easily be taken for a granular mass. This is, he thinks, the granular substance described by Kaiser and Niemack. Three layers are described in the mac- ulae and cristae acusticae: (1) a hair-cell zone in which there are two layers of crowded cells arranged perpendicularly to the surface ; (2) the nerves forming a “ plexiform stratum”’ at the base of the hair cells; (3) the supporting-cell zone with the cells placed vertically. On the basis of the fact that in his preparations the nerves never extended to the surface, he concludes that the hair cells are the medium through which the movements of the endo- lymph are conducted to the nerve fibers. He does not think that there is any intermediate substance connecting the nerves and hair cells, but that the peripheral portion of the cell pos- 66 MORRILL. [VoL. XIV. sesses different chemical or physical properties from its central portions, and that this will explain its physiological action. Cajal ('94) agrees in fundamental points with Retzius, van Gehuchten, and Lenhossék. He found some of the nerves end- ing free not far from the surface in varicose enlargements, while other similar fibers terminate in a very small number of cases outside the limits of the cristae acusticae. The nerve fibers were varicose. The branches distal to the bipolar cells he considers as protoplasmic processes, while the smaller nerve fibers extending internally from the bipolar cells are the true nerves. He did not find the network of nerve fibers below the hair cells described by Lenhossék, but thinks it probably due to the fact that they did not study animals at the same stages. Cajal studied foetal rats, while Lenhossék made his observations on rats several days old. Retzius ('94a) later studied successive stages in the reptiles, and arrives at the important conclusion that the sensory nerve fibers grow from within outward: “Weil es zeigt das Nerven- fasern von centraler Seite nach Peripherie hin wachsen und nicht von Aufang an von den Haarzellen entspringen oder mit ihnen zusammenhangen.”’ This statement, if confirmed, will explain Lenhossék’s finding the free endings so far from the surface. He also found, as in his earlier observations, some of the nerves ending in contact with the hair cells, but a greater number passed between the hair cells to end free near the surface. There was never a direct connection of nerve fiber and hair cells, although both may be blackened by the chromate of silver so that they appear to be connected. In a single instance he saw a nerve fiber extending into the general epithelium of the ampulla. Whether these fibers were sensory or not could not be determined. Branching of nerve fibers, after passing the proximal ends of the hair cells, was occasionally noticed. Retzius ('94b) confirms Ayer’s ('93) observation on multipolar cells in the cochlear ganglion in finding cells with three proc- esses, but did not find those with more processes. He thinks that Lenhossék lays too much stress on the horizontal distribu- No. 1.] EPITHELIUM OF MUSTELUS CANIS. 67 tion of the nerve fibers at the base of the hair cells. He found the nerves branching at any point and does not think there is a “nervum plexiforme.” 2. Recent work on nerve endings in frog's tongue.—The close similarity of the nerve endings found in the terminal discs of the frog’s tongue and those in the auditory epithelium make it important that a brief statement should be made of the results obtained by recent investigation. Fajersztajn (89) gives the following account of the distribu- tion of the nerves in the epithelium of the terminal disc of the frog’s tongue. After forming the subepithelial plexus, the fine nerve fibrillae become varicose and pass between the proximal cells of the disc, and in some cases extend vertically to the surface, where they end in distinct enlargements (boutonné) among the distal extremities of the cylindrical cells. The terminal enlargements do not generally differ in any way from the ordinary varicosities, but in a few cases are larger and present certain differences in structure. Ehrlich’s method, he says, renders it impossible to determine whether the terminal enlargements are found between the cells or rest upon the cell membrane; probably they adhere to the surface of the cells. The fibrillae which pass into the epithelium give rise to rami- fications, and the lateral branches terminate in enlargements. The terminal enlargement, intensely colored, is surrounded by a transparent vesicle. The arrangement of the nerves and their mode of branching, as found by Niemack (92) in the terminal discs of the frog’s tongue, are essentially the same as he found in the crista acustica of the same animal. In the subepithelial plexus he found many anastomoses; the nerve endings were rare in this region. From this network fibrillae passed between the cells to end free at the surface in knob-like thickenings. A second kind of nerve ending is found associated with certain cells. The end of the nerve is enlarged and attached to the proximal end of the cell. This attachment is destroyed by teasing the preparation, and the cell and nerve are separated. 68 MORRILL. [VoL. XIV. The cells associated with the nerve endings he considers as nerve cells. He found contiguity but no continuity. Niemack considers it possible that the two kinds of ending indicate two different functions. In his figures the nerve cells are repre- sented as deeply stained. Bethe ('94), in his study of the frog’s tongue and palate with a modification of Ehrlich’s method, found two kinds of cells reaching the surface of the terminal disc and several kinds of nerve endings: I. Free endings between the cells and reaching the surface. 2. Endings on or in the epithelial cells. (a) On cylindrical cells with three lobes (dreilappigen). (b) With round end plate on rod cells, forked cells, and deep cylindrical cells. These different endings may come from branches of the same nerve. The terminal enlargements were found by Bethe on the margins of the terminal discs and sensory elevations rather than in the center. He questions the accuracy of Niemack’s conclusion that a free ending is found between every two cells. The second kind of ending is found in the middle of the terminal disc and is generally enlarged and knob-like at the end. The terminal enlargement of the nerve is firmly adherent to the cell, and admits of the cell being moved about without becoming detached. With a one-sixteenth oil immersion it appeared to be intimately joined to the cell. No continuation of the terminal enlargement or end plate into the cell was observed. The nucleus was counterstained with alum cochineal and could be easily distinguished, except in the cylindrical cells, where it was hidden by granular matter. Bethe claims to be the first to demonstrate the threefold nature of the nerve endings. They show a distinct clover-leaf shape from the surface, but look like flat discs when seen from the side. The position of the nerve ending on the cell varies, being at No. 1.] EPITHELIUM OF MUSTELVUS CANIS. 69 different heights either above or opposite the nucleus. It was never found proximal to the nucleus in the cylindrical cells. The nerves end in a single rounded end plate proximal to the nucleus in the rod cells. The third kind of ending was found in connection with cells which do not reach the surface and in which the nucleus is placed at right angles to the long axis of the cell. Bethe does not think that the blue coloration of the cell is any indication of its sensory nature, as he found quite a variety of cells stained in that way; the color was superficial. He found nerves ending in connection with gland cells, ciliated epithelial cells, and in deep epithelium cells distin- guished by dark nuclei. On the ciliated cells the clover-leaf-shaped end plate was found in contact with the cell. Frequently a small branch of the nerve extends to the surface and ends free without a terminal enlargement. The dark nucleated cell with the round end plate is sparsely scattered through the epithelium. Bethe has seen varicosities form on the slide in living nerve tissue of the crayfish, and considers those seen in nerve preparations as artifacts. II. Methods. In recent study of the auditory epithelium, embryonic mate- rial has been treated by the rapid Golgi method, and adult tissue with Ehrlich’s method. In my investigation of the auditory epithelium of adult Mustelus, I was unable to secure any results with Golgi’s method, although many experiments were tried. At first Ehrlich’s method gave widely different results, but after considerable experimentation it was found that by using only active, healthy fish killed by decapitation as soon as removed from the water, it was possible to secure uniformly good results by using certain precautions. The ampullae were removed immediately and placed in enough normal salt solution to cover them. A Minot solid 70 MORRILL. [VOL. XIV. watch glass was used for this purpose. A %% solution of methylen blue in normal salt was added in sufficient quantity to produce a deep blue color, but not enough to render it opaque. Very little of the blue was required. The staining fluid was occasionally agitated by forcing air through it by means of a medicine-dropper. The temperature was kept between 80° F. and g0° F. The best results were obtained on the hottest and dryest days in August. A warm plate or thermostat was used to maintain the proper temperature when needed on cool days. At the end of an hour to an hour and a quarter the stain was removed by means of a pipette, and the specimens rinsed with normal salt solution to remove excess of stain. They were then exposed to the air for ten to fifteen minutes, care being taken to keep them moist with normal salt solution. At the end of this time the specimens were trans- ferred to the fixing fluid, which consisted of a saturated solution of picrate of ammonia in distilled water, to which one-third of its volume of normal salt solution was added. Two or three drops of 1% osmic acid was also added to every 10 cc. of the fixing fluid. The osmic acid prevented maceration and black- ened the medullated nerve fibers. After treatment with the fixing fluid for about an hour and a half, the ampullae were transferred to a saturated solution of loaf sugar in distilled water for one hour. The syrup was removed from the surface of the specimens with blotting paper, and they were placed in a satu- rated solution of pure gum arabic in water for fifteen minutes. The ampullae were then placed one at a time in a drop of the gum arabic solution on the plate of a freezing microtome, and after careful orientation were frozen. The freezing was accom- plished by means of liquid carbonic acid. The apparatus! used was modeled after the one designed by Dr. Mixter of the Harvard Medical School. The sections were cut with a plane iron mounted between two thin pieces of wood and held in the hand. This portion of the work was done as rapidly as possible, and even then it was sometimes necessary to refreeze to keep it sufficiently hard. The sections varied considerably in thickness, but averaged 1 Manufactured by The Bausch & Lomb Optical Co., Rochester, N.Y. No. I.] EPITHELIUM OF MUSTELUS CANIS. 71 about 50“. They were removed from the plane iron with a small camel’s-hair brush and transferred to dilute glycerine, to which sufficient picrate of ammonia solution had been added to give it a yellow color. The sections were easily straightened out by means of a brush or needles with the aid of a Leitz dissecting microscope, and the best ones removed to slides and mounted in dilute glycerine with a trace of picrate of ammonia. The specimens kept very fairly and bore transportation well. If desired, the cover glass can be cemented on with zinc white or with a mix- ture of equal parts of hard paraffin and, turpentine free, Canada balsam applied warm with a brush. My best preparations kept for several months without marked deterioration when no cement was used. The cell outlines are much sharper when the preparation is first mounted. The cells seem to swell in the glycerine and become more transparent, which often causes the nerve fibers to appear more distinctly. The preparations vary greatly under what appear to be exactly the same conditions. This may be due to different physiological conditions of the tissues, which it is as yet impossible to determine. As with the Golgi method, there are great variations in the good prepa- rations, some showing one detail much better than others. It is not difficult, however, to make out all of the main points of peripheral nerve distribution in nearly every well-stained preparation. It was found that if the tissues were nearly dead before being placed in the stain, or were allowed to remain too long in the stain, or the temperature was too high during the operation, isolated epithelial cells were deeply stained, or numerous deeply stained granules appeared about the base of the hair cells or over their surface. The nerves in this case often appeared as rows of disconnected deep blue beads. If too strong a solution of the methylen blue was used, varicosities were numerous along the course of the nerve fibers, and cells were also stained deeply. A dilute solution of the stain seems to act physiologi- cally, and the tissue is killed and fixed by the picrate of ammo- nia solution. When both cells and nerve fibers are deeply stained it is impossible to satisfactorily determine their relation, just as 72 MORRILL. [VoL. XIV. in the case with similar Golgi preparations. Geberg found that in his best preparations the hair cells were not stained. As was independently found by both Feist and Niemack, the blood corpuscles in the capillaries may stain in a way which is quite confusing, as they contain intensely colored granules which look like the larger varicosities of the nerves. If the fixing fluid is allowed to act too long, the epithelium is either macerated off or is so loosened that it is removed in the subsequent treatment. The osmic acid tends to prevent this maceration, but if too much is added it blackens the epithelial cells sufficiently to impair the clearness of the pictures shown. The sugar solution tends to prevent the disintegration of the epithelium or the rupture of the cells during freezing by pre- venting the formation of ice crystals in the cells. If the sugar is not removed from the surface of the ampullae, they are not held as firmly by the frozen gum arabic. Bethe’s method gave very good results, but for this particular tissue was not as satisfactory either in the time required or in the results obtained as the method outlined above. The papers of Dogiel (90) and Apathy ('92) were freely used in working out the method which was adopted. III. Jnnervation of Auditory Epithelium in Mustelus. This investigation was confined to the study of the ampullae. Large numbers of medullated nerves blackened by osmic acid were seen ascending to the cristae, but just before reaching its proximal surface the medulla disappeared. The nerve fibers could in some cases be seen through the lightly stained medul- lary sheath, and could be traced through the closely crowded capillaries found just inside the auditory epithelium. It was very difficult at first to do this, as the blood corpuscles were stained (as already observed by Feist, '90, and Niemack, '92) in such a way that there appeared to be an irregular mass of vari- cose nerve fibers crowded together at this point. The nerve fibers could be seen on both sides of it, proximally and distally. At last, as has been already stated, with more satisfactory preparations there was no difficulty in tracing the deep blue No: T.] EPITBELLSOM OR MUSTELOS CANTS, 73 nerve fibers through the network of capillaries, particularly at the edges of the crista, where the capillaries were less numer- ous and the outlines of the corpuscles could be clearly distin- guished. It was apparent that deeply stained granules (Figs. 1-3, 5) in the corpuscles resembled the varicosities so closely as to be easily mistaken for them when they were closely crowded together. The nerve fibers generally divided dichotomously, rarely into three branches. At the point of division or separation of the fibers, triangular enlargements were sometimes observed (Figs. 2, 8, 22), as has been so frequently noted by Retzius and many others. These enlargements were found to be due to the nerve sheath, which appeared to be stretched at this point (Figs. 2, 22). In many cases the nerve fibers could be distinctly seen through the walls of the sheath, and were easily followed. The nerves branched at different levels, as observed by Retzius, but were much more numerous at the base of the hair cells, as stated by Lenhossék, than at other points. Horizontal branches were very common at the base of the hair cells, and from them branches arose, which either ended in the character- istic enlargements in contact with the basal portions of the cells, or passed between them to end free near the surface in similar but smaller enlargements. In some cases the branch- ing fibers were so numerous as to appear to form anastomoses, but closer examination failed to demonstrate them (Figs. 1-4). A few nerve fibers were noticed which extended backward from the surface (Figs. 4, 6, 8). In other cases the fibers extended horizontally for a long distance in the middle portion of the epithelium (Figs. 2, 6). Varicosities were very rarely seen in well stained preparations and when seen, as observed by Ayers ('93), there was no increase in the size of the nerve fiber. They appeared to be due to a semitransparent, faintly stained sheath (Fig. 22). The lower or supporting cells, of which there were several layers, were not figured, the whole epithelium being represented in the drawings by a wash of yellow corresponding to the color produced by the picrate of ammonia. Only a few of the hair cells were drawn to show their relation to the nerve fibers. The 74 MORRILL. [VoL. XIV. closely crowded hair cells were found at two slightly different levels, as shown by the position of their nuclei. The hairs of the hair cells were often lacking, but in many cases were so perfectly preserved that the individual hairs could be distinctly seen with the oil immersion lens. It was noticed that the nerve fibers often took the stain more perfectly at the edges of the crista than elsewhere, no stain being seen at any other part in some cases. The terminal enlargement of the nerve fiber, deeply stained, was almost invariably surrounded by a clear, lightly stained vesi- cle, and was always present, whether the fiber showed varicosi- ties or not. No indication of the clover-leaf (dreilappig) nerve termination observed by Bethe (94) in contact with the cells was seen. The nerve endings in contact with the hair cells showed essentially the same structure as the free endings. Occasionally free endings were found in the central parts of the epithelium (Fig. 22), where the relation of the parts had not been disturbed. In some cases the nerve fibers branched at the base of the hair cell, the two parts closely adhering to the cell, and ending in enlargements at nearly the same level on opposite sides of the cell (Figs. 14, 16-18). In others the branches were not associated with the same cell (Fig. 12). Nerves ending at the very base of the cell (Figs. 15, 19, 20) were also observed. The hair cells were very rarely stained, while the nucleus was often faintly outlined, but never deeply stained. A considerable number of cells were observed in which, if the cell had been deeply stained, it would have been impossible to prove that the nerve fiber did not enter the hair cell (Figs. 5, 9, II, 13, 16, 17, 19, 20, 23). In cases such as those shown in Figs.9g and 13, where the nerve ends at the very base of the cell, no continuation of the fiber into the cell could be seen. In many specimens where the nerve fiber in contact with the cell was deeply stained, an effort was made to trace a connection between them by following the nerve fiber into the cell. It was always unsuccessful, although the outer parts of the cell were semitransparent. In a single case, where the cells had become separated from their neighbors, it looked as if the fibres could No. I.] EPITHELIUM OF MUSTELUS CANIS. 75 be traced into the cells (Figs. 23-25). A careful comparison of the size and relative position of these cells with others in the same section and in other preparations led to the conclusion that this was no exception to the arrangement found in other cases, and that there was no evidence of the nerve fiber enter- ing the cell. The section cut the hair cells obliquely, the proximal end, or base, being higher than the distal end. It is almost certain that parts of two or three cells are shown, and that the nerve termination is of the same kind as that shown in Figs. 14 and 17. It is interesting to note that the nerve endings so intimately associated with the hair cells are not in any case observed placed at a higher level than the nucleus. In some poorly stained preparations a granular mass was observed at the base of the hair cells, but it was found that the granules were observed at different levels in other specimens, and were almost entirely lacking in sections that showed the nerve fibers most distinctly. IV. Conclusions. (1) In good preparations there is no trace in Mustelus of Kaiser’s cup-like nervous mass at the base of the hair cells. (2) The varicosities on the nerve fibers are very rare, and when present are caused by the separation of the sheath from the nerve fiber. (3) The terminal enlargement of the nerve fibre is not like the varicosities, as it is always present. (4) The triangular enlargements observed at the points where the nerves branch are also due to the nerve sheath, through which the nerve fibers can be seen. (5) When the physiological effect of the methylen blue is obtained, the hair cells are not stained. (6) The staining of a cell does not necessarily indicate that it is a nerve cell. (7) When dying tissue is used, cells are frequently deeply stained, making it impossible to determine their relation to the nerve fibers. G| 6 MORRILL. [VoL. XIV. (8) No satisfactory evidence of anastomosis of nerve fibers was obtained. (9) Ehrlich’s method, under suitable conditions, may give as well defined pictures as Golgi’s, and leaves the cell in a better condition for studying the relation of the cell and nerve fiber, as the former is generally more transparent. (10) No unquestionable evidence of continuity of the nerve fiber and hair cell was found. (11) There are two kinds of nerve endings in the auditory epithelium of Mustelus, the greater number being free near the surface and the others in contact with the base of the hair cells. HAMILTON COLLEGE, CLINTON, N.Y. No. I.] EPITHELIUM OF MUSTELUS CANIS. 77 LITERATURE CITED, '92 APATHY, Dr. ST. Erfahrungen in der Behandlung des Nervensys- tems fiir histologische zwecke. Mitteil. 1. Methylenblau. Zezt. 7. wiss. Mtkros., Bd. ix, 1892. '92 AyvERS, Howarp. Vertebrate Cephalogenesis. II. A Contribution to the Morphology of the Vertebrate Ear with a Reconsideration of its Functions. Journ. of Morph., vi, 1892. '93. AYERS, HowaArD. The Auditory or Hair Cells of the Ear and their Relations to the Auditory Nerve. Journ. of Morph., viii, 1893. '94 BETHE, ALBRECHT. Die Nervenendigungen im Gaumen und in der zunge des Froschs. Arch. f. mikr. Anat., xliv, 1894. '94 CajAL, S. RAMON y. Les Nouvelles idées sur la structure du System Nerveux. Dr. Azoulay, Paris, 1894. '90 DoaIEL, A. S. Methylenblautinktion der motorischen Nervenendigun- gen in der Muskeln der Amphibien und Reptilien. Arch. f. mikr. Anat., xxxv, 1890. '89 FAJERSZTAJN. Terminaisons des nerfs dans les disques terminaux chez la grenouille. Archiv. de Zool. Expér. et Gén., ii Ser., tome viii, 1889. ‘93 GEBERG, A. Ueber die Endigung des GehGrnerven in der Schnecke der Saugetiere. 1885. '86 REICHENBACH, H. Studien zur Entwickelungsgeschichte des Fluss- krebses. Adbhandl. Senckenbg. Geselisch., Bd. xiv. 1886. '87 VAN BAMBEKE, C. H. Des Déformations artificielles du noyau. Arch. de Biol., t. vii. 1887. '89 KORSCHELT, E. Beitrage zur Morphologie und Physiologie des Zell- kerns. Zoolog. Jahrb., Abth. fiir Anat. und Ont., Bd. iv. 1889. ‘91 ZIEGLER, H. E. UND voM RATH, O. Die amitotische Kerntheilung bei dem Arthropoden. Zzolog. Centralol., Bd. xi. 1891. '92 CuENoT, L. Etudes physiologiques sur les Gasteropodes pulmonés. Arch. de Biol. t. xii. 1892. '92 IpE, M. Le Tube digestif des Edriophthalmes. Etude anatomique et histologique. La Cellule, t. viii. 1892. '94 RYDER, J. A. AND PENNINGTON, MARy E. Non-sexual Conjugation of the Nuclei of the Adjacent Cells of an Epithelium. Axatom. An- zeiger, Bd. ix. 1894. ’'95 LEE, A. BOLLES. La regression du fuseau caryocinétique. Le corps problématique de Platner et la ligament intercellulaire de Zimmer- mann dans les Spermatocytes des Helix. Za Cellule, t. xi. 1895. ’96 BeRGH, R. S. Ueber Stiitzfasern in der Zellsubstanz einiger In- fusorien. Anat. Hefte, Bd. vii. 1896. ‘96 KORSCHELT, E. Ueber die Structur der Kerne in den Spinndriisen der Raupen. Arch. fiir mtkr. Anat., Bd. xlvii, Heft 3. 1896. 108 MCMURRICH. EXPLANATION OF PLATE IX. Fic. 1. Surface view of the epithelium of the “midgut” of an adult Avma- dillidium. The drawing of this and of the other surface views is made with the objective focussed at a high level, so that the cells seem to be distinct from one another. (Zeiss, Obj. C., Oc. 2.) Fic. 2. Surface view of cell from “midgut” of Armadillidium. ch =optical section of the layer of chitin as it passes down between adjacent cells forming Carnoy’s “membrane secondaire”; 2 = nuclear membrane; 2#/ = nucleoli. (Leitz, Oil-imm. 5.) Fic. 3. Surface view of “ midgut ” epithelium from an Armadillidium which had fasted for 15 days. This figure shows the abundance of yellowish-green granules and also the arrangement of the nuclei which has called forth the theory as to their non-sexual conjugation. (Zeiss, C. 2.) Fic. 4. Surface view of two cells from the “midgut” of an Armadillidium which had fasted 10 days. Showing occurrence of vacuoles (v) and yellowish- green granules. The nuclei also show the clear zone of caryolymph within the membrane. Fic. 5. Section of two cells from adult Armadillidium. ch=\ayer of chiti- nous cuticle which remains unstained; ch’ that which stains with iron-lack hema- toxylin 4m = basement membrane; # = muscle tissue; VV = nucleus; 72/ = nucleolus; v= vacuole; sf= supporting fibres, one of which (s/’) is shown broken across. (Zeiss, D. 2.) : Fic. 6. Two cells from the “midgut ” epithelium of /dotea robusta, showing the appearance of intercellular bridges of protoplasm, the existence of two nuclei in what is apparently a single cell and the occurrence of intranuclear vacuoles. (Zeiss, D. 2.) Fic. 7. Epithelial cell from Armadillidium, showing a portion of the nucleus apparently fragmented off. (Zeiss, D. 2.) Fic. 8. Epithelial cells from Armadillidium, showing a nucleus which has undergone extensive fragmentation, some of the fragments having also wandered into an adjoining cell area. (Zeiss, D. 2.) Fic. 9. Nucleus of “midgut” cell of an Oviscus which had just left the brood pouch. (Leitz, Hom. Imm. 5.) Fic. to. Section of a nucleus of a “midgut” cell from an adult Ozzscus, showing the nuclear membrane zm and numerous nucleoli (#/) of various sizes. The chromatin is in the form of innumerable granules, and at the left side of the figure a little of the achromatic network is indicated. (Leitz, Hom. Imm. +5.) Fic. 11. Section of cell from the “ midgut ” epithelium of a specimen of Oxéscus 4mm. in length. It shows the finely reticulate cytoplasm and the supportive fibres (sf) projecting up only a short distance from the basement membrane (477). The clear space around one end of the nucleus is probably an artifact, and the chitinous membrane has been torn away from the surface. (Leitz, Hom. Imm. 7.) Fic. 12. Section through one of the large blister-like vacuoles (v) which occur abundantly in the “ midgut” of Armadillidium. ch =chitinous membrane ; 6a = basement membrane; m#==muscle fibres cut across; /V=nucleus. bn SoS I] ae e i rk r Journal of Morphology. Vol. Volume XIV. June, 1808. Number 2. JOURNAL OF MORPHOLOGY. EXPERIMENTAL STUDIES ON THE DEVELOP- MENT OF LIMB-MUSCLES IN AMPHIBIA. ESTHER F. BYRNES. THE muscles in the fins of elasmobranchs develop from muscle-buds which are formed from the ventral edges of the myotomes. Until very recently this method of formation of limb-muscles was supposed to occur not only in the different groups of fishes, where muscle-buds are best developed, but also in the higher groups of vertebrates. Muscle-buds have been shown to occur among fishes, in the teleosts as well as in the elasmobranchs; and among the higher vertebrates strong evidence of the presence of muscle-buds in the Lacertilia! has been given by Van Bemmelen? and Mollier.® In the remaining groups, however, the evidence in regard to the presence of muscle-buds is far from being conclusive. Another method of formation of limb-muscles has been described by Paterson,‘ and still more recently by Harrison.® 1 According to Corning, the ventral myotome-processes in the anterior limb- region in the lizard do not go into the limbs as muscle-buds. Corning believes that the so-called “muscle-buds ” are only those ventral myotome-processes which give rise to the muscles of the tongue. 2 Anat. Anz., Bd. 4, 1889. 4 Quar. Journ. Micr. Sci.. Vol. xxviii, 1887. 8 Anat. Hefte, 1895. & Archiv f. micr. Anat., Bd. 46, 3, 1895. 106 BYRNES. [VoL. XIV. In his work on the fate of the muscle-plate in the chick, Pater- son has been unable to find muscle-buds or structures homolo- gous to them taking part in the formation of the limbs. According to his interpretation, the limbs are derived wholly from the somatopleure. Harrison, in a recent contribution to the literature on the origin of the fin-muscles in teleosts, has conclusively shown that in the salmon neither muscle-buds nor myotome-derivatives are essential to the development of fin-muscles. In support of this view, Harrison finds that, while most of the fins of the salmon follow the rule in deriving their muscles directly from the myotomes, in the form of muscle-buds, the median and pectoral fins furnish exceptions to the rule. According to Harrison, the muscles of the middle portion of the dorsal fin of the salmon are developed from muscle-buds, while at the ante- rior end of the same fin all of the characteristic structures of the fin are developed out of a mass of mesenchyme cells, which are entirely independent of the myotomes. In the pectoral fins modification has gone still further. The ventral myotome- processes arising from the ventral edges of the myotomes in the region of the pectoral fins take no part whatever in the formation of pectoral fin-muscles, but, after becoming detached from the myotomes, unite with one another to form the coraco- hyoid muscle. All the characteristic structures of the pectoral fins, muscles as well as cartilage and connective tissue, develop directly from the somatopleure by the differentiation of mesen- chyme-like cells. These two views regarding the origin of limb-muscles can no longer be regarded as incompatible when both modes of muscle formation occur, not only in different fins in the same individual fish, but even side by side in the same fin. The starting-point in the more recent researches on amphib- ian limb-muscles has been the hypothesis that the muscles of the limbs are developed from myotome-derivatives which are homologous to the muscle-buds in the fins of fishes. According to Goette’s! account, the limb-muscles of Bombinator develop from cells derived from the outer layer of the muscle-plate. 1 « Die Entwicklungsgeschichte der Unke,” 1875. NO: 2-4] LIMB-MUSCLES [N AMPHIBIA. Oy Kaestner ! has failed to show that the myotomes take any part directly in the formation of the limbs in the Anura. Neverthe- less, if I have understood him rightly, he still believes that the limb-muscles are derived directly from the myotomes, although he has not seen the process, owing, as he believes, to the very early period at which it takes place. Still more recently, H. H. Field? described myotome-proc- esses taking part in the formation of the muscles of the anterior limbs in the Urodela (Amblystoma, Triton). He has also extended his observations to several of the Anura (Rana, Bufo) with similar results. It is unnecessary to discuss in detail the origin of limb-muscles in the higher groups of verte- brates, as this has been very fully treated elsewhere in the literature on the subject. It is sufficient to say that, with a single exception, in all the higher vertebrates in which true muscle-buds have not been found masses of proliferated cells, which are regarded as homologous to muscle-buds, have been described. It was with the hope of definitely determining, if possible, the source of the muscles in the limbs of some of the Amphibia that the present investigation was undertaken, at the suggestion of Dr. R. G. Harrison. I gladly take this opportunity of acknowledging my many obligations to Dr. Harrison for the friendly interest with which he has followed my work, and for having placed at my disposal much of his own prepared material. I also take pleasure in thanking Prof. T. H. Morgan, in whose laboratory most of the work was done, for his kind criticism. My thanks are also due to Prof. C. O. Whitman, of the Marine Biological Labo- ratory at Wood’s Holl, Mass., where the work was completed. Material. The material used in the present investigation consisted of Urodela, Amblystoma punctatum, and several species of Triton, and of Anura, Rana sylvatica, Rana palustris, and Bufo ame- vicanus, collected in the vicinity of Bryn Mawr, Penn. 1 Archiv f. Anat. u. Phys. (Anat. Abth.), Hefte 5 und 6, 1893. 2 Anat. Anz., Bd. 9, 23, 1894. 108 BYRNES. [VoL. XIV. Methods. Most of the embryos were killed in a saturated solution of corrosive sublimate to which five per cent acetic acid had been added. The embryos were subsequently sectioned and stained on the slide with Delafield’s haematoxylin, followed by a wash of picric alcohol. This method, when used on amphibian embryos, gives a very sharp differentiation of muscle-fibers, staining them a bright yellow color, while the chromatin in the nuclei remains a deep purple; the protoplasmic network remains faintly stained with the haematoxylin. Other double stains were used to detect the presence of muscle-fibrils, but they gave no better results than the method already described. I. NorMAL DEVELOPMENT OF THE MyoToOME-DERIVATIVES, ANTERIOR AND POSTERIOR LIMBS IN THE URODELA (Am- BLYSTOMA AND TRITON). Since the limb-muscles are derived directly from the myo- tomes in some of the lower vertebrates, and a similar process is believed to take place in the higher vertebrates, the develop- ment of the limb-muscles in higher forms must be studied in connection with the fate of the myotome-derivatives. Maurer! has already described the formation of myotome-processes in Amblystoma (Siredon) and has given a detailed account of their subsequent development into the muscles of the body-wall. He has not, however, considered the relation of the myotome- processes to the limbs. As my attention has been directed chiefly to the earlier stages in the formation of myotome-proc- esses and to their relation to the limbs, I shall, for the sake of completeness, give a brief account of the ventral myotome- processes up to the time when they first begin to develop muscle-fibrils. In Amblystoma all the myotomes of the trunk-region, ex- cepting the first and second, give rise to ventral myotome- processes which are alike in'structure. The myotome-processes develop first in the anterior part of the body, where they appear 1 Morph. Jahrob., Bd. 18, Heft 2, 1892. No. 2.] LIMB-MUSCLES IN AMPHIBIA. 109 as masses of embryonic cells densely packed with yolk-granules, and form projections from the ventral, outer borders of the myotomes (Pl. X, Fig. 1). Even in their earlier stages the myotome-processes appear as distinct diverticula whose cavities are continuations of the myocoel. As the myotome-processes elongate and the yolk- granules are gradually absorbed, the cells of the rudimentary ventral processes become arranged in a single row along the walls of the myocoel, as shown in Pl. X, Fig. 2. During the ventral growth of the myotome-processes, the cells lose their rounded, embryonic appearance and become elongated dorso- ventrally in the direction of growth. So pronounced is the dorso-ventral elongation of the nuclei of these cells that their characteristic shape often serves as a means of identifying the cells of the myotome-processes, even when they are closely crowded by other structures, as in the region of the anterior limbs (Pl. X, Fig. 4). As the myotome-processes elongate, the myocoel becomes greatly reduced, and finally wholly disappears, leaving double strands of nuclei in place of the thick-walled diverticula of the younger stage (Pl. X, Figs. 6, 7). While these changes are going on the myotome-processes in the fourth and fifth trunk-segments become separated from their respective myotomes by the developing pronephros. Throughout the length of the body the myotome-processes on each side, including those that have lost their connection with the myotomes, become united into a thin lateral sheet of muscle (‘the primary abdominal muscle”’). The primary abdominal muscles taper anteriorly into narrow strands which pass to the ventral side of the body, where they become attached to the hyoid cartilage. This anterior, ventral portion of the primary abdominal muscle becomes the sterno-hyoid muscle. In its mode of origin it closely resembles the coraco-hyoid muscle of the teleosts. Beyond this stage I have not followed the ventral 1 Harrison has shown that in the salmon the myotome-processes which are very early constricted off from the ventral edges of the anterior myotomes in the region of the pectoral fin do not go into the fin as muscle-buds, but unite with each other to form the coraco-hyoid muscle. Corning formerly described these detached myotome-processes in the pectoral fin-region as muscle-buds. Later he confirmed Harrison’s account. II1O BYRNES. [VoL. XIV. myotome-processes, as their subsequent fate has no bearing on the question of the origin of the limb-muscles. The details of their later development are given in Maurer’s paper on Siredon. Simultaneously with the early development of the myotome- processes the anterior limbs begin to develop. They originate as somatopleuric thickenings formed by an aggregation of mesenchyme-like cells around a slight fold of the epithelium bor- dering the coelom (PI. X, Fig. 1). H.H. Field has already called attention to this fold and its relation to the rudimentary limb. The anterior limb-rudiments develop some distance de/ow the level of the myotomes; hence, when the myotome-processes elongate ventrally they project toward the limbs. During the very early stages of development all the cells are so crowded with yolk-granules that the line of demarcation between the myotome-processes and the rudimentary limbs is often obscured. In favorable cases, however, a line of pigment bordering the myocoel outlines the extent of the process so that it can be followed (Pl. X, Figs. 3, 4). The apparent fusion of the myotome-process with the rudimentary limb-mass can often be explained as due to the fact that the myotome-processes are cut obliquely. Such sections often show no distinct space between the myotome-process and the limbs. The true relations be- tween the myotome-processes and the anterior limb-rudiments in Amblystoma are further obscured by the early appearance of the second pronephric funnel in the region of the limbs (Pl. X, Fig. 3). As the pronephric tubules increase in length they elongate at right angles to the direction of growth of the myotome-processes, and consequently push the myotome-proc- ess over against the somatopleuric thickening of the limb. The close proximity of the two structures seems to be due mainly to mechanical causes; 2z.¢., to the pressure exerted on the myotome-process by the elongating tubules. When the tubules become convoluted, later, they force their way in between the rudimentary limb and the myotomes, so that the connection between the myotomes and the ventral myotome-processes is broken (Pl. X, Figs. 6, 7). Maurer? has already shown these relations in the Urodela. 1 Morph. Jahrb., Bd. 18, 1, 1891. Fig. 7, Pl. VI. Noe 2] LIMB-MUSCLES IN AMPHIBIA. A It is the ventral myotome-process in the region of the second pronephric funnel which H.H. Field has called ‘“Unwirbel- knospe,”’ and from which he derives the pronephric capsule and the limb-muscles in Amblystoma. Posterior to the region of the pronephric funnels, the myotome-processes permanently retain their connection with their respective myotomes, and pass directly by the inner side of the limb without any appear- ance of fusion with it (Pl. X, Figs. 4, 5). These relations are even more clearly seen in Triton than in Amblystoma, for, while the body of Triton is relatively wider, it has a shorter dorso-ventral axis. Hence, as the ventral edges of the myotomes are nearly on a level with the anterior limb- rudiment, when the ventral myotome-processes are formed, instead of pointing toward the limb-rudiment, as in Ambly- stoma, the myotome-processes pass directly by its inner side (Pl. X, Fig. 11). In both Triton and Amblystoma the inde- pendence of the myotome-processes and the anterior limbs becomes more marked in older larvae after the limbs have begun to project slightly from the sides of the body. The yolk can be largely removed from young Amblystoma larvae so that whole preparations can be mounted in glycerine or balsam and thus be made perfectly transparent. In older embryos the myotome-processes are so closely applied to the walls of the coelom that it is often difficult to remove the yolk without completely tearing away the myotome-processes or breaking them. When the myotome-processes are removed, the anterior limb-rudiments a/ways remain intact as a uniform thickening of the somatopleure in which there is no evidence of any structure comparable with muscle-buds as they appear in fishes. This tearing away of the myotome-processes without disturbing the limb-rudiments seems to indicate an independ- ence of the myotome-processes and the limbs. Pl. X, Fig. 17, is intended to show these relations in the anterior limb-region of a young Amblystoma larva. When the ventral myotome-processes (z.¢., the ‘“ primary abdominal muscles’’) and the limb-rudiments in the anterior part of the body of Amblystoma are well developed, the myo- tome-processes and the limbs in the posterior part of the body hig 4 BYRNES. [VoL. XIV. are still in a very rudimentary condition. In the immediate region of the posterior limbs the myotome-processes are repre- sented only by afew embryonic cells at the ventral outer corner of the myotome (Pl. X, Fig. 13). — Fig. 15 represents a section through the posterior part of the body of a young Triton larva, some distance in front of the posterior limb. The myotome-process is represented by only a few cells at the ventral outer edge of the myotome. These cells are the forerunners of the primary abdominal muscle, with which they are connected in a more anterior section. One of the cells is shown in the act of dividing. Such cases of division are of frequent occurrence at the ventral edges of the myotomes in the posterior part of the body and are incidental to the growth of the abdominal muscle. Fig. 14 shows a section taken through the posterior limb- rudiment of a Triton embryo. The conditions shown at the ventral edge of the myotome in front of the limb (Fig. 15) differ in no way from those in the limb-region itself, except that in front of the limb the myotome-processes have begun to grow ventrally. On account of the close proximity of the pos- terior limb to the ventral edge of the myotomes, there is some- times an apparent connection between the two structures. Nevertheless, I believe this is only a coincidence, for, after having examined a large number of sections through the pos- terior part of the body, including the limb-region, I have been unable to find any special localized proliferation of cells from the myotomes to the limbs. Moreover, the number of dividing cells at the ventral edge of the myotomes in the limb-region is relatively very small. I cannot believe that in Amblystoma there is any ground for homologizing these single cells with muscle-buds when typical muscle-buds are absent. Among the higher groups of vertebrates, where muscle-buds, such as are found in the fishes, have not been shown to exist, the cells that are proliferated from the ventral edges of the myotomes have been very generally homologized with true muscle-buds. But the evidence given in these cases is not wholly convincing that the cells proliferated from the myotomes are really homologous to muscle-buds, or even that they give NG. 2°) LIMB-MUSCLES [IN AMPHIBIA. 113 rise only to muscle tissue. They are to be regarded rather as the ‘“ formative-tissue’’! cells of Goette and Ziegler. In the elasmobranchs, where ‘‘ muscle-buds”’ are best developed, Ziegler has shown that.cell-proliferation occurs independently of the formation of muscle-buds and in addition to it. The first indication of the posterior limbs is seen in a thick- ening of the somatopleure at the extreme posterior limit of the coelom. The limbs arise ventral to the myotomes and appear first as an aggregation of a few mesenchyme cells lying directly beneath the thickened ectoderm (Pl. X, Fig. 13). By the time the posterior limbs first begin to make their appearance the yolk-granules have been almost wholly absorbed from the mesoderm, leaving the large nuclei lying freely suspended at the nodal points of a protoplasmic network. In this network, which is almost colorless even in stained sections, the nuclear divisions can be easily followed. Since cell migration is of widespread occurrence in the formation of embryonic organs, evidence based upon nuclear division alone cannot be re- garded as entirely conclusive in determining the original sources of the cells that go to form any given tissue. Never- theless, whatever evidence can be deduced from karyokinesis points unmistakably to the somatopleure as the region of growth in the formation of the posterior limbs. This explanation is rendered all the more probable by the frequent proliferation of cells from the endothelium bordering the coelom directly in the limb-region. Comparing the total number of cases of mitosis at the ven- tral edges of the myotomes in the limb-region of eleven embryos with the total number of cases in the limb-rudiment, we find that there are 24 dividing nuclei at the edges of the myotomes to 242 in the somatopleure of the limb; or 1 in the myotome to every 10 in the somatopleure. When we consider that numerous cases of mitosis are also seen at the ventral edges of the myotomes throughout the length of the body and in the tail, the fact that isolated cases of cell division occur at the edges of the myotomes in the limb- 1 I have used “ formative tissue ” in Ziegler’s sense as referring to cells that are physiologically undifferentiated. 114 BYRNES. [VoL. XIV. regions can have little weight in establishing the dependence of the limb-muscles on the myotomes. II. NorMAL DEVELOPMENT OF THE MyYOTOME-DERIVATIVES, ANTERIOR AND POSTERIOR LIMBS IN THE ANURA (RANA AND BuFo). What has been said of the relation of the myotome-processes to the limbs in Amblystoma applies also in a general way to Rana and to Bufo. The Anura differ, however, from the Uro- dela in the structure of the ventral myotome-processes and in the early separation of the myotome-processes from the myo- tomes. Another point of difference between the two forms and one which has been taken advantage of in experimenting on Rana consists in the increased distance between the ventral edges of the myotomes and the limbs (Pl. X, Fig. 18). Kaestner! and Maurer? have already given a detailed account of the development of the muscles of the body-wall in the Anura, and Kaestner has attempted to show the relations of the myotomes to the limbs. Inasmuch, however, as the pres- ent account of the muscles of the limbs differs from any that has been given for Rana, I shall briefly review the early development of the myotome-processes during the time that they are in closest contact with the limbs. In Rana the myotomes give rise to short ventral processes which become constricted from the myotomes very early. Unlike the ventral myotome-processes of Amblystoma and Triton, the ventral myotome-processes of Rana contain no myocoel, but appear as solid outgrowths from the ventral edges of the muscle-plates (Pl. X, Fig. 18). The ventral processes of the anurans are first formed from the myotomes in the anterior part of the body. Soon after their formation they are constricted from the myotomes and begin to move toward the ventral body-wall. Later, the more posterior myotomes give rise to ventral processes which likewise become separated from the myotomes in the order of their formation. Throughout 1 Archiv f. Anat. u. Phys. (Anat. Abth.), Hefte 5 und 6, 1893. 2 Morph. Jahrb., Bd. 22, Heft 2, 1894. No. 2.] LIMB-MUSCLES [N AMPHIBIA. BES the length of the body these detached ventral myotome-processes participate in the formation of continuous lateral bands of muscle, which lie one on each side of the body, and which, as Maurer has shown, give rise to the abdominal muscles. In the tail-region the abdominal muscle-rudiment retains its connection with the myotomes (Pl. X, Fig. 12). It tapers anteriorly to a very delicate strand of muscle, which becomes attached to the posterior edge of the hyoid cartilage just above the heart. Further than this I have not followed the ventral myotome-processes. A full account of their later development has been given by Kaestner and by Maurer. The above account covers the period of growth when, if there had been any connection between the myotomes and the limbs, it would have been evident. The anterior limbs of Rana first appear as a thickening of the somatopleure just behind the gills. I have been unable to trace any connection between the anterior limbs and the myo- tomes in Rana. The two structures are so far separated from each other, not only as regards their actual position, but also as regards the time of their formation, that even if any intimate connection does exist between them it is impossible to follow it in normal embryos. According to Goette’s account, both the anterior and poste- rior limbs of Bombinator derive their muscles from the outer cells of the segmental plate. Jordan in his account! of the development of the anterior extremity of the Anura accepts the account already given by Goette, and treats only of the later stages of development after the differentiation of tissue has already begun. The relations between the myotomes and the limbs in the frog can be much more easily studied in the posterior than in the anterior limb-region. By the time the posterior limb- rudiments first begin to make their appearance the ventral myotome-processes in the posterior limb-region have just been constricted off from the myotomes, and have come to lie imme- diately ventral to them (Pl. X, Fig. 18). The rudiments of the posterior limbs first appear at the extreme posterior limit of 1 «“ Die Entwicklung der vorderen Extremitat der Anuren-Batrachier.” 116 BYRNES. [Vou. XIV. the coelomic cavity as aggregations of a few of the mesenchyme- like cells of the somatopleure. In the region of this meso- dermal thickening the ectoderm also becomes conspicuously thickened. Kaestner has already pointed out these relations in the development of the posterior limbs of Rana. After leaving its posterior attachment to the eleventh myo- tome, the abdominal muscle-rudiment (the ventral myotome- processes) skirts the anterior limit of the posterior limb, and as it passes forward and ventrally presses so close to the limb that it is often difficult to detect any boundary between the cells of the two structures (Pl. X, Fig. 12). Cross-sections through the posterior limb-region of Rana show the abdominal muscle elongated in the direction of the limb and having at times the appearance of a muscle-bud with a well-defined ven- tral boundary. Longitudinal sections, however (Pl. X, Fig. 12), show that this appearance is due only to the sectioning of an oblique structure, and that there are in reality no bud-like structures present. Directly in front of the limb-rudiment the abdominal muscle emerges at a level below the limb. The youngest larvae figured by Kaestner! are much too far advanced to show the closest connection between the posterior abdominal muscle-rudiment and the earliest rudiment of the posterior limbs. Pl. X, Figs. 12 and 18, show the normal rela- tions between the myotome, the posterior abdominal muscle, and the limb in a very young frog embryo in which the limbs are just beginning to develop. The only possible connection between the myotomes and the limb-rudiments in Rana is an indirect one through the abdominal muscle, which at a very early period comes into close contact with the limbs. But even here the connection between the limbs and the abdominal muscle is only apparent, as I hope to show in the chapter on experiments on Rana. Kaestner has been unable to demonstrate an ingrowth of any myotome-derivative into the limb-rudiment in Rana. He says : “So scheinen unsere Untersuchungen zu einem Resultat gefiihrt zu haben, welches im Widerspruch steht mit denen, die bisher an allen iibrigen Wirbelthier-Klassen gewonnen 1 Archiv f. Anat. u. Phys., Hefte 5 und 6, 1893. No. 2.] LIMB-MUSCLES IN AMPHIBIA. i gf worden sind. Wir sahen bei Froschlarven aus der undifferen- zirten Extremitatenanlage sowohl Knorpel als auch Musculatur hervorgehen, scheinbar ohne Betheiligung der Myotome. Aber dies auch nur scheinbar, denn vorausgesetzt, dass die Myotome der Froschlarven ebenso wie die der iibrigen Wirbelthier Klas- sen die Grundlage der Extremitatenmusculatur abgeben, so muss bei den friihesten Stadien von Froschlarven, die ich bisher beschrieben . . . jener Vorgang langst abgeschlossen sein.”’ My own odservations agree with Kaestner’s, but his assumption that the limb-muscles must be derived from the myotomes is, I think, not supported by the facts of development as shown by experimental study. From the study of normal amphibian embryos, urodeles, and anurans, I was led to conclude that the limbs are wholly somatopleuric in origin, that the myotome-processes take no part in the formation of the limbs, but give rise exclusively to the musculature of the body-wall, and that there is no special- ized proliferation of cells from the myotomes in the limb-region. I determined to further test the somatopleuric origin of the limb-muscles by experiment. The experiments were made in the spring of 1895, on young tadpoles (Rana sylvatica), and on young Amblystomas (Amblystoma punctatumy). III. ExpeERIMENTS ON AMBLYSTOMA AND RANA. The method of experiment was as follows: Very young embryos in which neither the myotome-processes nor the limb- rudiments had begun to develop, were cut from their capsules and placed in a watch crystal on filter paper moistened with water. Then with a hot needle the ventral halves of the myo- tomes of the posterior limb-region on the right side of the body were destroyed. A few similar experiments were made with cold needles. These, however, gave no definite results, owing to complete regeneration, and the use of cold needles was aban- doned. All the operations were performed under a dissecting microscope. Since in Amblystoma the posterior limbs arise in the twentieth segment, the attempt was made to destroy the ventral edges of the myotomes from the sixteenth to the 118 BYRNES. [Vou. XIV. twentieth, inclusive. The posterior limbs in the frog tadpoles develop in the eleventh segments. Here the injury extended from about the sixth trunk-segment to the tail. After the operation the embryos were transferred to large shallow dishes of water. Care was taken to change the water frequently within the first few hours after the operation, until the wounds had entirely healed over. After that no further precautions were taken. Most of the embryos survived the injury and were kept in the laboratory in an apparently healthy condition for periods varying from one to eight weeks. An effort was made to restrict the injury as far as possible to the myotomes, leaving the somatopleure of the limb-region uninjured. I hoped, by completely separating the limbs from the myotomes, to be able to test the power of independent growth of the limbs when all connections with myotome- derivatives had been destroyed. In Amblystoma the posterior limb-rudiments arise so close to the ventral edges of the myotomes that it is extremely difficult to destroy the ventral portion of the myotomes without involving the somatopleure of the limb-region more or less in the injury. On this account Amblystoma is not a very favorable object on which to study the origin of limb-muscles by the experimental method. Notwithstanding the distortion that the right side of the body of Amblystoma usually undergoes in consequence of the incidental injury to the somatopleure, the right limb often reaches a surprising development, very little, if at all, inferior to the limb on the normal (left) side of the body. Pl. XII, Fig. 37, represents a section through the anterior part of the posterior limb of an Amblystoma embryo killed four weeks after injury. The right myotomes have not regenerated their own tissue perceptibly, nor have they given rise to the abdomi- nal muscle-rudiment. In spite of the injury to the myotomes, the limb has reached a surprising development. The limb on the injured (right) side of the body is in every way similar to the one on the normal (left) side, not only in general size, but also in the appearance of its component cells. Pl. XII, Figs. 38 and 39, show the injured and uninjured sides, respectively, of an embryo, in the posterior limb-region, No. 2.] LIMB-MUSCLES IN AMPHIBIA. I19 six weeks after injury. Fig. 39 represents the posterior limb- region on the uninjured (left) side of the body. The myotome on the right side of the section has been almost wholly destroyed; the right side of the body has so contracted as to rotate the dorsal fin through an angle of nearly ninety degrees. Fig. 38 represents the posterior limb-region on the injured (right) side of the same embryo. The myotome, though not wholly destroyed, is greatly reduced in size, only a few muscle- cells remaining. The limb-rudiment has reached a conspicuous development, and is but little inferior to the one on the normal side. The slight reduction in size of the right limb is evidently due to some injury that resulted to the somatopleure of the limb-region when the myotomes were destroyed. Evidence of this is seen in the very general distortion of the whole right side of the section. Fig. 40 shows the right limb of an Amblystoma embryo killed forty-four days (six weeks and two days) after injury. In the limb on the normal (left) side of the body cartilage and muscle-fibrils are already present. In the limb on the injured (right) side the cartilaginous areas are distinctly marked out, although the cartilage is not so well developed as in the limb on the uninjured side. Differential stains fail to show the presence of muscle-fibrils in the right limb, although the muscle areas are distinctly marked out around the central core of carti- lage. Moreover, they correspond in every respect to the muscle- areas in the limb on the normal (left) side of the body. The entire mass of the right limb is less than that of the left limb, but the reduction in size, as well as the lesser degree of differ- entiation, in the right limb is evidently due to a general retardation of growth resulting from the injury to the right side of the body, and not to any difference in the zxds of tissue present. The permanently reduced size of the myotome bears witness to the extent of the original injury, and leaves little doubt that the region of growth of the myotome was completely destroyed. Even if the limbs were dependent on the myotomes for their muscle-tissue, it seems scarcely conceivable that the myotomes could, in the mutilated condition shown in the figures, contribute cells in sufficient numbers to enable the 120 BYRNES. [Vor. XIV. limb on the side of the injury to keep pace with the limb on the uninjured side of the embryo wethout first regenerating themselves. A few preliminary experiments were made on the tadpoles of Rana sylvatica, and these showed that the Anura offer much more favorable conditions for the experimental study of the limb-tissues than do the Urodela, as has already been stated. This is owing to the greater distance between the myotomes and the limbs in the Anura,—a condition which makes it possible to destroy the myotomes without necessarily involving the somatopleure of the limb-region in the injury. On the roth of April, 1895, eighty embryos of Rana palus- tris (7 mm. to 8 mm. in length) were taken from their capsules, and the ventral halves of the myotomes in the posterior limb-region were destroyed. On the 11th of April similar operations were made on forty more embryos from the same set of eggs. In all cases the operation was performed on the right side of the body. All these embryos (one hundred and twenty in number) survived the operation and were kept in the laboratory in an apparently healthy condition for periods varying from two days to six weeks. Some of these embryos showed the effects of the injury by a very perceptible shorten- ing of the dorso-ventral axis of the right side of the body and by a bending of the tail sideways through an angle of ninety degrees. From the 12th to the 23d of April some of these one hundred and twenty tadpoles were killed daily. After that, a few were killed at intervals of several days until the 4th of June, when all the remaining tadpoles of the set were killed. Normal embryos were also preserved as a check to the injured series. Many of the injured embryos have been disregarded as giving inconclusive results. None of the discarded embryos, however, furnish any evidence against the somatopleuric origin of the limb-muscles. Many of them show only the normal relations, in the posterior limb-region, between the myotomes, the abdomi- nal muscles, and the limbs. In some of the earlier experiments the injury was confined to the extreme posterior part of the body. Many embryos, thus injured, were kept for several No. 2.] LIMB-MUSCLES IN AMPHIBIA. Lat days and were then killed and sectioned. They show that the trunk-segments have entirely escaped injury, although the myo- tomes in the proximal part of the tail have often been reduced to one-half their original length and the whole right side of the tail has become greatly contracted. This apparent migration of the injury seems to be due to the gradual growth of the tail. As the tail lengthens, myotomes that in younger embryos seemed to belong to the trunk appear later as anterior tail-segments. Only those cases have been considered and figured in the present paper which show marked traces of the early injury to the myotomes and in which only a very slight injury, if any, has been sustained by the somatopleure of the limb-region. Normal frog embryos killed on the 1oth and 11th of April and used as a check to injured embryos show that at the time of the operation neither the primary abdominal muscle-rudiments nor the posterior limbs had begun to develop. Therefore, no myotome-derivatives could at this time have been given to the limbs. Pl. XI, Fig. 19, serves as a control for the injured embryos. On the right side of the section the myotome is reduced to almost one-third of its length, and the abdominal muscle-rudi- ment on the right side has been completely destroyed. The limb-rudiment is uninjured and is almost as well developed as the limb on the normal side of the embryo. Sections through the posterior limb-region of an embryo killed three days after injury show that the myotomes on the side of the injury have been greatly reduced throughout the limb-region. The abdominal muscle-rudiment is wanting in corresponding sections, and there is little evidence of any attempt at regeneration. The limb-rudiment, which consists of only a slight aggregation of mesenchyme-like cells, is present and normal. Another embryo killed three days after injury shows a double abdominal muscle-rudiment on the side of the injury. A small mass of cells constricted from the myotome lies a little below and to the inner side of the myotome. At the ventral, outer edge of the myotome there is a second mass of cells similar to the first and occupying the normal position of the primary abdominal muscle-rudiment. nae BYRNES. [VoL. XIV. Sections of an embryo killed four days after injury show that the myotomes have not been very greatly reduced, although the extreme ventral edges, together with the abdominal muscle- rudiment, have been destroyed. The somatopleuric thicken- ings of the limbs are equally developed on both sides of the body. Pl. XI, Figs. 19 and 20, represent sections taken through the posterior and anterior parts of the posterior limbs, respectively, of an embryo killed four days after injury. Fig. 19 shows the primary abdominal muscle on the normal side of the embryo, still in contact with the myotome. In Fig. 20 the primary abdominal muscle has become constricted off from the myotome on the normal side, and is approaching the extreme anterior limit of the limb-rudiment. There is as yet no evidence of regeneration of the abdominal muscle on the part of the injured myotomes on the right side. ; An embryo killed seven days after injury shows that the myotomes of the posterior limb-region have been reduced to one-third their normal length. Notwithstanding the injury to the myotomes, the abdominal muscle has begun to regenerate. Evidence of this regeneration is seen in the general tendency of the mesenchyme-like cells between the myotomes and the limb to elongate dorso-ventrally, and to fall into line along the path of the developing abdominal muscle. The right limb, though slightly smaller than the left, is well developed. The difference in the size of the two limbs in this embryo is due to the fact that the somatopleure, as well as the myotomes, has been injured. This is evident from the abnormal arrangement of the blood vessels on the side of the injury. Another embryo killed seven days after injury shows the right myotomes greatly reduced throughout the whole extent of the limb-region. On the side of the injury the abdominal muscle is beginning to regenerate. Both the posterior limb-rudiments are present and normal. Still another embryo killed seven days after injury shows the ventral halves of the myotomes in the right posterior limb-region completely destroyed. Near the limb the right abdominal muscle has begun to regenerate. Both the limb- rudiments appear normal. No. 2.] LIMB-MUSCLES IN AMPHIBIA. 123 An embryo killed eight days after injury shows the right myotomes reduced to one-half their original length. In the posterior part of the body, where the abdominal muscle and the myotomes remain permanently connected, the myotome has begun to regenerate dorso-ventrally. In front of this place of connection, however, the myotomes give little evidence of any dorso-ventral growth. The regenerating abdominal muscle has already developed well-marked muscle-fibrils, and both the limbs are normal. An embryo of an unknown species of frog killed nine days after injury shows a marked reduction in the size of the myotomes; it also shows a normal development of the limbs. Pl. XI, Fig. 41 a, shows a section through the posterior part of the posterior limb-rudiment of this embryo. On the normal (left) side of the body the abdominal muscle has not, as yet, been constricted from the myotome. The limb-rudiment is well developed. On the injured (right) side, the section shows the right myotome almost wholly destroyed, and with it also the abdominal muscle-rudiment. The injury has also involved the medullary tube, no trace of which remains in the limb- region. Notwithstanding the extent of the injuries, the right limb-rudiment is apparently normal. Fig. 41 6 represents a section through the anterior limit of the posterior limb of the same embryo. On the uninjured side of the body the abdomi- nal muscle-rudiment has not as yet come in contact with the limb. On the injured side the myotome is still greatly reduced, but shows a tendency to regenerate toward the limb-region. Although the myotome is not wholly destroyed on the right side of the embryo, it is so mutilated that it is scarcely con- ceivable that the few remaining muscle-cells could contribute cells to the limb, even if they did so normally. The limb- rudiments are, however, equally large on both sides of the body, as is shown in Fig. 44, which represents a camera drawing of the limb-region indicated in Fig. 41 a. Sections of an embryo killed eleven days after injury show that the outline of the body has not been distorted. The injured myotomes on the right side of the body extend ven- trally only to the level of the notochord. The abdominal 124 BYRNES. [VoL. XIV. muscle has regenerated so as to occupy an almost normal position at the anterior limit of the limb; it does not, however, extend throughout the entire limb-region, as is the case in normal embryos. The limb-rudiments are well developed and normal. In another embryo killed eleven days after injury the myotomes have been reduced to almost one-half their original length. The abdominal muscle-rudiment has regenerated, but is present in the limb-region only at the extreme anterior limit of the right limb. Both limb-rudiments are normal. An embryo killed thirteen days after injury shows that the myotomes of the posterior limb-region on the right side of the body have been reduced to one-third their original length. The right abdominal muscle, though present, is reduced in size and shortened. Notwithstanding the abnormal conditions in the myotomes and the abdominal muscles, the limbs are well developed and normal. Figs. 21-25 represent sections through an embryo killed twenty-six days after injury. Owing to the distortion that has resulted from the mutilation, the sections do not show cor- responding regions of the right and left limbs. The limbs extend, however, over an equal number of cross-sections, and are apparently in every way similar. Fig. 21 is taken through the mid-region of the limb on the injured (right) side of the body. . The myotome is reduced and the abdominal muscle- rudiment is wanting. The limb is well developed and normal. Fig. 22 shows a more anterior section through the most anterior part of the right limb, which is represented, in this section, by only a few cells in the dorsal wall of the coelom. The myo- tome has begun to regenerate ventrally, but has not as yet reached the level of the limb. Fig. 23 is taken directly in front of the limb-region. It shows the ventral growth of the myotome from which the abdominal muscle is to be, at least in part, regenerated. The relations of the abdominal muscle in still more anterior sections are represented in Figs. 24 and 25. These sections plainly show that the myotomes are still greatly reduced and that their ventral regions of proliferation must have been completely destroyed when the injury was first made. The limbs are, NOs, 23) LIMB-MUSCLES IN AMPHIBIA. I25 nevertheless, normal. The right limb is evidently entirely separated from the myotome or any of its derivatives; but it is, notwithstanding its isolation, in every way comparable with the limb on the uninjured (left) side of the embryo. The abdominal muscle has regenerated, but only in front of the limb. An embryo that had been injured on the 4th of April and killed on the 6th of May (thirty-two days after injury) shows but little trace of injury to the myotomes. The right myo- tomes are slightly shortened, but the relative distances between the myotomes and the limbs on the right and left sides of the body are about the same and normal. A reconstruction of the embryo shows, however, that, whzle the primary abdominal mus- cle ts present throughout the whole extent of the limb-region on the normal (left) side, on the injured (right) side the muscle ts present only at the anterior limit of the limb, and even there tt zs veduced to nearly half its normal size. The limbs are well developed and seemingly normal; they are, moreover, equal in size, and in both limbs the cells that will give rise to muscles are already clearly distinguishable from those that will form the cartilage. As yet, however, the muscle-fibrils have not appeared in either of the rudimentary limbs. Pl. XII, Figs. 46 and 47, show corresponding sections through the right and left limbs of this embryo. Sections of an embryo killed thirty-three days after injury show that the myotomes have been greatly reduced. The abdominal muscle-rudiment comes in contact with the limb only at its extreme anterior limit. Both limbs are equally well developed. One of the embryos killed thirty-four days after it was injured proved to be of particular interest. Pl. XI, Figs. 29-36, inclusive, show a series of sections taken through the limb-region of this embryo. The myotomes are but slightly shortened. Throughout the limb-region on the right side of the body there is xo indication whatever of the abdominal muscle-rudiment. Only at the most anterior limit of the limb does the muscle begin to make its appearance (Fig. 36), and then it is represented by only a single muscle-cell in which fibrils are well developed. Still more anteriorly, in front of the limb-region, the right abdominal muscle-rudiment remains 126 BYRNES. [VoL. XIV: greatly reduced, not only in thickness, but also in length. On the left side of the body the abdominal muscle has reached a very conspicuous development. Although the right limb is apparently free from contact with any of the myotome-deriva- tives, it has reached a development equal to that of the limb on ‘the normal (left) side of the embryo. The two limbs are in every way comparable, not only in size, but also as regards their regions of differentiation within the limb-bud, as shown im Pleura) a5. One of the embryos shows the myotomes in the posterior limb-region reduced to one-half their normal length, even after six weeks have elapsed since the operation. A few scattered muscle-cells lie between the lower edge of the injured myotome and the limb-region. Whether some of the original myotome cells have escaped injury or whether those now present have regenerated is not clear from the sections. The injury was an extensive one, however, and must have affected the growth of the limb-rudiment had the limb been dependent on the myo- tomes either directly or indirectly for its development. The abdominal muscle is present, though somewhat reduced. Directly in front of the limb-region the abdominal muscle is well developed. Both the limbs are normal. The results of these experiments both on Amblystoma and Rana confirm the conclusions already reached from the study of normal embryos; z.e., that the muscles of the limbs are devel- oped wholly out of the mesoblastic cells of the somatopleure, the myotome-processes taking no part in the formation of the limb-muscles. First of all, the myotomes after injury remain permanently reduced in size. When the myotomes are greatly shortened there is often a corresponding shortening or con- traction of the entire injured side of the body. In the later stages of these embryos there is no evident attempt on the part of the myotomes to regain their normal proportions. Even when the myotomes have been but slightly injured, only the extreme ventral edges being destroyed, there is generally some permanent! indication of the injury in a reduction in the size of the muscle-plates. 1 None of the embryos used in these experiments were kept longer than eight weeks. No. 2.] LIMB-MUSCLES IN AMPHIBIA. 127 Although the myotomes plainly show that they have been reduced by the operation, and the regions from which the myo- tome-processes develop have been destroyed, a rudiment of the primary abdominal muscle is always present, even though it is often greatly reduced in size. The presence of an abdominal muscle-rudiment, together with a greatly reduced myotome on the side of the injury, might seem to indicate that the primary abdominal muscle-rudiment had formed prior to the time of injury to the myotomes and had possibly escaped being de- stroyed when the myotomes were injured. Normal embryos killed on the same days that the others of the set were injured and used as a check to the injured series show that the ventral myotome-processes or primary abdominal muscle-rudiments are still in connection with the myotomes, even in the anterior part of the body, where the connection between the myotomes and their ventral processes is earliest lost. In the posterior limb-region of the “‘check’”’ embryos the abdominal muscle- rudiment is represented by only the extreme ventral edges of the myotomes, which had not begun to be constricted at the time of the operations. It is not probable that the abdominal muscle-rudiment in corresponding embryos always escaped being destroyed through- out the extent of the injury when the myotomes have been as greatly reduced as shown in Pl. XI, Figs. 24 and 25. I have made several sets of experiments on the tadpoles of Rana palustris to see if the primary abdominal muscle really does always regenerate after it has once been destroyed. These experiments consisted in destroying the ventral halves of the myotomes before the primary abdominal muscle had begun to develop. The injury was much more extensive in these experi- ments (made in the spring of 1896 and of 1897) than it was in the original ones (made in 1895), for it was not confined to the limb-region, although it often included the limb. The results of the experiments show that when the rudiment of the abdominal muscle is destroyed along with the ventral edges of the myotomes it always regenerates, the process of regeneration beginning within a few days after the injury. I have never found an embryo in which a rudiment of the 128 BYRNES. [Vou. XIV. abdominal muscle was wholly wanting. There is in nearly all of the injured embryos a dorso-ventral growth from the myo- tome in the posterior part of the body, where the rudiment of the abdominal muscle and the myotomes retain their connection with each other. This down-growth from the myotome, such as is shown in Pl. XI, Figs. 23, 24, and 27, occurs in connec- tion with the regeneration of the abdominal muscle, and not in connection with the regeneration of the myotome itself. Directly in front of this place of union of the abdominal muscle with the myotome (Figs. 23, 27), there is no cor- responding down-growth from any of the myotomes, but the rudimentary muscle is always present some distance below them (Figs. 25, 28). Since all the myotomes of the same embryo were injured at the same time, I expected to find them all simultaneously undergoing similar regenerative changes, but I have never found this to be true. Iam unable at present to satisfactorily explain the regeneration of the abdominal muscle, and can only suggest one of two hypotheses by way of explanation : either the abdominal muscle is formed anew (by the injured myotomes) from a second series of myotome-processes, or it is regenerated independently of the myotomes from an uninjured part of the muscle itself. On the hypothesis that the abdominal muscle regenerates from a series of down-growths from the injured myotomes, the anterior myotomes would have to regenerate their ventral myotome-processes before the posterior myotomes regenerated theirs, for the position of the regenerated muscle-rudiment in the body-wall is almost invariably od/zqgue ; the more anterior end being much further ventral to the myotomes than the posterior end of the muscle, which always remains in contact with the myotome from which it was originally derived. This hypothe- sis could account for the regeneration of the abdominal mus- cle in the posterior part of the body where the myotome and the muscle-rudiment are connected, but it cannot explain the regeneration of the abdominal muscle in front of this region. The oblique position of the regenerating muscle and the complete separation of the muscle from the anterior myotomes No. 2.] LIMB-MUSCLES [N AMPHIBIA. 129 are more readily explained by the alternative hypothesis ; z.e., that the abdominal muscle has regenerated from its own tissue in an uninjured part of the body, rather than from the injured myotomes themselves. This suggestion is made prob- able by the fact that the connection between the regenerating abdominal muscle and the ovzgzzal abdominal muscle-rudiment is always unbroken, the regenerating muscle always passing gradually into the normal muscle, which lies ventrally in a more anterior part of the body. This could hardly be the case if the regenerating muscle were formed by a second series of ventral processes from the myotome. Although the rudiment of the abdominal muscle regenerates sooner or later, the new muscle is often smaller than the corre- sponding normal one on the uninjured side of the body. It is also often much nearer the ventral edge of the myotomes than the one on the normal side. These experiments, showing that the abdominal muscle always regenerates, make it extremely probable that in the original experiments the rudiment of the abdominal muscle was actually destroyed, but that it has regen- erated and in some cases has reached almost its original size. Although the rapid regeneration of the rudimentary abdominal muscle often brings about normal relations between the muscle and the limb-rudiment, the injury often suffices to check the development of the myotomes and to keep them, as well as the myotome-derivatives, temporarily from coming in contact with the limb-rudiments. The most striking fact in connection with the injured embryos is that, notwithstanding the permanent reduction in the size of the myotomes and the consequent diminution in the size of the myotome-derivatives, the lémbs are normal. This normal development of the limbs can only be explained on the ground of independence of the limbs and the myotomes. If the limbs are normally dependent on the myotomes for so large a proportion of their entire mass as is represented by the mus- cles, why, when the source of the muscles has been destroyed, is there no corresponding diminution in the size of the limbs ? There is no reduction in the limbs corresponding to the reduc- tion in the muscle-structures, except in those cases where the 130 BYRNES. [Vou. XIV. injury to the myotomes has also involved the somatopleure immediately below them. In such cases, however, the reduction in the size of the limbs is due to interference with metabolic processes, and is not due to the exclusion from the limbs of masses of cells that are destined to provide them with any given tissue. Evidence that the limbs are not dependent on the myotomes for any such large proportion of their tissues as is represented by the muscles is given in Pl. XII, Figs. 42-47. All these cases, besides others which have not been figured, go to show that the limbs develop alike on both sides of the body, and nor- mally, even though the muscle-structures have been very largely destroyed. The regions in which the different tissues are going to develop become clearly outlined in the limbs long before differentiation into muscle and cartilage actually begins. The peripheral regions of the limb-rudiments give rise to muscles and contain many more nuclei than the central or cartilaginous region, and hence stain much more intensely than the rest of the section. Comparing the darker peripheral part of the limb- rudiment on the right (injured) side of the embryo with the limb on the normal (left) side, the different regions are found to exactly correspond, showing that there are neither quantita- tive nor qualitative distinctions between the two limbs. Even in Amblystoma, in which an actual reduction in the size of the limb on the right side of the embryo often occurs, owing to the necessary extent of the injury, the peripheral parts of the limb- rudiment are blocked out into the areas in which the various muscles of the upper limb are going to develop; and these regions correspond precisely to those in the limb on the normal side of the body. These facts show that even in those cases where there is a quantitative difference in the two limbs the difference is only one of size and not one of kzzds of tzssue present in the limbs. Should the constant regeneration of the abdominal muscle- rudiment be urged as an objection to the validity of the conclu- sions drawn from the experiments, it must be remembered that, although the muscle does regenerate, it nevertheless regenerates No. 2.] LIMB-MUSCLES [N AMPHIBIA. 131 in front of the limb and often does not come into contact with it. Even when the regenerating abdominal muscle does come in contact with the limb-rudiment it touches the limb only at its extreme anterior limit, and does not extend throughout the limb-region, as it does in normal embryos. Moreover, the limb on the side of the injured myotomes always keeps pace in development with the limb on the normal side, even from the beginning, its growth being wholly independent of whether the regenerating muscle reaches the level of the limb or not. The constant presence of normal limbs in the injured embryos and the fact that in normal embryos there is no local budding or proliferation of cells from the myotomes or from the abdomi- nal muscle in the posterior limb-region show pretty conclu- sively that the limbs in the amphibia are of somatopleuric origin. General Conclusions. The ventral processes from the myotomes in the Urodela (Amblystoma and Triton), and in the Anura (Rana and Bufo), go to form the ventral muscles of the body-wall. They do not go into the limbs as “ muscle-buds,”’ but pass to the median side of the limb-rudiments. In the Urodela the pronephros develops in the region of the anterior limb. As the pronephric tubules elongate at right angles to the direction of growth of the ventral myotome-processes, the pronephros pushes the myotome-processes over against the limb. The proximity of the two structures is a coincidence, and there is no fusion between the myotome-processes and the anterior limbs in the Urodela. There are no “muscle-buds” in the amphibia such as are found in the elasmobranchs and teleosts. In the poste- rior limb-region there is no local proliferation from the myotomes to the limbs. The anterior and posterior limbs in the Urodela and Anura arise as thickenings in the somatopleure. The thickening is formed zz sztw by the multiplication of mesenchyme-like cells, some of which owe their origin to the division of the endothelial cells. The myotome-processes as such take no part in the formation of the limbs. These results obtained from the study 132 BYRNES. [VoL. XIV. of normal embryos were afterward confirmed by experiments on Amblystoma and Rana. The myotome-processes were ex- cluded from the posterior limb-region on one side of the body by destroying the lower halves of the myotomes. Even under these conditions the posterior limbs were found to develop normally, although the myotomes and the myotome- derivatives were permanently reduced in size. After injury to the myotomes, the abdominal muscle regenerates, but the injury serves to delay its development and to keep it tempo- rarily away from the limb. The conclusions reached from the study of the limbs in the normal amphibia, and particularly from the experimental evi- dence, is that the limbs are of somatopleuric origin; z.¢., that the muscles as well as the cartilage and connective tissue of the limbs are formed from the somatopleure, and that the myotome- derivatives are not essential to the formation of muscles in the limbs. This conclusion brings into question the distinction that has been established between muscles derived from the mesothelium and those derived from the mesenchyme. I believe the results of the experiments on Amblystoma, and more especially those on the frog, must be interpreted as show- ing that in these forms, at least, the power to develop striated muscle has not been restricted to the myotomes; 2z.e., to meso- thelium, but that the mesenchyme-like cells of the somatopleure can and do give rise to voluntary muscles in the limbs. The experiments on the myotomes, therefore, furnish additional evidence to that already urged against the conception of a fundamental distinction between mesothelium and mesenchyme. No. 2.] LIMB-MUSCLES IN AMPHIBIA. ee LIST, OF - REFERENCES: BALFour, F. M. A Monograph on the Development of Elasmobranch Fishes. 1878. BARFURTH, D. Zur Regeneration der Gewebe. Archiv f. mikr. Anat. Bd. xxxvii. CorNING, H. K. Ueber die Ventralen Urwirbelknospen in der Brustflosse der Teleostier. JMZorph. Jahrb. Bd. xxii, Heft 1. November, 1894. CorninG, H. K. Ueber die Entwicklung der Zungen musculatur bei Reptilien. Avzat. Anz. (Gesellschaft). July, 1895. Dourn, A. Der Ursprung der Wirbelthiere und das Princip des Func- tionswechsels. 1895. FIELD, H. H. Die Vornierenkapsel, ventrale Musculatur und Extremita- tenanlagen bei den Amphibien. Azat. Anz. Bd. ix, No. 23. 1894. FIELD, H.H. The Development of the Pronephros and Segmental Duct in Amphibia. Bull. of the Museum of Comp. Zool. Pl. XXI, No. 5. FISCHEL, A. Zur Entwicklung der ventralen Rumpf und der Extremitaten- musculatur der V6gel und Saugethiere. Morph. Jahrb. Bd. xxiii, Heft 4. December, 1895. GoETTE, A. Die Entwicklungsgeschichte der Unke. 1875. HarRIsON, R. G. The Development of the Fins of Teleosts. Johns Hop- kins University Circulars. No.3. 1894. HarRRISON, R. G. Die Entwicklung d. unpaaren u. paarigen Flossen d. Teleostier. Archiv f. mikr. Anat. Bd. xlvi, Heft 3. December, 1895. JorpAN, P. Die Entwicklung der vorderen Extremitat der Anuren Betrachier. Juaug. Diss. Leipsic, 1888. KAESTNER, S. Ueber die allgemeine Entwicklung der Rumpf- und Schwanzmusculatur bei Wirbelthieren. Mit besonderer Beriicksichti- gung der Selachier. Archiv f. Anat. und Phys. (Anat. Abtheil.). 1892. nes S. Extremitaten und Bauchmusculatur bei Anuren. Archiv Jj. Anat. und Phys. (Anat. Abthetl.). Hefte 5 und 6. 1893. KOLLMANN, J. Die Rumpfsegmente menschl. Embryonen von 13 bis 35 Urwirbeln. Archiv f. Anat. und Phys. (Anat. A btheil.). 1891. Maurer, F. Die ventralen Rumpfmusculatur der Anuren Amphibien. Morph. Jahrb. Bd. xxii, Heft 2. December, 1894. Maurer, F. Der Aufbau und die Entwicklung der ventralen Rumpfmuscu- latur bei den urodelen Amphibien und deren Beziehung zu den gleichen Muskeln der Selachier und Teleostier. Morph. Jahrb. Bd. xviii, Heft 1. 1891. Maurer, F. Die Entwicklung des Bindegewebes bei Siredon pisciformis, etc. Morph. Jahrb. Bad. xviii, Heft 2. 1892. 134 BYRNES. [VoL. XIV. Mayer, P. Die unpaaren Flossen der Selachier. Mztth. a. d. Zool. Stat. z. Meapel. Bd.vi. 1886. MOLLIER. Die paarigen Extremitaten der Wirbelthiere. Anat. Hefte. Heft 16 (Bd. v, III). 1893. MOLLIER. Die paarigen Extremitaten der Wirbelthiere. II. Das Chei- ropterygium. lk cart. coel. 17. My. le RUG: L. Rud. L. Mus. My. Myoc. My. pro. TEL Vales WV BH IP) LP Pron. Reg. P. A. M. 5 JB} As SE REFERENCE LETTERS. PLATES X, XI, AND XII. Alimentary canal. Blood vessel. Cutis layer. Cartilage. Coelom. Injured myotome. Left limb-rudiment. Limb-rudiment. Limb-muscles. Myotome. Myocoel. Myotome-process. Primary abdominal muscle. Second pronephric funnel. Pronephros. Regenerating “ primary abdominal muscle.” Segmental duct. Somatopleuric fold. 26 5) 136 BYRNES. EXPLANATION OF PLATE X. (All drawings made with the camera excepting Fig. 17.) Fic. 1. Cross-section of young Amblystoma. Section taken through anterior limb-region, showing beginning of ventral myotome-process, somatopleuric fold of anterior limb, and 2d pronephric funnel. Fic. 2. Cross-section older larva just behind anterior limb-rudiment. Fic. 3. Cross-section older larva through anterior limb. Fic. 4. Cross-section through anterior limb posterior to 2d pronephric funnel. Fic. 5. Like Fig. 4. Shows myotome-process distinct from limb-rudiment. Fics. 6 and 7. Cross-sections through anterior limb-regions of older larvae. Myotome-process seen to median side of limb-rudiment. Connection lost between myotome and ventral myotome-process. Fic. 8. Cross-section in front of posterior limb-region in Amblystoma. Fic. 9. Cross-section behind posterior limb-region. Fic. 10. Whole cross-section of Amblystoma through anterior limb-region, showing regions indicated in Figs. 1-8. Fic. 11. Cross-section through anterior limb-region of Triton. Fic. 12. Longitudinal section through posterior limb-region of Rana. Section shows abdominal muscle-rudiment passing the limb-rudiment. Fic. 13. Cross-section through the posterior limb-region of Amblystoma. _ Fic. 14. Cross-section through the posterior limb-region of Triton. ' Fic. 15. Cross-section anterior to posterior limb-region of Triton. Shows rudimentary abdominal muscle at ventral edge of myotome. Fic. 16. Whole cross-section of Amblystoma through posterior limb-region, showing regions indicated in Fig. 13. Fic. 17. Whole preparation of anterior limb-region of Amblystoma. Fic. 18. Cross-section through posterior limb-region of Rana. (Cf Fig. 18 with Fig. 12.) al of Morphology Vol. XW: 138 BYRNES. EXPLANATION OF PLATE XI. (All outlines traced with the camera.) Fics. 19 and 20. Two cross-sections through the posterior limb-region of a tadpole killed four days after injury. Fig. 19 is more posterior than Fig. 20. Both show the ventral edge of the right myotome destroyed ; also the posterior abdomi- nal muscle-rudiment. The limb-rudiments are present and uninjured. Fics. 21-25 show a series of cross-sections taken through the posterior part of the body of a tadpole killed twenty-six days after injury. Fig. 21 is a section through the posterior limb. The right myotome is reduced and does not come in contact with the limb. Figs. 23-25 show the regenerated posterior abdominal muscle-rudiment. Fics. 26-28. Series of cross-sections through the posterior part of the body of an injured tadpole, showing the normal limbs, the reduced myotomes, and the regenerating posterior abdominal muscle-rudiment. Fics. 29-35 show cross-sections through the posterior limb-region of a tadpole killed thirty-four days after injury. The limb-rudiments are normal and of equal size. The myotome-derivatives are entirely wanting throughout the limb-region. Fic. 36 shows a section through the same embryo, but taken in front of the posterior limb-region. The posterior abdominal muscle‘is just beginning to form, but is greatly reduced. > x Journal of Morphology. Vol. XIV: 140 BYRNES. EXPLANATION OF PLATE XII. (All drawings made with the camera.) Fic. 37. Cross-section through the posterior limb-region of an Amblystoma larva killed four weeks after injury. Myotome greatly reduced. Right limb present. Fics. 38 and 39. Sections through the right and left posterior limbs, respectively, of an Amblystoma larva killed six weeks after injury. Fic. 40. Cross-section through posterior limb-region of Amblystoma. Muscle- regions beginning to develop in the right limb. Fics. 41 a and 41 6. Two cross-sections through the posterior limb-region of a tadpole killed nine days after injury. Fig. 41 a@ more posterior than 414. Right myotome almost wholly destroyed. Medullary tube wanting. Limb-rudiments equal and normal. Fics. 42 and 43. Cross-sections through right and left limb-rudiments of an embryo whose myotome-derivatives were destroyed. Both limbs are beginning to show a condensation of nuclei around the periphery to form muscles. Fic. 44. Section of Fig. 41 a, enlarged, showing relative number of nuclei in right and left limbs. Fic. 45. Enlarged section of Fig. 33 in Pl. XI. Posterior abdominal muscle wanting on right side of embryo. Right limb normal. Fic. 46. Cross-section through middle region of right posterior limb of tadpole killed thirty-two days after injury. Posterior abdominal muscle-rudiment wanting. Limb normal. Fic. 47. Middle region of left posterior limb of same embryo. Comparison of Figs. 46 and 47 shows the two limbs equal, although in Fig. 46 the myotome- derivatives are wanting. K rt bh: 1 ae Journal of Morphology VolAN ‘Wey ( oy My. ( Fo Nen sande, Reed. Cont. Cart. ; + DRL Rud ® be Ny \ \ nee eee Oyo) ; , 7 . = Tid Anse Werner 4 Wixter, Frock far AM ; ’ : Ww CONTRIBUTION, TO. THE MORPHOLOGY OF DERO’ VAGA: HOWARD S. BRODE. CONTENTS. PAGE TORTI ER © TW © Te © Nese ces cee cn te ee oe ON Nea ee OS pec ae ree eenceee ea 142 TIDES Pd Soggy RN Copa 2 Sa PAN SS ye a Oe ae a op ae ae oe te 142 Bs G@THAGSINI CAT ONS ainccet erates toss eedeate tea Ores ese na dete own eae eesccoarsuaseentdeestee 142 2 ete NPAC TAU ROAM ELSIE OR Weretacc ences Secret tars ke ect Anew on saetog cate Mextaseos sees eee 144 SPM V LUPO STGE CD) S frre oe reas gts betas ota cacao ath seat any casecb secon enh caaasanenestcussetosneteaccs 147 DATS Tie ast (@) ESE FAO) Te, ©) (Ges Vet er cere Se eB, oc wes SEs dando erent eee ese 148 I. THE BODY WALL AND EXTERNAL CHARACTERG............ccc:::cc0:000- 148 (SY IE 111227 IC Ai cee a Ee RE oe eR PEE Se 148 (2) SY 7ie7 ee ec eee NT eee SON ee ean or REE A see eee nose 149 (C) PLD EACH IES ose c cece ean Soaks Poche uea st vawssuct us tentennectutoeceseterececce neue 149 (DPB ileryt DUA GLAMES cee awa tock ciuee Seances c Sessartsuecebaneos sesoneceeens 150 CY ED EPAT A pe le Bem eRe US CR SEER aS EP 150 BO OLN CRIS She ZION te Sakae wee eee nor nee er eer re eee 151 (ap Aastoricahi soaks oe ce tee tt Bak Noes eee ES Oe I51 (DL 29CS CHEADLE sae Sk Papeete pre cnc ones ts ecenadeasue see opera eect apoatons 152 (OG) SACS TY TATED EDS TTL ao ee er aoc eae See SE 155 (A) RC Ome Dar Ati e Ben ae eens eee ne cree eceaneee naeh cbaey meee eaten coe 156 rm N SE ORGAN ooo o5 25 sactatspamdsaaus scnunoann opau szswnmapsbsiebleaauscaspsaeennpecestes 158 (A) LI CSCH IDLE eater aah cate eee tt epee eee crs tect cbr ones eee eee 158 (DD) Care Barts eee ear toes eee ane pa eet aoe eae nee tess te) LOO OL SOCRLETES ee an coer ere etn cc ese ee ean Ras ome Se ctr 160 (c) eA etameeritce SC7SCl OPUS ere aace eae ee cee sen ee eens ee 161 OLzOCH ete s tet Sle Ree tn As Eee te ee 161 POUCH CLES sete Mares pees Bin a cite coatencece hasensnipstescee saree petseodes 162 LEC CIES! Arstiae so eR NN rata gutta Lane siisuscvioust Doinabes set eorteenetea ste 162 VORTEC DF OLE S Be en eR eet ec ate eh ne lpr ce cue a Renee 162 ARISEN S O- CATE Ems CI AME VAT TUG DINU) 74 | tcc sesso ested ee ee ee 163 (ARET aston cleric r ee OD GE. ce phn ccna sal enc ean eee ps pier 163 WELDON UD LACE caste ect oe sere Lc ca seed tes vestennen suse abuse ccebaswas sate 167 (c) Laterpretation of Previous ObservatiOns .....2.-..2ec2eereee- 168 TV ee ENE) ORB a CAN © ONS DB RAC TO INS sees sccccna conse cee nese 169 I. ORIGIN OF METAMERISM..........-- sobechiie (ie ack ge ke eeaes eee st en .. 169 2. SEGMENTAL SENSE ORGANS IN ANNELIDS AND ORGANS OF SPECIAG SENSE IN HIGHER ANIMALS(..2.22.ccctccccectscnesesenonenes = 171 142 BRODE. [VoL. XIV. I. INTRODUCTION. Tuis paper embodies, in part, the result of three years’ work carried on in the Zodlogical Laboratory of the University of Chicago and at the Marine Biological Laboratory at Wood’s Holl, Mass. During the entire time the work has been conducted under the supervision of Dr. C. O. Whitman, Head Professor of Zodlogy at Chicago and Director of the Marine Biological Laboratory, at whose suggestion the work was undertaken. It has been a great source of pleasure to me to be under the direction of so able a teacher, and I am very deeply indebted to him for the interest he has taken in my work. I desire also to express my gratitude to the authorities of the University of Chicago for the favors which they have from time to time granted me. My early studies on this annelid were with reference to the process of multiplication by fission. In the course of this study it became evident that a very exact knowledge of the anatomy of one segment was necessary in order to understand fully the changes which take place when a fission zone forms in a seg- ment. This study led to the working out of thenervous system entire and the distribution of the sense organs, together with some points on the general morphology of the worm. I have made many observations on worms undergoing fission and hope at some future time to bring out a paper on normal and artificial fission and regeneration. II. GENERAL REMARKS. I. CLASSIFICATION. Dero vaga was originally described by Joseph Leidy in 18801 under the name Azlophorus vagus. The generic name Aulo- phorus was given by Schmarda? in 1861 to a form differing 1 Jos. Leidy, Notice of Some Aquatic Worms of the Family Naids, dmer. Vat., Vol. XIV, No. 6, 1880. 2 C. Schmarda, Neue wirbellose Thiere, beobachtet und gesammelt auf einer Reise um die Erde (1853-57), Theil I, Heft I, Leipzig, 1861. No. 2.] THE MORPHOLOGY, OF DERO VAGA. 143 somewhat from the form described by Leidy, while the latter form is very closely related to the other species of Dero and without doubt should be classed with them. Vaillant! and Beddard? classify the annelid as Dero vaga Leidy. - The following synopsis is taken from Beddard’s monograph: FAMILY NAIDOMORPHA. Definition. — Aquatic Oligochaeta of small size. Setae usually in four groups upon each segment, sigmoid, bifurcate, hastiform, and capilliform. Sexual reproduction at fixed intervals, between which asexual reproduction by fission occurs. Sexual organs (only known in a few types) are situated far forward, commencing even in the fifth segment. This family of Oligochaeta comprises the following recognizable genera : (1) Chaetogaster Baer. (5) Pristina Ehrenberg. (2) Amphichaeta Tauber. (6) Uncinats Czerniavsky. (3) Wazs O. F. Miiller. (7) Chaetobranchus Bourne. (4) Bohemilla Vejdovsky. (8) Dero Oken. GENUS DERO Oken. Syn. Proto Oersted. Uvonats Gervais. Xantho Dutrochet. Azlophorus Schmarda. JVazs O. F. Miller (in part). Definition. — Dorsal setae capilliform and hastiform,® commencing upon the sixth segment. Branchial processes present at hinder end of body. Eyes absent. Inhabit tubes. He notes eight species as follows: D. miilleri Bousfield. D. limosa Leidy. D. furcata Oken. D. perrieri Bousfield. D. obtusa D’Udekem. D. vaga (Leidy). D. latissima Bousfield. D. multibranchiata Stieren. DERO VAGA (Leidy). Azlophorus vagus J. Leidy. Am. Wat., 1880, p- 423. D.vaga L. Vaillant. Aznelés, p. 383. Definition. — Length about 8 mm.; number of segments, 25. Body ending in two long processes; branchiae rudimentary, only two slight processes. Dorsal setae bundles consisting of one capilliform and two pectinate setae.4 Perivisceral corpuscles present. Contractile hearts in VIII, IX, and X. fZab.— North America; Trinidad. 1L. Vaillant, Histoire naturelle des Annelés Marins et d’Eau douce, Tome III, Paris, 1889. 2 F. E. Beddard, Monograph of the Order Oligochaeta, Oxford, 1895. 8 Palmate setae may also be present in the dorsal bundles. 4 The setae in the dorsal bundles are more nearly palmate than pectinate, and there are usually two capilliform and two palmate in a bundle. Occasionally three of each are present. (Notes are mine.) 144 BRODE. [Vo. XIV. 2. NATURAL HIsTory. The family Naidomorpha includes many minute transparent worms varying in length from 1 to 15 mm. Some members of the family may be found in almost every collection of water plants from a pond or ditch. Members of the genus Dero may be recognized at once by the presence of digitiform processes at the posterior end of the body (Pl. XIII, Fig. 2), and the most apparent distinguishing character of Dero vaga is its habit of building a case for itself and pulling it about on the surface of the water (Pl. XIII, Pig. 1): Specimens may be found in ditches, ponds, and small lakes where there is an abundance of vegetation. For this work col- lections were made at Glacialis Pond near Cambridge, Mass., and at Wolf Lake, Ill. The surface of the Cambridge pond was almost covered with Lemna, and the worms were remark- ably abundant. The worms prefer a pond in the open field, but are found most abundantly in the shade of leaves of water plants near shore. In case there is a lack of Lemna the worms may be found in algae below the surface or even on the bottom of the pond. Dero vaga varies in length from 5 to 10 mm., according to the progress of the growth preceding fission. The width never exceeds .25 mm. The number of segments may vary from twenty-five to sixty. All segments excepting the first five have four bundles of setae. The first segment (prostomium) has no setae. Segments II-V have ventral setae only. The anterior end of the body is slightly enlarged, and during locomotion the pharynx is everted to form a sucking disc. The branchial apparatus at the posterior end of the body is made up of a disc with undulating edges, which occupies a dorsal position, and two well-defined digitiform appendages which are found near the ventral side. In addition to these parts there are two long, slender, outwardly curving appendages with en- larged tips which arise below the other parts (Fig. 2). All parts of this apparatus are covered densely with cilia, which, during life, are in constant motion. When the worm is undis- Near 2:i] THE MORPHOLOGY OF DERO VAGA. 145 turbed this whole apparatus is protruded from the case and is turned with the concave side of the disc uppermost. When the apparatus is contracted the disc is so folded as to give the appearance of four lobes, which, with the two well-defined ventral lobes, make up the half-dozen blunt papillae described by Leidy. The case (Fig. 1) consists of a thin hyaline tube covered over with dead Lemna leaves, statoblasts of fresh-water Bryozoa, Arcella shells, or small pieces of almost any substance which may be floating on the surface of the water in which they live. As the worm grows longer the case is also increased in length, and when fission is complete the worms place their heads together at the middle of the case and break it in two. Each worm goes away with one half of the old case. The cases of worms found on the surface of the water will float when the worm is driven out, while those found on the bottom will sink under similar conditions. Worms have been observed to change their position gradually from surface to bottom and from bottom to surface, according to the location of the food supply. Locomotion is effected in a jerking manner by extending the body some distance out of the case and attaching the anterior portion by means of the pharynx and the ventral setae and then contracting the body, pulling the case forward. At the time of the development of the sexual organs the worms have been observed to leave their cases and crawl about on the bottom of the dish. The food taken is apparently entirely vegetable matter and consists of desmids, algae, and at times the fronds of Lemna and Wolffia. Sexual organs are developed during the first two weeks in July. However, worms kept over winter in an aquarium showed sexual organs as early as April 1. A clitellum is formed which covers segments V—VII. Spermathecae occur in segment V and the atrium of the sperm duct is plainly visi- ble in segment VI. Egg masses fill a large part of the body cavity posterior to the clitellum. Eggs outside of the body have not been found and the manner of laying the eggs is unknown to me. 146 BRODE. [VoL. XIV. At all seasons of the year excepting two weeks in July the worms multiply by fission. This takes place slowly in the winter and very rapidly during the summer. The new head and tail form almost completely in the region of fission before separation takes place. In the summer fission occurs as often as three times a week. In a dish in which one worm was placed, eight were found at the end of one week. At the end of two weeks fifty were counted. The fission zone is formed near the middle of a segment and not between two segments, as has been described for some other Naidomorpha. In so far as I have observed, the regen- eration of head and tail in cases of fission occurs in the one somite in which the fission zone first appears. The number of segments formed in the new head is constant, being five, while the number in the new tail varies, — in fact there is no limit to the growth of the tail. Usually twelve to sixteen segments are visible before second fission occurs. When fission occurs in an anterior individual the zone appears in the first new segment of the previous fission zone. Three fission zones may be present in one worm during the period of rapid multiplication. More than three have not been observed by me. At the first outward signs of fission the worm has from thirty to forty segments and measures from 5 to 6 mm. in length. When the worms are ready to separate there are from fifty to sixty segments, and the length has increased to 10-11 mm. The number of the segment in which fission occurs is fairly constant, but is liable to vary. In all observed cases it occurs back of segment XVII and usually anterior to segment XXII. If a worm is divided by cutting, both parts will continue to live and in a short time will regenerate a new head and tail and form perfect individuals. There seems to be some limit, however, to the number of segments which will regenerate a new head or tail. My experiments have not been extensive enough to establish any rule regarding regeneration, but they have shown that the number of segments regenerated at the anterior end of the No. 2.] THE MORPHOLOGY OF DERO VAGA. TA] body is constant, z.e., only enough are regenerated to complete the five first segments. If two segments are removed a like number will be regenerated, but if seven segments be removed there will be but five new segments formed. In the latter case the regeneration proceeds much more slowly than in cases where fewer segments are removed. In order that the removed anterior portion of a worm regenerate a tail it seems to be necessary for it to have at least three or four segments in addition to the five in the cephalized part. In case a cut is made a short distance in front of a fission zone the part anterior to the zone may disintegrate while the posterior individual continues to live, and the normal process of regeneration is apparently hastened to adjust the worm to the new conditions. 3. METHODS. The following methods were found to give good results: Killing and Hardening. — Hot corrosive sublimate (saturated aqueous solution); hot acetic corrosive sublimate, Hermann’s fluid, and 1/10% osmic acid. Maceration. — 1/10% nitric acid, also a mixture of glycerine, acetic acid, and water, equal parts. The worms treated with the nitric acid were dissected with needles ground flat. Staining. — Of the ordinary stains Delafield’s haematoxylin, Bohmer’s haematoxylin, Grenacher’s borax carmine, and the triple stain of Heidenhain-Biondi-Erlichi gave good results. For the working out of the nervous system and sense organs the following methods were used: Gold Chloride. —The method employed was essentially the same as that given by Mr. C. L. Bristol in the American Natu- ralist for September, 1894. The live worms were killed in 10% formic acid and left for one minute, after which they were placed in 1% aqueous solution of gold chloride for ten minutes, and then left in 1% formic acid for from two to four hours. The gold chloride solution was kept out of bright sunlight while the specimens remained in it. The reduction in 1% formic acid was carried on for a portion of the time in sunlight. 148 BRODE. [VoL. XIV. Methylen Blue. — A solution of the stain was made by dis- solving a small amount of the powder in water; a very small drop of this was added to a drop of clear water on the slide in which was a live worm. A cover glass supported on wax feet was placed over this and the examination was made with the compound microscope. Within two hours the peripheral nerv- ous system would be well stained. Specimens were also killed by adding a drop of 2% formalin to the slide on which they were placed. The methylen-blue solution was then added. This gave the best results in the study of the sense organs, while the peripheral nerves took the stain only when the specimen was alive. Imbedding. — Specimens were imbedded in paraffin and were cut from 5 » to 15 pw thick. Drawing. — Drawings were made by aid of the Zeiss-Abbé camera lucida, and a number of points were worked out by means of reconstructions and composite drawings from camera sketches. The majority of the figures were drawn at a magnification of 400 diameters, and all were reduced one-half in reproduction. III. MORPHOLOGY. 1. THE Bopy WALL AND EXTERNAL CHARACTERS. (a) Segmentation. The number of segments in an individual depends upon its condition. If sexually mature it may have from twenty-five to thirty-five. This period is so short that the growth in length in the tail region is scarcely interrupted. If the worm is mul- tiplying by fission it may have as many as sixty segments. The first segment differs from the others in appearance. This difference may be due to its position and modified func- tion. The well-developed branchial apparatus at the posterior end of the body in all probability does not represent a segment, for in cases of fission it is not fully formed until many seg- ments have been marked off anterior to it. The growing zone, in which are located the cells corresponding to the teloblasts of the embryo, lies just anterior to this branchial area. The No. 2.] THE MORPHOLOGY OF DERO VAGA. 149 segments in the anterior region are shorter than those follow- ing, and all the segments have a more or less distinct secondary annulation. (b) Sezae. The first segment bears no setae. The second, third, fourth, and fifth bear each two bundles ventrally, containing from eight to twelve long slender setae with bifurcate tips. The following segments bear four bundles — two dorsally and two ventrally. The dorsal bundles contain two peculiar palmate setae and two capilliform setae. The ventral bundles contain four or five bi- furcate setae resembling those in the anterior segments, but being somewhat shorter and thicker. In sexually mature indi- viduals the ventral setae are absent in the sixth segment. In cases of fission and regeneration the ventral setae form before the dorsal, and the setae in the anterior segments whether in the head or tail are always the first formed. (c) Epidermis. The epidermis is composed principally of hexagonal colum- nar cells, the width of which is about twice the height. These cells have large nuclei and are covered externally by a thin chitinous cuticle. In addition to these cells there are found sensory cells and gland cells. The sensory cells are spindle-shaped and have large nuclei compared to the size of the cell. The nuclei stain much more deeply than those of the ordinary epidermal cells. From the outer end of the cell projects a long, stiff hair or bristle (Figs. 10, 14-16). The sensory cells may occur singly or in groups. In the latter case the epidermis shows a hemi- spherical elevation at that spot. The sense organs will be discussed in more detail later on in this paper. The gland cells are goblet-shaped and often are found with granular contents. They are more abundant in the head region than elsewhere. The epidermis is thicker on the ventral side than on the dorsal and the cells multiply rapidly where a fission zone forms and at the posterior end of the worm. The epidermis of the 150 BRODE. [Vor. XIV. prostonium is very much thickened and is thickly set with sensory cells. (d) Epzdermal Glands. In addition to the scattered gland cells mentioned above there are some other modifications of the epidermis which very probably are glandular in their nature. The clitellum covering segments V—VII during the time of sexual development has not been specially studied in this form, but the structure and function is in all probability much the same as has been described in other Oligochaetes. Near the posterior border of every segment beginning with segment VI there is a marked band of cells in the hypodermis (Pl. XIV, Fig. 10, g/d.) which when treated with ordinary reagents appear as empty cells surrounded by a substance taking a stain somewhat more deeply than other parts of the epidermis. | Another peculiar band (Pl. XIV, Fig. 10, @.gr.) appears in specimens stained with methylen blue. It is situated just posterior to the anterior girdle of sense organs and consists of very deep-staining dots regularly arranged on all segments back of the first five. Connecting the dots in the band there appears a narrow groove in the epidermis. I have not made a careful study of these structures, and my reason for mentioning them is on account of their metameric arrangement. (e) Muscles. There are two layers of muscles in the body wall, an outer circular and an inner longitudinal layer. The muscles are all of the so-called Nematoid type. The circular muscles (Pl. XIV, Fig. 18, ¢.mus.) are arranged in a single layer just beneath the epidermis. The nucleated plasma parts are gathered in the two lateral lines (Pl. XIV, Fig. 18, 2.7.) of the worm lying in the breaks between the dor- sal and ventral halves of the longitudinal muscles. This pecu- liar arrangement of nuclei was first made out by Hesse,! and I 1R. Hesse, Beitrage zur Kenntnis des Baues der Enchytraeiden, Zezt. f. wiss. Zool., Ba. LVII, 1893, p. 6. No. 2.] THE MORPHOLOGY OF DERO VAGA. pS i have been able to corroborate his statements so far as concerns Dero and Tubifex. The longitudinal muscle layer (Pl. XIV, Fig. 18, ¢.#zus.) lies next the circular and is composed of fibres which are not arranged in groups, as in Lumbricus, but are isolated. The plasma parts with their nuclei show on the inner side of the layer. 2. NERvous SYSTEM. (a) Hestorical. Observations have been made on Dero by a number of inves- tigators, but little has been written concerning the nervous system. Perrier! described the nervous system of Dero obtusa (D. perriert), but confined his observations to the dorsal gan- glion and the ventral cord. No account was given of lateral nerves. Reighard? described the nervous system of D. vaga and gave an account of the nerves arising from the dorsal ganglion and commissure. No mention was made of lateral nerves from the ventral cord. He noticed the “lateral line,’ but could not trace it as far forward as the dorsal ganglion. In 1885 Anton Stole? in a paper on the anatomy and his- tology of D. digitata Mill. describes and figures the nervous system in greater detail. The dorsal ganglion in the form studied has a greater length from front to back than has that of D. vaga. The ganglia of the ventral cord correspond in shape to those of D. vaga. He figures some very minute nerves from the anterior face of the dorsal ganglion between the two large nerves and also some from the inner sides of the com- missures near their posterior place of fusion. He cites similar structures in Stylaria described by Vejdovsky. I am inclined to doubt the nervous nature of these fibres, for muscles are found in similar positions and I have been unable to find nerves 1 Edmond Perrier, Histoire Naturelle du Dero obtusa, Archives de Zoologie expérimentale et générale, Tome I, 1872, pp. 83-85. 2 J. Reighard, On the Anatomy and Histology of Awlophorus vagus, Proc. Am- Acad. Arts and Scz., Vol. XX, 1884, pp. 101-104. 8A. Stole, Dero digitata O. F. Miiller: Anatomickaé a Histologicka Studie. SB. Bohm. Ges., 1885, pp. 65-95. 2 pl. 152 BRODE. [VoL. XIV. so situated in D. vaga. He does not figure the nerves from the commissure corresponding to those found by Reighard. He found that as many as three nerves were given off from the ventral cord in a segment. These correspond in position to some of the nerves found in D. vaga. (b) Descriptive. The nervous system of Devo vaga consists of a central ganglionated cord with lateral nerves and a sympathetic system which covers the pharynx. Central Nervous System.— The central cord is made up of a dorsal ganglion, the so-called “ brain,” and an indefinite number of ventral ganglia, corresponding in number to the segments of the body. The dorsal ganglion is united with the first ventral ganglion by a commissure, which passes around the alimentary canal. The dorsal ganglion (Pl. XIII, Figs. 3, 6, dg.) is situated in the first segment (preoral lobe) and consists of two pear- shaped masses united at their larger ends, while the smaller ends are drawn out to form the commissures. The ventral ganglia in the first four setigerous segments are crowded together so that no space is left between them. However, the characteristic lobed appearance clearly shown in the follow- ing ganglia may also be traced in this part of the cord. The ventral cord consists of two large bundles of fibres on which are found at intervals of one segment the masses of ganglion cells which form the ganglia. An intermediary nerve is also clearly visible. The typical ganglion (Pl. XIII, Figs. 6, 8) consists of a series of four distinct enlargements. The second is the largest and is near the middle of the ganglion. The entire mass, with the exception of the posterior enlargement, lies in one segment and occupies a position a little posterior to the middle. The poste- rior enlargement occupies a position close to the dissepiment, and apparently has been pushed back so as to lie in the next segment. At the tail end of the worm or at a fission zone, the ventral cord is in connection with the epidermis (Pl. XIII, Fig. 7), from No. 2.] THE MORPHOLOGY OF DERO VAGA. 153 which it is constantly being formed. I have observed no specimens in which this connection did not exist. Sexually developed forms as well as those undergoing fission were observed. In consequence of this continued growth the ganglia vary in number. A cross section of the ventral cord through the widest part is shown highly magnified in Pl. XIV, Fig. 17. The nerves emerging from the cord (/.z.) are those which pass through the setae bundles. The ganglion cells (g.c.) are arranged in three groups, two lateral and one ventral. The fibrous portion (/2.) is divided by faint clear spaces into three parts. In the lower portion of the middle part, the cross section of the intermediary nerve (z.z.) may be seen, and in the dorsal part of the fibrous portion are three giant fibres (gf). Two muscle bands (#us.) appear, one on either side of the fibrous bundle, and a blood vessel (d.v.) lies closely applied to the dorsal surface of the cord. The examination of dissected specimens shows large irregular cells scattered along the dorsal surface of the cord. In the tail region and at a fission zone these cells are very much more abundant. Without doubt they correspond to the “ chorda cells’ of Semper,! and are probably identical with the “ neo- blasts” described by Miss Randolph? in Lumbriculus. In describing the lateral nerves I shall begin with the simpler condition found in the body segments, and proceed later to describe the nerves in the so-called “head.” As has been mentioned before, each segment of the body contains a ganglionic swelling of the ventral cord. This gan- glion reaches its maximum width at a point posterior to the middle of the segment, and a portion of the swelling extends through the dissepiment into the next following segment. Four pairs of lateral nerves are given off from each ganglion (Pl. XIII, Figs. 3, 6). These nerves pursue essentially the same course. On leaving the cord they pass obliquely downward and away from the cord, and pass through the longitudinal muscle layer 1C. Semper, Die Verwandtschaftsbeziehungen der gegliederten Thiere, III, Strobilation und Segmentation, Avéeit. a. d. Zool.-Zoot. Inst. Wiirzburg, Bd. III, 1876, p. 186. ? Harriet Randolph, The Regeneration of the Tail in Lumbriculus, Journ. of Morph., Vol. VII, 1892. 154 BRODE. [VOLE ADV. and come to lie among the circular muscle fibres. At the point where the nerve enters the muscle layers a branch is given off which passes to the ventral side of the body, while the main trunk passes dorsally. The nerves pursue a straight course around the body, and no branches have been noticed from any of the nerves in the body region. The first nerve from the ganglion (/.7z.1) passes around the body near the middle of the segment. It is the second largest of the four, and apparently innervates the muscles and other organs of the viscera. The second nerve (/.z.?) passes through the setae bundles, and is the largest nerve of the group. It innervates the greater band of sense organs found on the poste- rior part of the segment. The third and fourth nerves (/.7.3, /.n.4) are smaller than the first and are nearly equal in size. The third passes into the dissepiment, and very probably innervates the muscles of its walls. The fourth lies in the following seg- ment, and supplies the sense organs in the lesser band which encircles the body close to the anterior end of the segment. This arrangement of nerves I have traced forward through all the segments up to the first. The nerves from the dorsal ganglion are four in number, and, like those from the following ganglia, are placed three in the first segment and one in the next following or second segment. This latter, fourth nerve, has a course correspond- ing to that of the following nerves, while the three anterior nerves have a varied course. The first nerve (z.1) is large and leaves the ganglion dorsally and laterally at a point near where the commissure begins its downward course. It grows out a short distance as a single nerve, and later breaks up into three branches. The first runs almost straight forward, while the second bends below the first and follows the anterior wall of the proboscis, approaching the corresponding branch from the other nerve of the pair at the median line. The third branch passes forward and downward, and extends to the epidermis. Each of these branches subdivides into smaller branches near the body wall. The second and third nerves (z.?, 2.3) arise close together a short distance below the first. The second soon divides into two rami and these pass to the ventral and No. 2. ] THE MORPHOLOGY OF DERO VAGA. I55 lateral walls of the proboscis. The third nerve passes down- ward and slightly forward, and lies close to the anterior wall of the buccal cavity. From the distribution of the first three nerves it seems probable that some fibres at least in each of them are sensory. The fourth nerve (z.4) is situated at some distance from the third, but I have been able to trace ganglion cells from the dorsal ganglion as far down the commissure as the place of origin of this nerve. (c) The Sympathetic System. Two systems of visceral nerves have been described under the name “sympathetic nerve.” Leydig! distinguished the two by the names “sympathetic” and “vagus.” The system covering the pharynx he called the vagus nerve, and applied the term “sympathetic” to the system distributed over the intestine. Other writers speak of his vagus as the ‘“‘ sympathetic of the head” or simply as “‘ sympathetic nerve.” The sympathetic in this latter sense has been described in many annelids, both marine and fresh-water. Beddard says:? ‘«‘T have never found it to be wanting in any earthworm where I have looked for it.” Vejdovsky has described this system in Chaetogastridae and also mentions having found a ganglion on the posterior part of the pharynx of a young specimen of Wazs elinguis. So far as I know, this is the extent of the work on this system in the Naidomorpha. The pharynx of Devo vaga occupies the first four setigerous segments, and is composed of two very unlike regions. The dividing line is the boundary between the second and third setigerous segments. The lumen of the anterior half is divided into a dorsal and a ventral part by an infolding of the wall on either side (Pl X4IT, Pig:\5). The main branches (Pl. XIII, Fig. 5, 2.5.2.) of the sympa- thetic (vagus of Leydig) nerve lie in the groove between these two divisions and extend back to the ganglia (Pl. XIII, Fig. 4, 1 F. Leydig, Ueber den Bau des thier. Korpers, Tiibingen, 1864. 2 Beddard, /.c., p. 20. ; 156 BRODE. [VoL. XIV. s.g.), which are situated on the sides of the pharynx at the junc- tion of the anterior with the posterior region. The connecting nerves are distributed over the dorsal part of the pharynx, the whole system lying just beneath the epithelium of the canal. The main trunks (s.z.) are given off from the inner sides of the oesophageal commissure and from points a little below the brain. Their course is slightly dorso-lateral until they reach the pharynx, where they divide into two branches; the larger (m.s.n.) takes the position in the groove above alluded to, and the smaller extends upwards and back over the side of the pharynx and unites with the larger branch at the ganglion. At the place of branching, a large commissural nerve joins the two main trunks. A large nerve also extends over the dorsal side of the pharynx, uniting the two ganglia. Between these two commissures there are about fifteen smaller nerves, which also extend over the pharynx, anastomosing with the upper longitudinal trunks. The ganglia (s.g.), two in number, are double, the ganglionic mass being divided into an anterior and a posterior half. These parts are elongated dorso-ventrally, and the anterior mass is smaller than the posterior. Sensory cells are found in the pharyngeal epithelium, which in some cases are apparently connected with the branches of the sympathetic nerve. These cells are especially numerous along the two main nerve trunks. No connection has been found to exist between the sympa- thetic system and the nerves from the ventral cord, and no indication of a sympathetic nerve in Leydig’s sense has been observed. (d) Comparative. The nervous system in all Oligochaetes consists of a dorsal ganglion lying above the alimentary canal, and a ventral ganglionated cord connected with it by a circum-oesophageal commissure. The dorsal ganglion may be found in the first segment or it may be pushed back to lie in the third segment. In the devel- opment of the earthworm the brain is formed in the first No. 2. ] THE MORPHOLOGY OF DEKO VAGA. E57 segment, and, as development proceeds, it is pushed back to the third. From this we may infer that the former condition is more primitive than the latter. The shape of the dorsal ganglion differs in different species. In Dero vaga it is very simple, much more so than in other Naidomorpha and in Tubificidae, and resembles somewhat the dorsal ganglion in Lumbricidae. From descriptions of the nerves given off from the dorsal ganglion the number varies greatly. This may be due largely to insufficient investigation and difference in interpretation as to the origin of the nerves found. I think it highly probable that the number of nerves given off from the dorsal ganglion in any case will be found to correspond with the number given off from each ventral ganglion. This correspondence exists in Dero and Beddard ! states that a similar condition exists in Spirosperma. In most earthworms there appear to be three nerves from the dorsal ganglion and three from each ventral ganglion. The third nerve in each segment, however, sends off a branch which goes to the dissepiment. Further investi- gation is necessary before we can compare the nerves of the dorsal ganglion of the earthworm with the lateral nerves from the ventral cord. I believe that the arrangement of nerves which I have described for Dero vaga will be found to hold good for the majority of forms in the families Naidomorphaand Tubificidae. The observations of Vejdovsky? and Stolc3 on Tubificidae point in this direction. While Vejdovsky figures five nerves to a segment, it is not improbable that the two going to the dissepi- ment are really one. Stole describes one to the dissepiment and three others within the segment. 1 Beddard, Z.c., p. 20. 2 F. Vejdovsky, System und Morphologie der Oliogochaeten, Prag, 1884, p. 85. Pl. VIII, Fig. 2. 3 A. Stole, Llyodrilus coccineus Vejd., Ein Beitrag zur Kenntnis der Tubificiden, Zool. Anz., Bd. VIII, 1885, p. 641. 158 BRODE. [Vou. XIV. 3. SENSE ORGANS. (a) Descriptive. In common with other Oligochaetes the epidermis of Devo vaga is richly supplied with sense cells. These cells may occur isolated or in well-defined groups of from five to seven cells each. The isolated cells are scattered irregularly over the entire body and are more numerous on the anterior segments. On the first five or six segments there are at least three hundred of these cells to a segment. On the tenth segment and on the following segments the number is near fifty. The groups of cells, which I shall speak of as sense organs, have a very definite arrangement. They are found on every segment of the body and, excepting the first five segments, are arranged in two bands encircling the segment. The lesser band is found near the anterior part of the segment and the greater band passes through the setae in the posterior part of the segment. In the first segment the sense organs are very numerous and are irregularly distributed. The largest organs are on the anterior and dorso-lateral borders of the segment. At the dividing line between the first and second segments there is a band of organs, the exact number in which is not easily determined on account of the great number of isolated sense cells which occur on these segments. However, enough can be seen to determine that the organs are less numerous here than in the double bands in the next following segments, and, furthermore, that such organs as can be made out corre- spond in position to those in the lesser bands of the trunk segments. Segments II-V at first sight seem to possess but one band of organs, which occupies the position of the greater band in the other segments. Closer study shows that these organs are not exactly in the same line around the seg- ment; and, taking into account the fact that the number of organs in one of these bands corresponds to the number in the two bands of the other segments, I think we are justified in assuming that this band is a double band formed by the union of the greater and lesser band. No. 2.] THE MORPHOLOGY OF DERO VAGA. 159 There are twelve well-defined organs in the greater band, six large and six small (Pl. XIII, Fig. 9; Pl. XIV, Figs. 11-13). In the lesser band there are eight organs, two large and six small. These organs have a very constant, regular arrangement in each band so that the organs in corresponding bands form longitudinal rows which extend the whole length of the body, — in all twenty rows. The rows containing the larger organs are the two median- dorsal rows (m.s.0.), the two dorso-lateral (d-/.s.0.) occurring just below the dorsal setae, the two lateral rows (/.s.0.) occupying a position just below the so-called “lateral line,” and the two ventro-lateral rows (v-/.s.0.) which are found immediately above the ventral setae. The lateral rows only are made up of organs occurring in the lesser bands. The sense organs in the living worm appear as hemispherical elevations of the epidermis and are set with hairs. The diam- eter of these elevations varies from .o175 to .025 mm., and the elevation above the surface is about .0125 mm. The hairs have a length of .o175 mm. After the animals have been subjected to the killing reagents, the organs are found to be, in most cases, level with the surface of the epidermis. The organs are very simple in their structure, consisting of sensory cells alone. Covering cells have not been observed. The sense cells (s.c.) are elongate with enlarged middle portion and have large nuclei which stain deeply with the ordinary reagents, but remain clear when treated with gold and silver. Each cell bears a long stiff hair. The inner end of the cell in many cases rests directly on the lateral nerve from the ventral cord (Pl. XIV, Fig. 16, /..); otherwise no direct connection has been observed between cells and nerves. The sense organs in the greater band occur on the second lateral nerve (/.7.2), while those in the lesser band are found in connection with the fourth lateral nerve (/.z.4). The first well- defined band of organs on the body is innervated by the fourth nerve from the dorsal ganglion. 160 BRODE. [VoL. XIV. (b). Comparative. Oligochaetes. — In this discussion of the sense organs in the various forms I shall confine my attention to those organs which are distributed over the surface of the body, generally termed “touch organs,’ and which are diffusely or metamerically arranged. Vejdovsky } summarizes the discoveries made prior to 1884, and classifies the various forms of ‘touch organs” as touch papillae (“Tast papillen ’’), touch. hillocks (‘ Tasthiigel”’), and cup-shaped organs (‘‘becherformigen Organen”’). The “touch papillae’ are organs similar to those found in Chaetogaster. The “touch hillocks”’ are non-retractile elevations such as are found on WVazs appendiculata. The “cup-shaped organs’”’ have been described in Lumbriculidae and Lumbricidae. These organs which Vejdovsky has classed in three groups are much alike. ‘Touch papillae” and the “cup-shaped organs’’ seem to be names applied to organs both of which may appear either as papillae or as insinkings of the skin. They are essentially identical in structure. The “touch hil- locks ” differ from the other organs in that they are not retrac- tile. They usually occur in forms which have a covering of débris over the surface of the body. The sense organs project through this covering. Miss Randolph? has described organs in some Tubificidae which she considers intermediate between the “touch papillae ”’ and the “touch hillocks.’’ Bousfield® considered that the genus Ophidonais should be united with that of Slavina on the ground of similarity of sense organs. In reply to this, Stolc4 asserts that the organs of Ophidonais differ much from those of Slavina, the former being “touch papillae,” while the latter are “touch hillocks.” However, it can hardly be said that they 1 Vejdovsky, Z.c., pp. 96-99. 2 Harriet Randolph, Beitrag zur Kenntnis der Tubificiden, Jenatsche Zeit. f. Naturg., Bd. XX VII, N.F. XX, p. 465. 3 E. C. Bousfield, Slavina and Ophidonais, Jour. Linn. Soc., XIX; Jour. Royal Mic. Soc., 1886, p. 445. + A. Stolc, Beitrage zur Kenntnis der Naidomorpha, Zool. Anz., Bd. IX, 1880, P- 503- NOE 2] THE MORPHOLOGY OF DERO VAGA. 161 differ in histological structure. The only difference lies in the fact of the different external appearance of the organs. How- ever, since it is probable that all the Oligochaetes possess “touch organs,” the fact of their presence alone cannot be used as a character in determining relationships in genera and species. (c) Metameric Sense Organs. Oligochaetes. — Segmentally arranged sense organs have been described in Wazs appendiculata and Nats lurida. In the former they are said to be arranged in a band passing through the setae. There are from fifteen to twenty organs in a band. In the latter they are said to be in two bands made up of six to eight organs each. The arrangement of organs in WV. appendiculata, as figured by Vejdovsky,’ is very similar to that in Dero vaga in the band passing through the setae. Miss Randolph? has described metameric organs in two species of Tubificidae where they are arranged in two bands, one through the setae and one near the dissepiment. In one species there is a showing of a third band. She does not mention the number of organs in a band nor give their arrange- ment. Vejdovsky * mentions a single pair of organs in every seg- ment in Lumbriculus, Claparedilla, and Rhynchelmis in the lateral-line region. In Benhamia, Eisen* figures a band of sense organs about the middle of every segment. Miss Langdon ® and Hesse® both describe three bands of sense organs to a segment in Lumbricus. Hesse finds the number of sense organs to be the same on the two lateral halves of the body, there being as many as one hundred in the middle 1 Vejdovsky, Z.c., Taf. III, Fig. 17. 2 Harriet Randolph, /enaische Zeit. f. Naturg., Bd. XXVII, n.F. XX, pp. 463- 476. 3 Vejdovsky, Z.c., p. 98. * Gustav Eisen, Pacific Coast Oligochaeta, II, Cal. Acad. Scz., Vol. II, No. 5: Pl. XLVII, Fig 20. ° Fanny E. Langdon, The Sense Organs of Lumbricus agricola, Journ. of Morph., Vol. XI, pp. 193-234. ° R. Hesse, Zur vergleichenden Anatomie der Oligochaeten, Zeit. f. wiss. Zool., Bd. LVIII. 162 BRODE. [Vou. XIV. or largest girdle. These organs vary in size, but he does not describe any arrangement of organs in rows longitudinally. Polychaetes. —In this group of annelids we have the well- known “ lateral organs”’ of capitellids which have been described at length by Eisig.1 These number one pair to a segment and are situated in the lateral-line region of the body, and in a band passing through the setae. Similar organs have been found by Meyer? in Polyophthalmus, and in addition to these, in this form, there are sense organs resembling eyes on twelve of the body segments. ‘These are in a line with the other organs, but are situated near the anterior border of the segment. Eisig finds in addition to the “lateral organs,’ which are highly developed, some organs of a simpler character which are scattered diffusely over the body. These he calls ‘cup-shaped organs.” He thinks that for the present we can assert no rela- tionship between these two kinds of organs excepting the fact of their origin from the epidermis. Leeches. — The metameric sense organs, sensillae, of leeches have been described by Whitman.® In Clepsine there is a band of fourteen organs around the anterior portion of every seg- ment. These organs are so situated as to form longitudinal rows extending the whole length of the leech. In six rows the organs are larger than in the others. In Clepsine the organs of the median dorsal row gradually change from tactile to visual organs in the anterior segments. Vertebrates. —In fishes and larval batrachians we find scat- tered “cup-shaped organs,” and also more highly developed organs which are arranged in certain definite lines, and in young forms often are arranged metamerically. These organs are considered by some writers to be essentially the same, —the variations being due to difference in development rather than to a structural unlikeness. The suggestion has been made by several workers that there is a homology existing between these 1 See Bibliography, p. 173, Nos. 8 and 9. 2 Eduard Meyer, Zur Anatomie und Histologie von Polyophthalmus pictus Clap., Archiv f. mikros. Anat., Bd. X XI, 1882, pp. 797-799. 8 C. O. Whitman, The Metamerism of Clepsine, Festschr. f. Leuckart, Leipzig, 1892, pp. 385-395: No. 2.] THE MORPHOLOGY OF DERO VAGA. 163 organs and the sense organs of worms, but from the present status of our knowledge of the subject we cannot draw any valuable conclusions. 4. THE SO-CALLED “ LATERAL LINE.” (a) Azstorical. This structure has been a problematic one for twenty years, and I think that at last the true nature of the “lateral line”’ of annelids has been discovered. With this thought in mind I give below an extended historical account setting forth the various views that have been held regarding it, and have tried to interpret the observations of various investigators on this cell cord. To Carl Semper is given the credit of the discovery of the so-called “lateral line” in annelids. In his extended work on the relationships of segmented animals! published in 1876 he described and figured this structure and discussed its probable significance. He characterized it as a row of cells belonging to the side tract of the worm, lying between the two lateral bands of mus- cles and extending from the tail, where it originates from the ectoderm, to the front of the head, where it passes into the oesophageal collar. In the case of individuals undergoing fis- sion, the cord forms in the developing head a sensory plate which gives rise to a part of the brain and commissure, and as it seemed to him might even give rise to muscular fibres. He found the cell cord to exist in Chaetogaster, Nais, Tubi- fex, and Psammoryctes. In a species of the last genus he figures the connection of the cord of cells with the commissure near the brain.? Semper considers that this annelid lateral line may be com- pared to the lateral line of the lower vertebrates, and thinks that there is a resemblance between it and the nerve of the lateral- line system of fishes and larval amphibians, his reasons being 1C. Semper, Die Verwandtschaftsbeziehungen der gegliederten Thiere, III, Strobilation und Segmentation, Arbeit. a. d. Zool.-Zoot Inst. Wiirzburg, Bd. III, 1876. 2 Semper, /.c., Taf. XI, Fig. 3, s7. 164 BRODE. [Vo. XIV. its origin from the epidermis, its position between the dorsal and ventral muscles, its separation from the epidermis by the pushing in of muscles, and its connection with the oesophageal collar. He thinks that no such physiological importance can be ascribed to it, as in the fishes with its sense organs, but its morphological significance is unmistakable and perhaps greater than has yet appeared. Semper makes the very interesting observation that the origin of the eyes of Nais is confined to the lateral line, and raises the question as to the lateral trunk eyes of Polyophthalmus being also limited in their development to the lateral line and its transformations. Biitschli! misinterpreted Semper’s observations and supposed that the lateral lines of which he spoke were merely two of the many interruptions in the longitudinal muscle layer. Eisig 2 discusses the “lateral line” of Naids and comes to the conclusion that the homology instituted by Semper between this formation and the lateral line of vertebrates does not exist. His inference from the account of Semper’s discovery is that in Nais a problematic cell cord occupies a position similar to that occupied by the lateral-line system in vertebrates. Eisig’s reasons for concluding as he does are based on the fact that by the lateral line of vertebrates we mean the whole lateral-line system, and that Semper’s cell cord cannot be com- pared to any one part of the system. Semper’s intimation that it is comparable to the lateral nerve is not in harmony with what is known of the development of this organ in vertebrates. However, considering the fact that Semper found the eyes of Nais to occur in this line and thought it probable that the trunk eyes of Polyophthalmus would be found similarly situated, we must admit that he had some ground for making the com- parison. The “Anlage”’ of the lateral-line system is a cord of cells which grows down the side of the body and differentiates later into the different organs of the system. Now, if the lateral 1 Q. Biitschli, Untersuchungen iiber freilebende Nematoden und die Gattung Chaetonotus, Zezt. f. wiss. Zool., Ba. XX VI, 1876, p. 401. 2 Hugo Eisig, Die Seitenorgane und becherformigen Organe der Capitelliden, Mitth. a. d. Zool. Sta. Neapel, Bd. I, 1879, pp. 322-325. No. 2.] THE MORPHOLOGY OF DERO VAGA. 165 line described by Semper is a cell cord in which sense organs are found, the similarity to the vertebrate “ Anlage”’ is striking, to say the least. In a preliminary paper Vejdovsky 1 notes the occurrence of a “lateral line” or lateral cord in Anachaeta, and thinks it probable that it functions as a sympathetic nerve, and, being connected with the commissure, suggests that it might be called a “vagus nerve.” Timm? has noted the occurrence of this cell cord in Phreoryctes, and Bilow finds it in Lumbri- culus, and thinks it is undoubtedly nervous in its character. Rei- ghard* found the cells in Dero, but could not trace the cord as far forward as the commissure. Stolc 5 called it a lateral nerve and thought that it probably arose from the two large cerebral nerves. Vejdovsky® in his valuable monograph considers that this structure is of general occurrence in the Oligochaeta, and _ pro- poses that the term ganglion cell cord (‘ ganglienzellstrange ’’) be applied to it in view of the fact that its homology with the lateral line of vertebrates has not been thoroughly established. He says that the connection of the cord with the commissure is made out with great difficulty. In many worms the cord in the anterior segments is differentiated into fibres which con- nect with the brain,’ while in the higher forms the undifferen- tiated cells merge into the brain or rather into the commissure near the point where it leaves the brain. The cells which make up this cord are mostly unipolar, and only in Enchytraeidae did he find bipolar and multipolar cells. Lateral branches, some- times composed of ganglionic cells and sometimes of fibres, are given off from the main cord. 1 F. Vejdovsky, Vorlaufige Mittleilungen iiber die fortgesetzten Oligochaeten- studien, Zool. Anz., 1879, p. 184. 2 R. Timm, Beobachtungen an Phreoryctes menkeanus Hoff. und ais, Arbeit. a. d. Zool.-Zoot. Inst. Wiirzburg, Bd. VI, 1883, pp. 131, 132. 3 C. Biilow, Die Keimschichten des wachsenden Schwanzendes von ZLumbriculus variegatus, etc., Zeit. f. wiss. Zool., Bd. XXXIX, 1883, p. 75. 4 J. Reighard, Anatomy and Histology of Awlophorus vagus, Proc. Am. Acad. Arts and Sct., Vol. XX, 1884, p. 104. 5 A. Stolc, Dero digitata, etc., p. 91. 6 F. Vejdovsk¥, System und Morphologie der Oligochaeten, 1884, pp. 93, 94. " Le, Vat Als Fiesty: 166 BRODE. (Vou. XIV. He mentions! having found, in Lumbriculus, Rhynchelmis, and Claparedilla, “cup-shaped organs” in the epidermis situ- ated on the ganglion cell cord and in the region of the break in the longitudinal muscle layer. They agree in position with the lateral organs described by Eisig in the capitellids. These organs are in the epidermis, have cilia, are arranged one pair in each segment, and are retractile. I donot understand from his work that they are in the ganglion cell cord, but that they are in the epidermis above it. In his monograph Eisig “affirms his former statement regard- ing the problematic character of Semper’s “lateral line,” and cites Vejdovsky’s statement that it is a sympathetic nerve. He thinks that the “‘ cup-shaped organs”’ described by Vejdovsky if more fully investigated may be found to be homologous to those described by him in Capitellidae. An entirely different view is held by Hesse,? whose investi- gations have led him to decide that the two lateral lines are composed of the nucleated plasma parts of the circular muscles which are collected into these two lines. His early observa- tions were made on Nais and Fredericia. In a later work* he extends his observations to Chaetogaster, Stylaria, Tubifex, Limnodrilus, and Lumbriculus, and thinks that this peculiarity is common to all Oligochaetes. In the excellent monograph of the Oligochaetes recently issued by Beddard,°* this structure is referred to as the lateral nerve or ganglionic chain, and Vejdovsky’s interpretation, that it may be compared to the sympathetic system of higher forms, seems to be preferred. However, Hesse’s view is stated, without comment, in a footnote. Summing up briefly these observations, we conclude as follows regarding this structure: 1 F. Vejdovsky, /.c., p. 98. 2 H. Eisig, Monographie der Capitelliden des Golfes von Neapel, etc., Fauna wu. Flora d. Golfes von Neapel, 1887, p. 510. 3 R. Hesse, Beitrage zur Kenntnis des Baues der Enchytraeiden, Zeit. f. wiss. Zool., Bd. LVII, 1893, p. 6. 4 R. Hesse, Zur vergleichenden Anatomie der Oligochaeten, Zezt. f. wiss. Zool., Bd. LVIII, 1894, pp. 396, 397, 401, 402. 5 F. E. Beddard, Monograph of the Oligochaeta, Oxford, 1895, pp. 20, 21. No. 2.] THE MORPHOLOGY OF DERO VAGA. 167 (1) In most, if not in all, Oligochaetes there exists on either side of the body in the lateral line a row of peculiar cells extending the entire length of the worm. (2) Sense organs may occur in or near this line. (3) In some forms this cell cord apparently merges into the brain. (4) In case of multiplication by fission a proliferation of cells either in or near this line takes place. As to the probable significance of this cell cord the following views have been advanced: (1) It is a homologue of the lateral-line system of the lower vertebrates (Semper). (2) It is a part of a sympathetic nervous system (Vejdovsky). (3) It is a problematic cell cord of doubtful significance (Eisig). (4) It is an aggregation of the nucleated plasma parts of the circular muscle cells (Hesse). (b) Descriptive. ’ The “lateral line” in Devo vaga was noted at the beginning of my work on that form, and I have made a careful study of it in order to determine its true nature. Starting with the idea that we have here a cord of cells which may be compared to the “ Anlage”’ of the lateral-line system of lower vertebrates, I have been led to accept the view advanced by Hesse, and am now confident that it cannot be interpreted as a nervous struc- ture. In this form the “lateral line”’ occupies the break between the dorsal and ventral half of the lateral muscle areas and extends from the head segment to the growing zone at the tail, where it increases in width and is lost in the embryonic tissue present in that region. The parts of the cells which are clearly visible are elongate pear-shaped and extend horizon- tally into the coelom, forming a strand composed of two or three rows of nuclei. Pl. XIV, Fig. 18, shows the cells as they appear in a thick (15 w) cross-section of the worm. The nerve given off from the ventral cord is the fourth lateral. The muscles extending from the vicinity of the strand to the 168 BRODE. [VoL. XIV. alimentary canal form a part of the dissepiment, while those immediately below the strand extend back of the dissepiment, and many of them run to the body wall close to the large sense organ of the lesser band. There is a possibility that these muscles (Fig. 18, /./.as.) may correspond to the muscles of the lateral organs found by Eisig in the Capitellidae and by Meyer in Polyophthalmus. In the dissected specimen shown in Pl. XIV, Fig. 19, the connection between the nuclei and the muscle fibres may be seen, and also the position of the sense organs relative to the strand.. A frontal section is shown in Fig. 20, and a more highly magnified portion of the same in Big. 21. (c) Luterpretation of Previous Observations. Since the cells forming the “lateral line’ are without doubt muscle cells, it seems necessary to attempt an interpretation of the observations made by several workers who held to the nervous character of the structure. In the first place, all have said that it was in connection with the brain or commissure, but no one has figured a definite connection such as we should expect. Vejdovsky finds that - the cells may merge into the central nervous system or may be united to it by fine anastomosing fibres. I have been able to trace the cells up to, or forward of, the brain and have found fine muscle fibres extending from the brain to the lateral wall of the body near the “lateral line.’ Even should a nerve branch be found going to these cells, it would not prove that the cell cord was a nervous structure, but would rather indi- cate, as suggested by Hesse, the connection of the nervous system with the circular muscles. However, I do think the muscles are not all innervated from the ganglion in the head segment. Vejdovsky’s term “ ganglion cell”’ will hardly apply to these cells from what has been shown to be their true nature. Another point opposed to Vejdovsky’s view is that the cells have their long diameter at right angles to the cord, and not in the line with the cord (Pl. XIV, Fig. 19). No. 2.] THE MORPHOLOGY OF DERO VAGA. 169 As regards the same author’s view that this cell cord repre- sents the sympathetic system, I can find no nerves extending from the line to the alimentary canal, to the setae, and to the nephridial openings, but I do find muscle fibres in these posi- tions (Pl. XIV, Fig. 18), and some of them I at first mistook for nerves, but later was convinced that they were muscles. In my early investigation I thought I had found sense organs in this line, but later I found the organs in the epidermis just above and below the line. The line of cells also increases in width near the setae and at the dissepiment, but this is due to a massing of muscles in these regions, and not to the presence of sense organs. Vejdovsky also states, in describing the large sense organs of WV. appendiculata, that the “lateral ganglion cell cords”’ give off branches encircling the segments, and that the organs are in these bands and are innervated from them, and not from the central nervous system.! In Dero there is a band of cells somewhat resembling the cells of the lateral line which encircles the posterior part of each segment (Pl. XIV, Fig. 10), but I have clearly made out that the sense organs do not occur in this band, but occur before and back of it. Gold chloride does not stain this band, and it in all probability is composed of gland cells. I do not wish to deny that there are any nervous elements in connection with this line, but I do believe that all the elements that have heretofore been considered nervous are not of that character, and this being the case it certainly bears no relation to the lateral-line system of vertebrates. IV. THEORETICAL CONSIDERATIONS. I. ORIGIN OF METAMERISM. The facts above presented have a very important bearing in support of the colonial theory of the origin of metamerism. Stated briefly, this theory proposes to account for the origin of segmentation in animals by supposing that they arose from un- segmented forms through the process of multiplication by fission. 1 Vejdovsky, /.c., p. 97. 170 BRODE. [VoL. XIV. In illustration I may cite the case of Microstoma. This is a small Turbellarian which multiplies by means of fission. This unsegmented worm possesses an alimentary canal extending the length of the body, a so-called “brain” with two lateral nerve trunks and well-marked sense organs situated at the anterior end of the animal. -The process of fission is such that the animal may show, according to von Graff, as many as sixteen individuals of vary- ing ages in one chain. These subsequently separate, forming so many complete individuals. Before separation occurs we have a chain of individuals with a common alimentary canal and a nervous system extending the whole length of the chain. Each individual shows one or two pairs of sense organs at its anterior end. The individual mouth openings have not as yet pushed through. Should this temporary condition become permanent, we should have a segmented form resembling in some essential features an annelid worm. In the different forms of animals in which fission occurs we find several modes of fission. In the form mentioned each individual proliferates continuously. In some forms the pro- liferation is confined to one individual, while in others each individual in turn takes part in the proliferation. This last mode could be applied to the segmentation of an annelid. Supposing that annelids arose in this manner, we should expect to find the segments of the body practically homodyna- mous, with the more perfectly developed and highly specialized segments at the anterior end of the worm. In Dero, as in many other forms, there is in the adult form a marked “cephalization.”’ Apparently the first five segments are formed after the trunk segments are laid down. A study of the embryological development of the worm may clear away this apparent objection to the theory, as Professor Whitman has shown in the case of Clepsine, that, while differentiation does not become apparent in the head as early as it does in the trunk, the segments forming the head are really laid down before those of the trunk region. In support of the theory I have shown that in Dero there is a ganglionic swelling and four lateral nerves for each segment No. 2.] THE MORPHOLOGY OF DERKO VAGA. £7! of the body, and, furthermore, that there is a gradual shading off from the first ganglion to those in the middle region of the body. The sense organs I have been able to trace in lines extending the whole length of the worm. Posterior to the fifth segment the glandular bands and dotted grooves occur on every segment. I have not been able to trace them to the first seg- ment. The first five segments contain certain organs which are necessary for the proper maintenance of the life of the worm, and it is not improbable that this differentiation may account for a partial obliteration of the metameric features in this region. 2. SEGMENTAL SENSE ORGANS IN ANNELIDS AND ORGANS OF SPECIAL SENSE IN HIGHER ANIMALS. ’ From the discussion of the so-called ‘lateral line”’ in anne- lids it now appears that there can be no homology between annelids and vertebrates based on this structure. It will be well, however, to call to mind other lateral-line homologies which have been suggested by several writers who take the segmental sense organs as a basis for comparison. In 1879 Eisig threw doubt on the homology instituted by Semper, and considered that the segmentally arranged sense organs of the Capitellidae were homologous with the sense organs of the lateral-line system found in fishes and larval batrachians. In 1887 he affirmed his former view and cited in support of his homology the work of Meyer on Polyophthalmus and Beard+ on sense organs of the lateral line in vertebrates. Professor Whitman in 1884? and again in 1889? suggested that the segmental sense organs of the leech are identical with the lateral-line organs of the vertebrates. He further suggests “that the segmental sense organs of annelids have formed the 1 J. Beard, On the Segmental Sense Organs of the Lateral Line, etc., Zool. Anz., Bd. VII, 1884, pp. 123-140. 2C. O. Whitman, Segmental Sense Organs of the Leech, 4m. Wat., Vol. XVIII, 1884, pp. 1104-1109. 3 Jézd., Some New Facts about the Hirudinea, Journ. of Morph., Vol. I, 1889, PP- 592-595: 172 BRODE. [Vou. XIV. starting-point for the development of the organs of special sense in the higher animals, not excepting even the eyes of ver- tebrates.”! He also advocates the view that “the ancestral segmental sense organs were not limited to a single pair of lateral lines, but to several paired lines symmetrically placed on the dorso-lateral and ventro-lateral surface.” ? Objections to the homology held by the investigators above mentioned have been brought forward by a number of men, including Balfour, who in regard to Capitellidae says: “I am not inclined to think that there is a true homology between these organs and the lateral line of Vertebrata.” ® It must be admitted that in the adult form there is a notable difference between the systems of organs in annelids and ver- tebrates. If we consider the annelid organs as the starting- point, we find some points of similarity, such as the metameric arrangement and the general plan of structure of all the organs, which may prove of much importance in the further study of the lateral-line system. THE UNIVERSITY OF CHICAGO, June, 1896. 1C. O. Whitman, Journ. of Morph., pp. 592, 593- 2 [bid., p. 595- 3 F. M. Balfour, Complete Works, Vol. III, p. 535, note. Neils Ba THE MORPHOLOGY OF DERO VAGA. 173 IT. 12. 18. 19. LIST OF PAPERS REFERRED TO. BaLrour, F. M. Complete Works. Edited by Foster and Sedgwick. London, 1885. BEARD, J. On the Segmental Sense Organs of the Lateral Line, etc. Zool. Anz. Bd. vii. pp. 123-140. 1884. BEDDARD, F. E. A Monograph of the Order of Oligochaeta. Oxford, 1895. BOuSFIELD, E. C. On Slavina and Ophidonais. /. Zznn. Soc. Vol. xix. pp. 264-268. /.R. M.S. p.445. 1886. BuLow, C. Die Keimschichten des wachsenden Schwanzendes von Lumbriculus variegatus, etc. Zeit. f. wiss. Zool. Bd. xxxix. 1883. BUTSCHLI, O. Untersuchungen iiber freilebende Nematoden und die Gattung Chaetonotus. Zezt. f. wiss. Zool. Bd. xxvi. 1876. EISEN, G. Pacific Coast Oligochaeta. II. Mem. Calif. Acad. Sci. Voloi No: 5. 1896; E1sic, H. Die Seitenorgane und becherférmigen Organe der Capitel- liden. Mitth. a. d. Zool. Sta. Neapel. Ba. i. 1879. Etsic, H. Monographie der Capitelliden des Golfes von Neapel, etc. Fauna u. Flora d. Golfes von Neapel. Ba. iii. 1887. HEssE, R. Beitrage zur Kenntnis des Baues der Enchytraeiden. Zeit. f. wiss. Zool. Bd. lvii. 1893. HEssE, R. Zur vergleichenden Anatomie der Oligochaeten. Ze7t. ve wiss. Zool. Bd. lviii. 1894. LanGpon, F. E. The Sense Organs of Lumbricus agricola Hoftm. Journ. of Morph. Vol. xi. pp. 193-234. 1895. LeEIpy, J. Notice of some Aquatic Worms of the F amily Naids. Amer. Nat. Vol. xiv. pp. 421-425. June, 1880. Lrypic, F. Ueber den Bau des thierischen Kérpers. Tubingen, 1864. MEYER, Epuarp. Zur Anatomie und Histologie von Polyophthalmus pictus Clap. Archiv f. mikros. Anat. Bd. xxi. pp. 769-823. 1882. PERRIER, E. Histoire Naturelle du Dero obtusa. Arch. de Zool. Expér. et Gén. Tomei. 1872. RANDOLPH, HARRIET. The Regeneration of the Tail in Lumbriculus. Journ. of Morph. Vol. vii, No. 3. 1892. RANDOLPH, HARRIET. Beitrag zur Kenntnis der Tubificiden. /ena- wsche Zettschr. Bd. xxvii, N.F. xx. 1893. REIGHARD, J. On the Anatomy and Histology of A ulophorus vagus. Proc. Am. Acad. Arts and Sct. Vol. xx. 1884. 174 20. 21. 22. 23. 24. AN 26. 27. 28; 29. 30. a1, BRODE. [Vou. XIV. SCHMARDA, C. Neue wirbellose Thiere beobachtet und gesammelt auf einer Reise um die Erde. 1853-1857. Theil I, Heft I. Leipzig, 1861. SEMPER, C. Die Verwandtschaftsbeziehungen der gegliederten Thiere. III. Strobilation u. Segmentation. Arédezt. a. d. Zool-Zoot. Inst. Wirzburg. Bd. iii. 1876. Stoic, A. Dero digitata O. F. Miiller: Anatomické a Histologicka Studie. SB. Bohm. Ges. pp. 65-95, 2 pl. 1885. Stoic, A. Llyodrilus coccineus Vejd.: Ein Beitrag zur Kenntnis der Tubificiden. Zool. Anz. Bd. viii. pp. 638-643, 656-662. 1885. Stoic, A. Beitrige zur Kenntnis der Naidomorphen. Zool. Anz. Bd. ix. pp. 502-505. 1886. Timm, R. LBeobachtungen an Phreoryctes menkeanus Hoffm. und Nats. Arbeit. a. ad. Zool-Zoot. Inst. Wiirzburg. Bd. vi. 1883. VAILLANT, L. Histoire naturelle des Annelés Marins et d’Eau douce. Tome iii. Paris, 1889. VEJDOvSKY, F. Vorlaufige Mittheilungen tber die fortgesetzten Oligochaetenstudien. Zool. Anz. 1879. VEjJDovsky, F. System und Morphologie der Oligochaeten. Prag, 1884. WHITMAN, C. O. Segmental Sense Organs of Leeches. Am. Wat. Vol. xviii. pp. 1104-1109. 1884. WHITMAN, C. O. Some New Facts about the Hirudinea. Journ. of Morph. Vol. ii. 1889. WHITMAN, C. O. The Metamerism of Clepsine. Festschr. f. Leuck- art. pp. 385-395. Leipzig, 1892. No. 2.] bu. c.mus. com. ag. a.gr. ais. a-l.s.0. as. ep. fo. Lule gb. sf. ZW. 2d. Zd.mus. mus. Wee Lae Vi ln? ln3A THE MORPHOLOGY OF DERO VAGA. 175 REFERENCE LETTERS. blood vessel. circular muscle layer. commissure. dorsal ganglion. dotted groove. dissepiment. dorso-lateral row of sense organs. dorsal setae. epidermis. fibrous part of ventral cord. ganglion cell. glandular band. giant fibres. intermediary nerve. lateral line. lateral line muscle. longitudinal muscle layer. lateral nerve. first lateral nerve from ven- tral ganglion. second lateral nerve from ventral ganglion. third lateral nerve from ven- tral ganglion. fourth lateral nerve from ventral ganglion. 1.5.0. mM. 72.0. ppc.mus. 5.7. lateral row of sense organs. mouth. median row of sense organs. main sympathetic nerve. muscle. first nerve from dorsal gan- glion. second nerve from dorsal ganglion. third nerve from dorsal gan- glion. fourth nerve from dorsal gan- glion. nephridial opening. plasma part of circular mus- cles. pharynx. sense cell. sympathetic ganglion. sympathetic nerve. sense organ. setae sac. ventral cord. ventral ganglion. ventro-lateral row of sense organs. ventral setae. 176 FIG. FIG. FIG. FIG. Fic. ke? 5: BRODE. EXPLANATION OF PLATE XIII. Dero vaga, worm in case. Branchial apparatus at posterior end of body. Lateral view, showing central nervous system and lateral nerves. Lateral view of sympathetic system — outer coat of pharynx removed. Cross section of worm through pharynx. Gold chloride preparation, showing sympathetic and lateral nerves. Fic. 6. Dorsal view of nervous system in anterior segments. FIG. 7. Dorsal view of nervous system at growing zone. Fic. 8. Dorsal view of frontal section of nerve cord in one segment. Gold chloride preparation. Fic. 9. Lateral view of anterior portion of a worm, showing sense organs. Lith. Anst.u Werner Winter Frankf. y - Fic. Fic. FIG. organs. FIc. organs. FIG. PIG. Io. II. 12. 13: 14. cals BRODE. EXPLANATION OF PLATE XIV. Lateral view of one segment much enlarged. Diagram showing connection of nervous system and sense organs. Diagram of cross section of worm through the lesser band of sense Diagram of ‘cross section of worm through greater band of sense Sense cells. Gold chloride preparation. (X 2000 reduced %.) Sense organ seen in cross section of worm prepared with acetic corrosive sublimate. (X 2000 reduced %.) Fic. 16. Sense organ as seen in frontal section of worm prepared with Her- mann’s fluid. (X 2000 reduced ¥4.) Fic. Fic. FIG. Fic. Fic. 17. 18. 19. 20. 21. Cross section of ventral cord. (X 2000 reduced %.) Cross section of a worm near dissepiment. One segment dissected to show “ lateral line.” Frontal section through “lateral line.” (X 400 reduced %.) Portion of same more highly magnified. (X 2000 reduced 4.) * #. ny Brode Del. 7 > Lah. Anst.ic Werner &Winter, Frankfurt. EXPLANATION OF PLATE XV. Diagram showing anterior segments of a worm cut on ventral side and spread out to show arrangement of sense organs. \ 1) Ou ! sp t w-lso Lith Anst x Werner sWinter, Frankfurt oat HS. Brode Del. THE ORIGIN AND BEHAVIOR OF THE CENTRO- SOMES IN THE ANNELID EGG. A. D. MEAD. CONTENTS. PAGE Te GENER AUB occ cec opm accceneces gente sonsecuecnenonnataonecadeenansosavaccknursnatesnnanntnsndpreedscc=nnccasnantieee 181 (a) Zhe Morphological Relation of the Centrosome to the Other Organs of FY CHUL ne oA ere ac cE ce P Ep © eae oe eee ry eee Ape ccE cee rere Eon -> 182 (b) Zhe Function of the Centrosomes in CHIBI EROTLO TE rere ane ee ene 188 IJ. DESCRIPTIVE........--- Soe eS cat ee ee reper beet coe crececr ee atsew sec aeceoctee 192 (a) Collection and Preparation of Material .......--2-..------- SP eee Aen ee 192 (b) Method of Fixing and Staining ...-ce-cceecorene veces cennnennnen cesennne nae 193 (c) Origin of the Maturation-Spindle 2-12 21--veeerenenneennnnnnnee nena 193 (d) Observations upon the Unfertilized Living Eggs... 2 LOT (6) Fertilization ..secccccneenenseeeccreennenceeeeeceeecninnniesnecnmncenanane ceennesaeease tease 198 VG, Spo U Se pe ete ce eee ence eee: rer ere pee eee raaereceeber soceet beer bene beeectcic AO) I. GENERAL. No protoplasmic structure has been the object of greater interest or incited more investigation during the last few years than the centrosome. The fact that it remained undiscovered until a comparatively recent date, and that, with modern technique, it has been found to occur in cells representing many different tissues in a variety of animals and plants, would alone place the centrosome in a conspicuous position in the literature on the cell; but the difficulty and uncertainty often attending the demonstration of this structure, its minute- ness, the variety of phases under which it manifests itself, the diversity and supreme importance of the functions attributed to it, either as the bearer of hereditary qualities or as the organizer and director of cell-activities, have stimulated re- search and discussion to an extraordinary degree. The literature on the centrosome has developed two general problems toward the solution of which the results of these observations are contributed. 182 MEAD. [Vou. XIV. (a) The Morphological Relation of the Centrosome to the Other Organs of the Cell. This problem demands consideration of the doubt as to the very existence of the centrosome as a definite cell-organ, and of the uncertainty as to the zdenzzty of this organ in case it exists. The possibility that bodies described as centrosomes are fre- quently artifacts does not rest solely upon the negative evidence of numerous observers who fail to find centrosomes where they might be expected to occur, but upon the fact that similar bodies can be produced by the coagulative action of certain reagents. The classic centrosomes, which dance the quadrille in the egg of Strongylocentrotus lividus (Fol, '91) are not found by later workers in the eggsof closelyrelated sea-urchins. Inhis first paper on the “ Fertilization of Toxopneustes,” Wilson main- tains that in well-preserved material ‘“ there is absolutely noth- ing to be identified as a centrosome,” though irregular clumps — fortuitous groups of granules—closely similar to those described by Fol as “centrosomes ’”’ may be produced by the destructive action of picro-osmic acid. Eismond (Azat. Auz., X, 7, 8) and others have suggested that the centrosomes or “centrioles”’ have been, at least in some cases, produced by the clotting action of reagents, and do not represent actual cell- organs. But in view of the fact that so many investigators have demonstrated the centrosome in many different cells and with different reagents, and that in some instances this struc- ture can be followed through a constant and continuous series of changes, including growth and division, it is safe to maintain that the centrosome is an actual organ of the cell. While the existence of the centrosome has been established beyond a reasonable doubt, there still remains the puzzling question of zdentity. Structures widely different in appearance have been called centrosomes, and the same structure has been designated by this and by various other names. Much confu- sion, therefore, arises from the terminology, though the dis- crepances can by no means be assigned to this cause alone, for there are differences of interpretation in regard to the morpho- logical limitations of the structure itself. No. 2.] CENWTROSOMES IN THE ANNELID EGG. 18 J Watasé’s endeavor to bring into relation the various proto- plasmic structures, cyto-microsomes, “centrosomes,” Zzw7schen- korper, rod-like “centrosomes” of the pigment-cell, contraction- band of the muscle-cell etc., is an attempt to extend, not the terminology, but the homology of the centrosome. Which of the three concentric structures — centrosphere, centrosome, or centriole — at the centre of the aster in Echinus (Boveri) is the morphological equivalent of the centrosomes of Strongylocen- trotus (Fol), the large centrosphere of Toxopneustes (Wilson), the scattered centrosomes in the leucocyte (Heidenhain), the centrosphere-like centrosomes of Crepidula (Conklin), the minute centrosomes of Myzostoma (Wheeler), Chzetopterus (Mead), and Thalassema (Griffin), must, terminology aside, remain a matter of interpretation based on structural and physiological characters. It is usually assumed that that structure in the aster which persists through the various stages of mitosis is to be consid- ered the centrosome, whether it be a large reticular area (Crepidula), a minute dot frequently surrounded by such an area, or a structure midway between the two (Echinus). On this principle Wilson, in his paper on the “ Archoplasm, Centro- some, and Chromatin of the Sea-urchin Egg,” raises his pre- viously described “ central mass”’ of the aster of Toxopneustes to the morphological value of a centrosome, though he has since, on other grounds, altered this interpretation. ‘‘ What, then, shall we identify in the sperm-aster of Toxopneustes as the ‘centrosome’ in Boveri's sense, z.¢., as the structure that divides to form the dynamic centres of the ensuing cleavage? I think the only structure that can answer to this definition is the central mass of the aster, z.e., the substance of the original middle-piece, without regard to its subsequent morphological differentiation.” It was on precisely these grounds that, in a preliminary paper on the “ Fecundation of Chzetopterus,”’ I identified the minute dark granules in the centres of the asters as centrosomes, and endeavored to show that the centrosomes which were developed in connection with the sperm-aster persist and, by successive divisions, furnish the centrosomes of each cleavage-spindle up 54 MEAD. [Vou. XIV. to the 8-cell stage. Having requested some of my preparations for examination, Wilson says in referring to them (AZ/as of Fertilization, p. 20): “The central mass of the aster undoubtedly contains at this period (early phase of the cleavage-amphiaster) one or two deeply staining centrioles, which in this case may possibly have the morphological value of centrosomes.” How- ever, reéxamination of all the stages with new material supports my earlier interpretation that these structures have unquestion- ably the morphological value of centrosomes. More recently Wilson also has discovered two deeply stain- ing “ centrioles’ in the aster of the egg of the annelid Nereis, and Griffin, working under his direction, has found the same in Thalassema. Griffin has also showed that these granules, arising in the sperm-aster, persist through the first cleavage- amphiaster, divide and give rise to the centrosomes of the succeeding amphiasters. In view of these results, which indi- cate that ‘“‘the true centrosome certainly corresponds to the central granule or centriole,’ Wilson is inclined to modify again his interpretation of the identity of the centrosome, and to believe that further research will bring out the “ minute central centrosome ’”’ in Toxopneustes, and in all similar cases where they appear to be absent. I am indebted to Dr. F. R. Lillie for permission to refer to some extremely interesting unpublished observations on the egg-centrosomes of Unio which, doubtless, will throw a great deal of light upon the question of zdentzty. From the stage in which it has the appearance of a minute, deeply staining dot, the centrosome is traced, step by step, through a metamor- phosis in many of whose phases its identity would not ordi- narily be recognized. The problem of the morphological relation of the centrosome to the other organs of the cell involves also the important ques- tions of its origin and its persistence. This question, both sides of which have the support of eminent authority, has been stated by Watasé (94) in the following terms: ‘‘ According to the one view (1) the centrosome is a permanent or ultimate organ of the cell, an organ sw generis, and coexistent with other organs No.2.) CENTROSOMES IN THE ANNELID EGG. 185 of the cell, as the nucleus and the cytoplasm. According to the other view (2) the centrosome is a derivative structure, arising by the modification of some preéxisting element in the cell, as the chromosome, nucleolus, or the cytoplasm.’ The theory that the centrosome is a permanent and ultimate organ of the cell finds direct support in the observations of a number of investigators, which prove beyond doubt that the centrosome is capable of self-division and growth, and that it may persist from one generation to another. In Chzetopterus the centrosomes can be seen distinctly in every phase of mitosis, and are always surrounded by an aster, even in the resting stage. On the other hand, in certain animals, the centrosomes are not always demonstrable in the asters. Sometimes both the asters and the centrosomes vanish for a while and then reappear (Wheeler, Lillie, MacFarland, etc.). It is conceivable, however, that, though invisible, the centrosomes may be present in the cell, for they have been found to lie naked in the cytoplasm, bereft of rays (Heiden- hain, '93). To maintain that the centrosomes are absent, simply because they are not demonstrable, is, of course, to base an assumption upon negative evidence, a procedure especially dangerous when applied to the centrosome, inasmuch as this structure is, at best, very minute, and comparatively difficult to demonstrate, even when its exact position is indicated by the presence of an aster. But, admitting that the centrosome is “a persistent morpho- logical element having the power of growth, division, and persistence in the daughter cells,’ and even admitting that it exists incognito, it remains to be proved that it arises only by division from a preéxisting centrosome and that it does not cease to exist as a morphological element in cells which subsequently possess centrosomes. Watasé has pointed out that the transition between centro- somes and other structures of the cytoplasm should be sought, not among the most highly developed typical centrosomes, such as are to be found in mitosis, but among structures — centro- somes or homologues of centrosomes — which are less persistent 186 MEAD. [Vou. XIV. and pass more easily from one phase into another; for example, the Zwischenkorper and the contraction-band in muscle-cells. But the origin and behavior of the asters and centrosomes in the maturation of Cheetopterus directly support the interpre- tation that the centrosomes arise de novo out of the cytoplasm and are resolved into it, and that they are not, therefore, per- manent organs of the cell, like the chromosomes, although some of them show a considerable degree of persistence. Leading up to the formation of the first maturation-amphi- aster, there are formed within the egg a large number of distinct asters (seventy-five more or less), two of which — the primary —come to lie at the poles of the maturation-spindle.! The view held by the majority of recent workers that the aster is ‘formed under the influence of the centrosome,” or the admis- sion that the centrosome is in any way a constant feature or necessary adjunct of the aster (and without this hypothesis there is no force in the permanent-organ theory) offers a dilemma: either (a) this cell (the odcyte of the first order) contains a very large number of centrosomes which have arisen by the division of a preceding centrosome, or (0) it contains a large number of centrosomes which have arisen de nxovo out of the cytoplasm. If one accepts the first alternative, he must imagine a satisfactory explanation of the origin of the many centrosomes from a preceding element of the same kind, and of their distri- bution to the various portions of the cytoplasm, and must also account for the total disappearance of by far the larger portion of them. It is hard to reconcile such wholesale disappearance with the notion of the centrosome as a permanent and ultimate organ of the cell. Multiple asters, similar to those in Chzetopterus, have been described by Carnoy in Ascaris during the formation of the second polar globule, and by Reinke (94) in the peritoneal cells of the larval salamander. Reinke groups the multiple asters into three classes,— primary, secondary, and tertiary mechanical centres, — according to the degree of their develop- ment. The primary mechanical centres, which contain a tru¢ centrosome, arise by the coalescence and further development 1 Possibly several coalesce to form each aster (see p. 195). NiGir2.5] CENTROSOMES IN THE ANNELID EGG. 187 of the secondary and these in turn arise in a similar manner from the tertiary centres. Watasé has seen in the egg of Macrobdella “a series of thirteen asters, ranging from a minia- ture aster, with a microsome for its centre, to the normal aster, with a veritable centrosome.” Morgan ('96) found numerous “artificial astrospheres’”’ in sea-urchin eggs kept in sea-water to which 1% % salt had been added. The salt, he believes, stimulates the living eggs to pro- duce these structures. It is interesting that the multiple asters appear in the eggs of Chaetopterus immediately after they have been deposited in sea-water. The latter probably contains more salt than the fluids of the body-cavity of the worm. R. Hertwig (95) has shown that minute quantities of strich- nine stimulate the production of asters (even amphiasters) in the maturated unfertilized egg of the sea‘urchin. No centro- somes, however, were found. Osterhout (97) has recently described in Equisetum the origin of the amphiaster from multiple asters, though no cen- trosomes were demonstrable. In the egg of Unio, during the metaphase of the second maturation-spindle, a supernumerary aster appears, remains for a short period, and then vanishes. The interesting question presents itself, — If this supernumerary aster contains a centrosome, from what preceding centrosome does it arise? If it does not contain a centrosome, the latter is not a necessary feature, much less the originator, of the aster. Referring to the observations of Reinke, Watasé, Morgan, and Hertwig, Wilson says: ‘ All these observations are of high interest in their bearing on the historical origin of the centro- some; but they do not prove that the centrosome of the normal aster ever arises by free formation. On the whole, the evidence has steadily increased that the centrosome is to be classed among the permanent cell-organs; but whether it ranks with the nucleus in this regard must be left an open question ”’ (oGell7 py 226) 1 In the appendix to the second edition of this book on the “ Cell,” Wilson is inclined to adopt a conclusion nearly related to that which he maintained in an earlier paper ('95), by reason of recent observations which “throw grave doubts upon the hypothesis of the universal autonomy and genetic continuity of the centrosome.” 188 MEAD. [Vou. XIV. The multiple asters in the egg of Cheetopterus are certainly normal; they are demonstrable in the “ving eggs, are brought out by various reagents, are a constant feature of every egg at a certain stage of its development, and always undergo a con- stant and continuous series of consecutive changes. Therefore, as far as the phenomena in Cheetopterus prove the free forma- tion of the centrosome at all, they prove it in the normal aster; and the same can be said of Reinke’s observation. (b) Zhe Function of the Centrosomes in Fertilization. The classic papers of Boveri ('87,'91) and Fol (91) formulated and brought into prominence twodistinct theories of fertilization: that of Boveri rests upon thesupposition that thecentrosome is the dynamic centre of the cell and initiates cell-activities. It implies also that the centrosome is a permanent and ultimate cell-organ, handed down from one generation to another by means of the spermatozoon. The gist of the theory appears in a paragraph from his earlier paper: ‘‘ The ripe eggs possess all the organs and qualities necessary for division, excepting the centrosome, by which division is initiated. The spermatozooén, on the other hand, is provided with a centrosome, but lacks the substance in which this organ of division may exert its activity. Through the union of the two cells in fertilization, all the essential organs necessary for division are brought together; the egg now contains a centrosome which, by its own division, leads the way in the embryonic development.”’ He writes also: “The end of fertilization is the union of the germ nuclei and the equal distribution of their substance, while the active agent in this process is the centrosome.... It is the centrosome alone that causes the division of the egg, and is, therefore, the fertilizing element proper.”’ To these conclusions Wilson subscribes.! Fol maintained, on the other hand, that, in fertilization, the centrosomes of the cleavage-amphiaster arise by the fusion of sperm-centrosomes and egg-centrosomes, just as the cleavage- nucleus is formed by the union of the sperm-nucleus and the egg-nucleus. The centrosome at either pole of the amphiaster 1 Wilson, “ The Cell,” p. 140. * No: 2.) CENVTROSOMES IN THE ANNELID EGG. 189 is composed of both male and female elements; for the original sperm-centrosome and egg-centrosome divide into two, and each daughter-centrosome derived from the sperm fuses with one of the daughter-centrosomes derived from the egg. The obvious inference which has been drawn from these phenomena is that “fertilization consists, not only in the adding together of the two pronuclei derived from individuals of different sexes, but also in the fusion of four half-centres derived from the father and the mother into two new bodies, the astrocentres ” (centro- somes).! Obviously, if the permanent presence and fusion of the sperm- and egg-nuclei indicate that these structures are the vehicles of hereditary properties, the same function may be predicated of the centrosomes. The results of Guignard’s researches on the fertilization of the lily and Conklin’s work on the gasteropod Crepidula agree in all essential respects with those of Fol on the sea- urchin. According to almost every other observer, however, either the sperm-centrosome or the egg-centrosome, or both, disappear before the formation of the cleavage-amphiaster— a phenomenon manifestly incompatible with Fol’s interpretation of their functions in fertilization. The fusion of the sperm- and egg-centrosomes in fertilization is not of universal occurrence, and the inference that these structures are vehicles for the con- veyance of hereditary qualities is unwarranted. But the observations which discredit Fol’s interpretation do not confirm, in every case, Boveri’s theory of fertilization. Wheeler showed that in Myzostoma there is no indication that a centrosome is brought in by the spermatozoon or that a centrosome or aster subsequently develops in connection with the sperm-nucleus. On the other hand, the egg-centrosomes derived from the second maturation-spindle accompany the egg- pronucleus as it approaches that of the sperm. During the approach of the pronuclei the egg-centrosomes move away from each other as though they were to form the poles of the cleavage-amphiaster; but during a certain brief period “it is extremely difficult or even impossible to make out the egg- centrosomes.” It is probable that these bodies — certainly not 1 Fol (91), p. 274; Conklin ('94), p. 18. 190 MEAD. [VoL. XIV. the sperm-centrosomes — enter into the formation of the first cleavage-spindle. Miss Foot’s (97) account of the origin of the cleavage-cen- trosomes in Al/olobophora fetida does not accord with Boveri's theory of fertilization nor with that of Fol. ‘ The egg attrac- tion sphere is present during the two maturation divisions, but after the second polar body is formed and the female pronucleus begins to develop it totally disappears. The sperm attraction sphere is present until the head of the spermatozo6n begins to develop into the male pronucleus, when it also totally disappears. Both spheres are absent during a relatively long period (2.c., while the growing pronuclei are developing) ; and when the two pronuclei have attained their maximum size and are in contact, two attraction spheres again appear in the cytoplasm and the cleavage-spindle is formed.” Lillie (97) has observed a peculiar behavior of the centrosomes and asters in Unio. After undergoing extraordinary meta- morphoses, the egg-centrosomes alone enter into the formation of the cleavage-amphiaster, as they do in Myzostoma; a con- spicuous comet-like aster with a centrosome develops in con- nection with the sperm-nucleus, but totally disappears before the pronuclei come together. MacFarland’s (97) results on Pleurophyllidia are similar to those of Miss Foot on Allolobophora in that both egg- and sperm- centres are apparently absent during a certain period preceding the union of the pronuclei. All these observations form a serious obstacle in the way of accepting Boveri’s theory of fertilization, but there is a further and perhaps more serious difficulty in the insecurity of the hypotheses underlying the theory itself; wzz., (a) that the cen- trosome is a permanent organ of the cell, and (4) that it initiates and directs the cell-activities. We have already referred to the question of the validity of the first of these hypotheses (p. 185), and comparison of the normal fertilization phenomena of vari- ous forms leaves room for doubt as to the validity of the second. The eggs of various animals attain to different stages of matu- ration before fertilization takes place; some remain in the germinal-vesicle stage (with no visible centrosome or aster) No. 2.] CENTROSOMES IN THE ANNELID EGG. IQI and await the entrance of the spermatozoon (Nereis), some remain in the metaphase of the first maturation-amphiaster (Chzetopterus), while others proceed with the formation of the polar globules and the reconstitution of the egg-nucleus before fertilization (sea-urchin). If it is the function of the centro- somes upon being brought into the egg “to organize the machinery of mitotic division,” 1 its task must be very different in different eggs; for in one it must first organize the machinery for the two maturation-divisions, in another it finds this machinery -already organized but in a state of rest, and in a third it has to organize only the machinery for the first cleavage- mitosis. In Cheetopterus the behavior of the sperm-centrosomes is strictly orthodox, according to Boveri’s doctrine ; they are the centrosomes of the first cleavage-spindle, and give rise by division to those of the succeeding spindles, while the egg-cen- trosomes totally disappear. Nevertheless, it does not seem necessary to conclude that it is by virtue of the presence of the sperm-centrosomes, rather than of the sperm-nucleus, that the maturation-processes are resumed, even if we grant that the spermatozoén brings in the centrosomes. An adequate demonstration that the sperm-centrosomes are actually carried into the egg by the entering sperm is, moreover, extremely difficult to make, and it is perfectly possible, as Miss Foot has urged, that in many cases these structures arise de novo out of the egg-cytoplasm in the vzczuity of the middle-piece of the spermatozoon. The foregoing considerations lead to the conclusion that the centrosomes in fertilization are neither vehicles of hereditary qualities nor the active agents which organize the machinery of mitotic division, but that they may be, like other centro- somes, ‘the expression rather than the cause of cell-activities.”’ 1 Wilson, “ The Cell,” p. 171. 192 MEAD. [VoL. XIV. II. DESCRIPTIVE. (a) Collection and Preparation of Material. The observations recorded in the following pages were made upon the eggs of Cheptopterus pergamentaceus Cuvier, pro- cured at the Marine Biological Laboratory, Woods Holl, Mass., during the summers of 1894, 1896, and 1897. These extraordinary annelids are found below low-water mark in leathery tubes. The latter are U-shaped, ten to fifteen inches long, about an inch in diameter in the widest part, and are buried beneath the mud except half an inch at either end. After being removed from the tubes the animals may be kept alive for a few days in an aquarium; they are quite helpless in their new environment, and usually lie on their side, keeping up continuously the rhythmical respiratory movement of their wing-like body-folds, which, under natural conditions, would serve to create currents of water through the tubes. When disturbed at night, they emit a phosphorescent light, apparently dependent upon a secretion from epidermal glands, for the water in which they have been kept becomes itself slightly phosphorescent. The sexes are readily distinguished, the body-wall being nearly transparent and the posterior segments distended with eggs or spermatozoa. The large parapodia hold the sexual products, and any number may be cut off without injury to the worm. The eggs may be fertilized at any time during the day or night and will develop normally, provided the sperm is added within a few hours after they have been removed from the ovaries. This is true of every individual collected during the months of July and August, and indicates that the eggs are probably carried in the body for many days after they are perfectly mature and ready to be fertilized. If the eggs are kept in sea-water for half an hour or more and not fertilized, all except the smaller ovarian eggs are found to have the first maturation-spindle well formed, in its definitive posi- tion, and always in the same stage of development, 2.¢., the metaphase or equatorial-plate stage. But, if the eggs are ex- No. 2.] CENTROSOMES IN THE ANNELID EGG. 193 amined after having remained only a few minutes in sea-water, they are all found to contain the germinal vesicle and no spindle. It is evident, therefore, that sea-water in some way stimulates the eggs to the production of the maturation- spindles. (b) Method of Fixing and Staining. The best preparations were obtained by fixing the eggs in Boveri's picro-acetic acid, and staining with Heidenhain’s iron- alum hematoxylin, followed by orange G. The slides were left in 4% iron-alum for half an hour, rinsed, and left in 4% hematoxylin for twelve hours. After drawing the color with iron-alum, the slides were dipped in an aqueous solution of orange G or Bordeaux red. Hermann’s fluid, Flemming’s fluid, and a mixture of Hermann and formalin also gave satisfactory results, though the staining was not so brilliant. Sublimate-acetic usually wrought havoc in the region of the centrosphere, though the astral rays were not destroyed. The sections varied in thickness from three to seven and one-half microns. (c) Origin of the Maturation-Spindle. The figures in Plate I represent sections of the unfertilized eggs during the growing-period and up to the formation of the first maturation-spindle. As may be seen in Fig. 1, the smaller eggs lie nearest the lumen of the ovarian tubule. They are characterized by their relatively large nuclei and their com- pact cytoplasm which stains, throughout the egg, a nearly uni- form'deep purple (PI. XVI, Bigs. 1,\2; 6; and:2). The older eggs, usually more remote from the lumen of the tubule, are larger, the increase in size being due in great measure to the accumulation of yolk, the distribution of which is accompanied by noteworthy changes in the appearance of the cytoplasm. The latter takes on the appearance of a reticu- lum composed of beaded strands which stain purple, while within the meshes lie the pale yellow yolk-granules. Up to the time when the egg has attained about two-thirds its full size, only a part of the protoplasm presents the loose reticular appearance; the rest remains as dark purple masses, which I 194 MEAD. [VoL. XIV. consider to be equivalent to the mebenkerne or paranuclei of various authors (Fig. 1, c,d, f). These masses are not homo- geneous, but resolve themselves into a cytoplasmic network, of which the meshes are much compressed, and the strands usually parallel with the surface of the nucleus, though at the periphery of the masses they fray out and become continuous with the open network which contains the yolk (Fig. 1, g, Fig. 3, par.n., Fig. 4, par.n.). Frequently sections show but one mass of this sort, crescentic in outline, with the concavity toward the nucleus, and occasionally some of the constituent fibres, instead of running parallel with the nucleus, are rolled up spirally. As arule, however, the paranucleus is fragmented and numerous portions are found in a zone between the nucleus and the periphery of the egg. The substance within the meshes of the paranuclear reticulum does not take the yellow, but the blue or the purple stain. As the egg accumulates more yolk and increases in size, the paranuclei, through a process of continuous ravelling, become resolved into the network which now presents a nearly uniform appearance throughout the cytoplasm. The last traces of the fragmented paranucleus are recognizable even when composed of only two or three strands (Fig. 4). The reticulum can be traced with ease to the extreme periphery, where it forms what, in section, appears to be a distinct beaded line, running entirely around the egg. Im- mediately inside this outer “pellicle’’ is a narrow zone con- taining a single layer of yolk-granules regularly arranged (Fig. 9), and in this zone the strands of the cytoplasm are comparatively few. The nuclear membrane is continuous with the cytoreticulum and presents a similar granular appearance. During the growth of the ovum the character of the reticulum is constantly changing in respect both to the shape of the meshes and to the thickness of the component strands. In later phases of the paranucleus, and immediately after its disappear- ance, the reticulum is particularly easy to demonstrate. The strands are, at this time, thick, richly stained, and seem to be composed of a series of granules arranged in linear order, while the meshes are small and, in section, nearly circular in outline. No. 2.] CENTROSOMES IN THE ANNELID EGG. 195 But although the structure of the reticulum, the peripheral egg membrane (pellicle), and the nuclear membrane are easily demonstrable and beautifully clear throughout the cytoplasm, there is, as yet, 2o trace of anything suggesting a centrosome or an aster. As the egg grows larger, however, the outlines of the meshes become polygonal rather than circular (Fig. 6), and show rather pronounced nodes. Eggs which have reached this stage of development, when placed in sea-water continue to develop as far as the formation of the first maturation-spindle. The tendency of the fibrils of the network to straighten becomes accentuated, so that many of them extend in straight lines for a distance several times the diameter of the single meshes (Fig. 6). Moreover, these longer fibrils radiate from common centres, and in this way there arise in the cytoplasm a number of miniature asters (Figs. 6,7). At first the asters possess only two or three rays, but the latter soon increase in number and in length at the direct expense of the remaining network. The formation of asters continues until a climax is reached, when one can count no less than seventy-five distinct asters scattered about through the cytoplasm of a single egg. They are most numerous in the zone formerly occupied by the paranucleus. These structures correspond closely to the “ secondary mechani- cal centres” of Reinke, and, for reasons which appear further on, I have called them secondary asters) All stages in the development of the asters out of the polyg- onal network may be represented in a single section, yet often many of the larger asters are approximately equal in size, and, though distinct from one another, are frequently so close together that their rays intercross (Fig. 7). The nuclear membrane now presents a peculiar appearance, being drawn out into numerous sharp points—a phenomenon which is probably correlated with the development of the multiple asters (Fig. 7). The period of development characterized by the multiple asters is not of long duration. Two of the asters gain pre- dominance over the others in point of size, and continue to grow larger, while the others gradually evanesce (Fig. 8). 1 Mead ('97a). 196 MEAD. [VoL. XIV. These two primary asters (‘primary mechanical centres,” Reinke, '94) arise at some distance from the wall of the germi- nal vesicle, and usually about ninety degrees from each other, though they may be nearer together or even farther apart. I am not prepared to say at present whether the primary asters are formed by the further growth and specialization of two of the secondary asters or by the union and coalescence of several. The nuclear membrane regains its regular contour when the multiple asters have vanished, except for a deep sinus in the vicinity of each of the two primary asters (Figs. 8,9). A well- defined centrosome, staining dark brown, now appears in the centre of each aster, surrounded by an area of lighter color (centrosphere) from which the large granular astral rays diverge in all directions. These are the centrosomes and asters of the jirst maturation-spindle (Figs. 9-13). The centrosphere always stains brown, though very much lighter than the centrosome. The rays from the two asters give the appearance of actually pushing in the nuclear membrane, though the latter remains for a while intact. The rays of the two primary asters are many of them coarse and quite extensive (Fig. 9). They do not, however, reach the periphery, but break up into the network which extends through- out the whole cytoplasmic portion of the egg. Eventually the nuclear membrane disappears, though the region corresponding to the germinal vesicle still takes a stain different from the rest of the protoplasm and has also a different texture (Pigs: 10;"11). Between the two asters a spindle is formed which remains for some time near the vanishing germinal vesicle, but at right angles to the radius of the egg. The rays from the asters enter the region of the vesicle, the chromosomes gather at the equator of the spindle, and the latter gradually swings around to its definitive position, perpendicular to the surface of the egg (Figs. 10-13). The nucleolus, meantime, breaks up into a number of pieces which remain for a time in the vicinity of the spindle, but gradually degenerate and disappear. The centro- somes divide very early before the spindle begins to move No. 2.] CENTROSOMES IN THE ANNELID EGG. 197 toward the surface, and appear as two distinct dots in the midst of a clear yellow centrosphere at either end of the spindle. The fibres of the central spindle differ in color from the other fibres of the asters, staining yellowish brown with orange G (or red with Bordeaux) much like the centrospheres, while the other rays are purplish. The chromosomes, moreover, do not, at first, lie directly between the two poles, but upon the surface of the central spindle (Fig. 10); later they seem to insinuate themselves between its fibres (Fig. 11). The remains of the germinal vesicle are evident for some time after the spindle has assumed its definitive position, but gradually fade away (Figs. 12-15). Having reached the meta- phase the spindle remains without apparent change until the egg is fertilized. The astral rays extend for a long distance into the cytoplasm, intercross with one another, and break up at the ends into the cytoreticulum, which is now much more delicate and finer than during the earlier stages when the paranucleus was disappearing. It is difficult to understand why the process of karyokinesis should be suspended at this time, for the apparatus of division is apparently ready, the asters are well developed, the chromo- somes in position at the equator of the spindle, and the centro- somes have divided in anticipation of the next mitosis (Figs. 13-16). (d) Observations upon the Unfertilized Living Eggs. During the past summer I have examined a large number of living eggs to confirm the results obtained from the study of preserved material. Full-grown eggs taken from the body- cavity of the female and examined under slight pressure in- variably show a germinal vesicle with even contour, and inside the vesicle the nucleolus. The cytoplasm is, at first, uniformly opaque, but in two or three minutes a number of light points appear, and soon the whole cytoplasm is studded with a multi- tude of secondary asters. Since the yolk is repelled from the centre of the asters, the latter appear as transparent spots in an opaque field. After about four minutes two of the asters (primary asters) become especially distinct and the others 198 MEAD. [VoL. XIV. gradually evanesce. The two arise separately, sometimes near together, sometimes far apart. When one of these living primary asters is examined under a high power, the yolk-granules can be seen to move away from the centre with a trembling, vibratory motion. Later the areas free from yolk extend to the region between the two asters, which represents the central spindle, and within about ten minutes after the egg is placed in sea-water the whole amphi- aster migrates to its definitive position at the periphery of the ege. The changes in the contour of the germinal vesicle and its final disappearance can also be followed in the living egg. All these phenomena afford a complete confirmation of the results already obtained from the study of preserved material. (e) Fertilization. The spermatozoon of Chzetopterus has a bullet-shaped head and a long vibratile tail. When first teased out into sea-water, it remains motionless for a few minutes, but soon becomes extremely active. Apparently it may penetrate the egg at any point on the surface, though it usually enters nearer the vege- tative pole. Polyspermy is rare. The entrance of the sper- matozoon initiates profound changes in all parts of the egg. The latent activity of the maturation-amphiaster is revived ; the polar globules and the female pronucleus are formed while the sperm is but a very minute and inconspicuous body in a distant portion of the egg. Other changes are begun in the vicinity of the spermatozo6én itself. After it has penetrated a little distance, a diminutive aster with two centrosomes lying close together and surrounded by a minute centrosphere may be seen near it (Fig. 19). These two centrosomes are the sperm-centrosomes, though in Che- topterus I am not sure that they are actually carried in by the spermatozoon. However this may be, the sperm-centrosomes separate as the head of the spermatozoon enlarges to form the male pronucleus, and, as they separate, the rays diverging from them become more and more extensive (Figs. 21, 22, 25, 30, 31, 34, 36-38). No. 2.] CENMTROSOMES IN THE ANNELID EGG. 199 Besides moving apart, the centrosomes migrate toward the centre of the egg, the male pronucleus accompanying them, sometimes on one side and sometimes on another, but always near at hand. Their final position is near the centre of the egg, on the side toward the polar globules. The central spindle, which has developed between them, lies at right angles to the egg-axis (Figs. 30, 36-38). After the centrosomes have sepa- rated a certain distance, the centrosphere disappears and the rays diverge from the centrosomes themselves. A lightly staining band—the incipient central spindle of the first cleavage- amphiaster — extends from one centrosome to the other (Figs. 36-39). The rays of the sperm-asters become more and more exten- sive at the expense of the cytoreticulum until, at the time of the union of the pronuclei, they often extend to the extreme periphery and incorporate nearly all the cytoplasm of the egg. They are not straight but curved, those from either centrosome taking different directions, so that in certain portions of the egg the rays cross one another (Figs. 36—40). Since the central spindle and two centres of radiation lie on one side of the male pronucleus and in close proximity to it, a conical space on the opposite side of the pronucleus is left free from the rays (Figs. 38-40). In this space and between the rays in other portions of the egg, especially near the periphery, strands of cytoplasm may be seen running in various directions (Figs. 39, 40). The rays themselves are usually branched at the outer ends. The yolk-granules do not approach very near to the centres of radiation, apparently because the rays are too numerous or because the asters repel them. A few granules, however, are found in the conical space opposite the amphiaster and near the pronucleus (Fig. 38). The sperm-head, or male pronucleus, having grown to its full size, finally takes a slightly eccentric position in that radius of the egg which, if extended, would pass through the polar glob- ules, while near it lie the male centrosomes and the huge amphiaster just described (Figs. 34—40).} 1 In the late stages (Figs. 36-38) there are variations in the relative position of the male pronucleus and amphiaster which show a certain degree of independence 200 MEAD. fVOL. Ah. While these phenomena, directly connected with the develop- ment of the male pronucleus and amphiaster, have been going on, the first maturation-amphiaster, whose activity was resumed upon the entrance of the spermatozoon, has brought about an apparently independent series of changes in another part of the egg resulting in the formation of the polar globules. Beginning with the formation of the polar globules, the living ege undergoes a constant series of form changes, these being most pronounced in eggs taken from worms which have been but a short time (one or two days) in the aquarium. While the first polar globule is being formed, the egg, at first spherical, becomes distinctly flattened at the animal pole. This reminds one of the flattening of the adjacent surfaces of the cleavage- blastomeres which occurs shortly after cell-division. The egg resumes its spherical form, but, after the extrusion of the second polar globule, becomes pear-shaped, the smaller end at the animal pole. It again assumes for a short time the form of a sphere. A protuberance then appears upon the vegetative hemisphere. The successive stages in the devel- opment of this protuberance — yo/k-lobe — are uniformly syn- chronous with the various phases of the first cleavage-mitosis.! The lobe first becomes noticeable during the.early metaphase of the spindle, and reaches the height of its development during the telophase. Meanwhile, the first cleavage-furrow cuts the egg into two unequal cells and the lobe remains attached to the larger one, into which it is later resorbed. These phenomena are of especia] interest in connection with certain experiments which I have made upon unfertilized eggs. Soon after the maturation-spindle resumes its activity the nine chromosomes divide and the daughter-chromosomes migrate toward the two poles of the spindle, while the double centro- somes at the inner end of the spindle move apart and a small central spindle is formed between them (Figs. 14-20). The centrosphere fades away and the rays diverge directly from the two centres, as was the case in the evolution of the sperm- in the behavior of both; but, notwithstanding these variations, the axis of the amphiaster is in every case at right angles to the egg-axis. 1 The same is true of the yolk-lobe in mollusks; cf Crampton. No. 2.] CENTROSOMES IN THE ANNELID EGG. 8OT amphiaster at a corresponding stage. The centrosomes at the outer end, however, do not move further apart, but are carried into the first polar globule with the nine daughter-chromosomes, and there degenerate (Figs. 19-21). During the early phases of mitosis the amphiaster presents several interesting features. The yellowish brown fibres of the central spindle may be seen between the chromosomes after they have divided. Between the halves of each chromosome is a distinct white band with no such fibres, but a few minutely beaded, almost black lines (Fig. 1 7). The spindle does not taper to a point at either end, but is truncated, and at the truncated ends a ring of extremely minute dots, like the centrosomes in color but very much smaller, are brought out in many of the clearest preparations (Figs. 14, 15). These dots are probably nine in number and appear to be the foci of pen- cils of rays extending to the chromosomes. The latter are clearly seen in transverse sections of the spindle at a little distance from the equator. In the very’ late stages of mitosis a delicate Zwischenkorper is formed at the junction of the polar globule and egg, but it soon vanishes and has nothing to do with the formation of the second maturation-spindle. The daughter-chromosomes at the inner end of the spindle, which at first lie in a circle, later take on an elliptical arrange- ment, while the adjacent centrosomes continue to move apart and eventually lie one at either end of the ellipse (Figs. 22— 24). The central spindle, which was formed between the two centrosomes at a very early stage, has now reached consider- able size, and we have the incipient amphiaster of the second maturation-spindle. The centrosomes at the poles of this aster are identical with the two which lie close together at the inner end of the first maturation-spindle (Figs. 14-17), and these are derived by division from one of the centrosomes of the primary asters (Figs. 8-13). The chromosomes soon are drawn into the equator of the spindle, and the latter gradually swings around to a position vertical or nearly so, directly under the first polar globule (Figs. 25-29). Again, a centrosphere develops around 202 MEAD. [Vor. XIV. each centrosome, and the rays of the aster —which have never been absent — diverge from it, rather than directly from the centrosome. The centrosome at the inner end of the spindle often, perhaps always, divides, but the daughter-centrosomes are not so large as the corresponding ones in the first spindle, and always remain close together (Figs. 28, 29). The chromosomes, during the metaphase of the second matu- ration-amphiaster, are frequently dumb-bell shaped (Figs. 26, 27), and sometimes in four parts, as shown in Fig. 28. The succeeding phases are similar to those of the first maturation- ° amphiaster described above. The peculiar “wake” left in the midst of the fibres of the central spindle by the receding halves of the chromosomes is evident during the anaphase, and by this means one can ascertain which of the daughter-chromo- somes were together (Fig. 29). It is found in some instances that one of the chromosomes is drawn much nearer the pole of the spindle than its counterpart.} The second polar globule is formed directly under the first, which is thus pushed away (Figs. 31-33), and it contains the centrosomes and the nine daughter-chromosomes (Fig. 31). At about the 32-cell stage, both polar globules are ingested by the apical cells.2 The Zw¢schenkorper, which is developed during this mitosis, persists for a considerable time, as may be seen by comparing Figs. 31, 32, 35, 37, and 39. As the spindle vanishes the chromosomes which were at the inner end elongate and bend so as to become V-shaped (Figs. 31-33). They then group themselves in a hollow hemisphere whose concave side is directed toward the centre of the egg so as partially to obscure the centrosome and its vaguely defined centrosphere, though at this period the radiations from the aster in question are extensive and very distinct, many of them cross- ing those of the male aster. The chromosomes ultimately surround the centrosome so that the astral rays diverge from their very midst (Figs. 33-35). Gradually the chromosomes become vesiculated and, as the nine vesicles begin to migrate toward the male pronucleus, they 1JIn this figure the lithographer has slightly displaced the outer centrosomes and the chromosome nearest the pole. 2 Cf. Mead, '97b. No. 2.] CENTROSOMES IN THE ANNELID EGG. 203 continue to grow and press against one another, still including the female centrosome, whose position is indicated by the point of convergence of the rays (Figs. 35-38). The latter gradually become fewer and less distinct and finally vanish altogether. I believe that the rays of the female aster, which were so strongly developed in the earlier stages of the reconstitution of the pronucleus, become resolved into a cytoplasmic network, which in part may be incorporated into the system of rays belonging to the male amphiaster. I have seen during this period of disintegration a number of extremely minute asters (secondary or tertiary mechanical centres) between the periph- ery of the egg and the female pronucleus. The vesicles completely coalesce to form a female pronucleus similar in size and general appearance to the male pronucleus with which it later comes into close apposition (Fig. 39). These phenomena suggest the interpretation that the chro- mosomes at the inner pole of the second maturation-spindle are not only drawn toward the egg-centre, but that the latter con- tinues for some time to be the centre of attraction and thus groups the chromosomic vesicles about itself and holds them together. The disappearance of the female aster is simultane- ous with the coalescence of the vesicles. After their fusion there is no further need of this attractive influence and the aster disappears. Apparently, also, the male centres exert an attractive influence upon the group of vesicles as a whole, though it is not ordinarily strong enough to dissociate the group by drawing the nearest vesicles to the centre in advance of the others. In one case, however, this phenomenon seems actually to take place (Fig. 36). But, whether the grouping of the vesicles is a function of the female centrosome or not, it would seem utterly preposterous to presume that this waning structure suddenly emerges from the midst of the fusing vesicles, divides, and unites with the male centrosomes. Before the two pronuclei meet, the vesicles of the female are completely united and resemble the male pronucleus in all respects, though they always lie on the side toward the polar globules. The two pronuclei come together and flatten against 204 MEAD. [Vou. XIV. each other between the poles of the male amphiaster, forming a spherical nucleus — the first cleavage-nucleus (Figs. 39, 40).! The male asters reach the height of their development as the pronuclei come together, and the two centres, connected by a spindle, are already widely separated. Each centre contains a clearly defined and easily demonstrable centrosome, which is soon surrounded by an incipient centrosphere, from which the protoplasmic rays extend throughout the whole egg (Figs. 39, 40). The pronuclei elongate, while the poles of the spindle con- tinue to move further apart, and later the nuclear membrane gradually disappears. An actual fusion of the pronuclei does not take place (Figs. 40, 41), and, even after the membrane has vanished, the chromosomes derived from the egg and from the spermatozoon respectively are often seen to be in separate groups (Fig. 43). As the cleavage-nucleus begins to elongate, the rays of the amphiaster become less extensive, breaking up into a network in the outer portion of the cytoplasm, and when the nuclear membrane disappears, centrospheres become more strongly developed around the centrosomes. The chromosomes arrange themselves in an equatorial plate, and the metaphase of the first cleavage-amphiaster is established (Figs. 43, 44). The centrosomes at the poles of the spindle by this time have each divided in anticipation of the next mitosis, and the centrospheres have increased in size. Several nucleoli lie scat- tered among the chromosomes in the equatorial plate (Fig. 45). Like the chromosomes they stain with haematoxylin, but are easily distinguished by their irregular shape and arrangement (Fig. 46). The chromosomes split longitudinally according to the hetero- typical method (Figs. 44, 45), and the daughter-chromosomes recede toward the opposite poles (Fig. 46). In the cross- section of the spindle I have often counted the chromosomes, and found them always to be eighteen in number (Fig. 47), as would be expected, since there were nine in the maturation- spindle (Fig. 18). When the daughter-chromosomes have sepa- rated to some distance, the nucleoli are seen in their original position midway between the poles of the spindle (Figs. 46, 1 The signs ¢ and ? in Figs. 40, 41, and 43 should be transposed. No. 2.] CENTROSOMES IN THE ANNELID EGG. 205 462), where they remain during the succeeding stages of mitosis (Figs. 48-52). The centrospheres reach their greatest development toward the end of the anaphase, though the radiations at this time are not so extensive (Figs. 462-48). The centrosomes, which divided very early, continually move apart within the centro- sphere without in the least altering the regular contour of the latter. In the early stages a line joining the two centrosomes would nearly coincide with the spindle-axis, but as they move apart they also swing around through an angle of about 9go° (Figs. 44-48). The distance between the centrosomes within the centrosphere is in definite and constant relation to the successive phases of mitosis, and one can predict the position of the chromosomes from the examination of the centrosomes, and vice versa. The centrosphere is smaller in the end of the spindle which belongs to the smaller cell (Figs. 46, 464, 48). The dark brown centrosomes are especially distinct during these stages in contrast to the light yellow centrosphere. In some preparations a minute halo surrounds each centrosome (Fig. 46), and in the later stages of the anaphase, before the centrosome disappears, a band of fibres, the incipient central spindle of the next mitosis, is demonstrable between them (Figs. 46%, 47). ’ When the rod-like chromosomes have reached a position near the centrosphere they gradually become vesiculated. Each chromosome at first appears as a double row of granules, sepa- rated by a longitudinal cleft (Fig. 48), but later the regular arrangement of the granules is lost, and each chromosome con- tinues to swell up and forms an oval vesicle (Fig. 49) resembling a miniature nucleus. I have several times counted the vesicles, and always found them to be eighteen. They arrange themselves in the form of a disc, and for some time the fibres of the cen- trosphere may be seen between them (Fig. 49). As they grow still larger they coalesce, not into a single mass, but into several irregular masses which themselves fuse to form a spherical nucleus (Figs. 50, 51). The cytoplasmic portion of the egg becomes pone ered during the later phases of the reconstitution of the nuclei, and 206 MEAD. (Vou. XIV. in the line between the two new cells there appears a series of black granules, the foci of pencils of connecting fibres extend- ing in both directions to the nuclei (Fig. 51). These bodies later become aggregated at one place and form a large brown Zwischenkorper (staining like a centrosome), from which the rays diverge. In side view the Zwzschenkorper may always be seen to lie below a line connecting the centres of the nuclei; z.¢., nearer the vegetative pole. At this stage the nucleolar frag- ments of the original cleavage-nucleus lie in the larger of the two cells (Figs. 51, 52). Here they remain for a while, gradu- ally becoming smaller and less distinct, and vanish entirely before the karyokinetic figures are formed in the two blasto- meres. The centrosomes, which divided at an early stage and sepa- rated as the karyokinesis progressed, continue to move apart during the reconstitution of the nuclei, and a spindle develops between each pair. While the chromosomes are being trans- formed into vesicles the centrosphere at either end of the spindle disappears (Fig. 49), and the rays which now diverge from the centrosomes increase rapidly in length and thickness, and reach their maximum development in a late stage of the reconstitution of the nucleus, as is represented in Figs. 51 and 52. They extend to the periphery of the egg and are easily distinguishable between the closely packed yolk-granules at the lower pole. They can be traced even through the substance of the yolk-lobe which develops on the lower hemisphere when the first cleavage-spindle is in an early stage (metaphase) and eventually becomes a part of the larger of the two blastomeres (see Fig. 50).!. The rays diminish in extent as the new nucleus assumes its definitive spherical contour; the centrosomes, mean- while, take their respective positions, nearly 180° apart, near the surface of the nucleus (Fig. 53). The reconstitution of the nuclei and the accompanying phe- nomena proceed simultaneously in the two blastomeres, and each is now in a stage of karyokinesis exactly comparable to that of the original odsperm after the union of the pronuclei. 1 In a previous account of this phenomenon I said that the yolk-lobe was sepa- rated from the blastomere. This was a mistake, which I corrected in a later paper. No. 2.] CENTROSOMES IN THE ANNELID EGG. 207 One cycle of karyokinesis has been completed, and the next cycle which is concluded with the formation of the four blasto- meres is essentially similar. Around each centrosome there develops a centrosphere (Fig. 53). The nuclear membrane, beginning at the portion nearest the centrosome, breaks down (Fig. 54), the chromosomes group themselves in the equatorial plate (Figs. 55, 56), and the typical anaphase is established. During the succeeding stages, the chromosomes in each blasto- mere divide longitudinally and migrate toward the poles of the spindle, the nucleoli drop out into the cytoplasm, and the cen- trosomes divide and move apart within the growing centrosphere (Figs. 57, 58). I have followed the karyokinetic processes to the formation of sixteen cells. All the phenomena are essentially similar to those in the preceding cycles of division. The thirty-two cen- trosomes of the 16-cell stage arise by the successive divisions of the original sperm-centrosome, while the centrospheres, on the other hand, appear and vanish in each mitosis. III. SUMMARY. During the growing-period of the oocytes a deeply-staining paranucleus is developed which contains a reticulum continuous with that of the surrounding cytoplasm; but, before the oocyte has attained to its full size, this structure becomes entirely resorbed into the general cytoreticulum. Ripe eggs may be carried in the body-cavity of the worm for several days before they are laid. During this time neither centrosome nor aster can be distinguished, though the reticu- lum is unusually distinct. In a few minutes after the eggs have been deposited in sea-water, however, a large number of asters are developed by rearrangement of the cytoplasmic network. Two of these asters (primary asters) continue to develop and finally lie at the poles of the first maturation-spindle, while the others gradually vanish. Distinct centrosomes are not demon- strable except in the primary asters. If they are present in the multiple asters, it is extremely difficult to reconcile their presence with the theory that the centrosome is a permanent 208 MEAD. [Vou. XIV. cell-organ; for the theory would require, in this instance, that no less than seventy-five centrosomes should arise by the division of the two centrosomes of the cell of the preceding generation, and that these centrosomes should be distributed throughout the larger portion of the egg-cytoplasm. If they are not present in these asters, the centrosomes are only an occasional and not a constant or an essential feature of the aster. The centrosomes of the two primary asters evidently arise de novo out of the cytoplasm, and are typical in every respect; they lie in the midst of the astrospheres, grow, divide, and persist in the next cell-generation. Normally the maturation of the unfertilized egg proceeds no further than the metaphase of the first maturation-spindle, but upon the entrance of the spermatozoon the karyokinetic activity is immediately resumed and the maturation is completed. I do not know whether the spermatozo6n actually brings the sperm- centrosomes into the egg or not; at any rate, they are demon- strable in the midst of a minute aster which lies close to the sperm-head soon after the latter enters. From this time the development of the sperm-aster into the cleavage-amphiaster, and the mitotic divisions, resulting in the formation of the polar globules, proceed simultaneously in different parts of the egg, and appear to be independent phenomena. The primary centrosomes lie at the poles of the first matu- ration-spindle, and the daughter-centrosomes, arising by the division of one of these, move apart and form the poles of the second maturation-spindle. During the reconstitution of the egg-nucleus and its approach to the sperm-nucleus, the egg- centrosome remains in the midst of the fusing vesicles, its posi- tion being indicated by the point of convergence of the rays of its waning aster. Not only the fact of its disappearance, but the fact that, when last seen, the centrosome is in the midst of the group of vesicles, renders it in the highest degree improb- able that the egg-centrosome takes part in the formation of the cleavage-amphiaster. Moreover, the sperm-centrosomes may always be seen at the poles of the incipient cleavage-amphiaster, and they become more and more conspicuous up to the time of ey No. 2.] CENTROSOMES IN THE ANNELID EGG. 209 the fusion of the pronuclei. During the metaphase of the cleavage-amphiaster the sperm-centrosomes divide, and the daughter-centrosomes at either pole move apart (an incipient central spindle developing between them) and form the poles of the cleavage-spindles of the two blastomeres. This process is repeated in each subsequent mitosis, and the centrosome can be demonstrated, lying in the midst of an aster, at every phase of mitosis, even including the so-called “resting stage.” It follows, therefore, that the centrosomes of the cleavage-cells are derived directly from the sperm-centrosomes, — a fact irrecon- cilable with Fol’s theory of the “ quadrille.” The behavior of the sperm-centrosomes is in harmony with Boveri's theory of fertilization, but is not necessarily a confir- mation of it; for the karyokinetic activities which are revived upon the entrance of the sperm are those leading to the forma- tion of the polar globules. The machinery for these mitotic divisions is already organized, and it is quite as likely that the stimulus which starts it going emanates from the sperm-nucleus as that it emanates from the sperm-centrosomes. There are nine chromosomes in each maturation-amphiaster and eighteen in the cleavage-spindle. In the metaphase of each cleavage-amphiaster the numerous nucleoli lie scattered among the chromosomes and remain at the equator of the spindle until they completely degenerate. As mitosis progresses, the chro- mosomes split longitudinally and the halves move toward opposite poles of the spindle, where they form new nuclei. During the telophase of each mitosis a distinct Zzwzschen- korper is present, which gradually fades away as the reconstituted nuclei approach the “ resting stage.”’ It is a pleasure to acknowledge the many courtesies extended to me by Dr. Whitman and other officers of the Marine Biological Laboratory, and to express my appreciation of the kind assistance of my friend Dr. W. M. Wheeler. BROWN UNIVERSITY, September 26, 1897. 210 96. oe ’87. '93. 87. 91. 1a: ae 96. 195: ion. Bk 96. ‘ox 195. io: MEAD. [Vou. XIV. BIBLIOGRAPHY. AUERBACH, LEOPOLD. Untersuchungen tiber die Spermatogenese von Paludina vivipara. Jena. Zezt. Bd. xxx, Nr. 4. BAMBEKE, CH. VAN. A propos de la Délimitation Cellulaire. BzdZ. de la Société belge de Micros. Tome xxiii. BENEDEN, E. VAN et NEyT, A. Nouvelles recherches sur la féconda- tion et la division mitosique chez l’Ascaride mégalocéphale. 2x7. Acad. Roy. Beligue. 3me sér., Tome vii. Bianc, H. Etude sur la fécondation de l’ceuf de la truite. Ber. nat. Ges. zu Freiburg. Tome viii. BOVERI, THEODORE. 2. Ueber die Befruchtung der Eier von Ascaris megalocephala. Sztzungsber. Ges. Morph. und Phys. Miinchen. Bad. iii. BovERI, THEODORE. Befruchtung: JJerkel und Bonnet’s Ergebnisse. Bd. i. BOVERI, THEODORE. 2. Ueber das Verhalten der Centrosomen bei der Befruchtung des Seeigeleies, nebst allgemeinen Bemerkungen iiber Centrosomen und Verwandtes. Verh. d. phys.-med. Ges. zu Wiirzburg. N.F. Bd. xxix, Heft i. CONKLIN, E. G. The Fertilization of the Ovum. A7zol. Lect. Marine Biological Laboratory, Woods Holl. Boston, 1894. CRAMPTON and WILSON. Experimental Studies on Gasteropod Development (H. E. Crampton). Appendix on Cleavage and Mosaic Work (E. B. Wilson). A. 2xtm. Vol. iii, Part I. FARMER, J. B. 1. Ueber Kernteilung in Lilium-Antheren, besonders in Bezug auf die Centrosomenfrage. flora. 1895. Foi, H. Le Quadrille des Centres. Un episode nouveau dans Vhistoire de la fécondation. {3 Fic. VIII. 236 CLAY POLE. [VoL. XIV. between the second and third body segments. Fabre ('55) and others describe the ova as developing exclusively on the ventral surface of the ovarian sac, which reaches back into the hind body segments. Two strings of ovules, “ placentaires,” extend throughout the length of the ovary, united to the membrane of the sac. Each egg is enclosed in a separate follicle, which eventually breaks, allowing the eggs to fall into the unpaired ovary, formed by the fusion of two primitively distinct sacs. Heathcote (ss) describes the ovary of the just-hatched /wlus terrestris aS an unpaired sac enclosing a double line of ovules; earlier there were two distinct sacs; a fusion followed. Accord- ing to Schmidt (94, ’95), who agrees with Grassi (’86), the ovaries in Scolopendrella are paired, with paired anterior outlets opening upon the fourth segment. Each ovum is enclosed in a distinct follicle. Schmidt (95) and Kenyon (95) both agree in their description of Pauropus, the lowest form of myriapod; they represent it as having an ovary of the typical diplopod type. It consists of a large unpaired sac lying on the median line below the gut, crowded with ova, the largest of which are forced forward and sideways, leaving a central and posterior mass of small ova. A small unpaired oviduct opens in the third segment a little to the right of the mid-ventral line. No evidence of double strings of these ova has been observed, but only mature or nearly mature females were studied. The ova are inclosed in follicles within the ovarian sac. Briefly summarizing the conditions found in the myriapods: in the Chilognatha the ovary is an unpaired sac, the germinal epithelium is placed chiefly at the hinder end of the body, and the developing ova pass forward through the successive seg- ments as they ripen. The oviducts begin usually in about the fourth or fifth segment and open in paired outlets on the second or third. It is evident that a paired condition primitively existed, and that the unpaired ovary is the result of fusion of two sacs. The primitively paired condition is still indicated by the devel- opment of ova in the young animal and by the paired openings. In the Symphyla, Scolopendrella, the paired condition persists in the adult, the opening appearing on the fourth body segment. The Pauropoda show unpaired ovaries with a cephalic, asym- No. 2.] ANURIDA MARITIMA. 235 metrical opening in the third body segment, and no traces of paired origin unless the eccentric opening may be such. In the Chilopoda, paired conditions are lost; no traces of them have been found; the opening is posterior and unpaired also. One more point of interest in connection with the structure of the myriapod ovary has been advanced by Lubbock (61). He makes a fundamental distinction between the principles of fol- licle formation in insects and myriapods. In the latter the follicle projects zz¢o the ovary, while in the former it projects Jvom the ovary. The importance of this distinction may not be great, but the comparisons already started between myriapod and hexapod ovaries may explain it. Comparing Figs. I-VIII the fol- lowing line of development can be traced. Beginning the series with the generalized condition shown in Fig. VIII, Glomeris, and disregarding the anterior opening, Anurida is easily derived by a slightly greater localization of the germinal epithelium and non-fusion of the ovarian sacs. Japyx (Fig. V) can be derived directly from Glomeris by localizing the points of origin of ova still more sharply, and, arranging the ova in strings, by retaining a connection between the maturing eggs instead of dropping them at once into the ovarian sac, posterior unpaired openings having succeeded paired anterior ones. Machilis (Fig. IV) is the result of a condensation of the conditions started in Japyx; the tubules are elongated and crowded together. From the method of development, the germinal epithelium is at the ends of the tubules. Ladzdura riparia shows a further concentration of tubules, which in this case number only five. To gain the most highly developed hexapod ovary, it is only necessary to increase the number of tubules, which could be done primarily or secondarily. Heymons (91), in his studies on Blatta, shows the process of origin of the numerous tubules found in the adult; from a mass of undifferentiated cells arise by rearrange- ment the “ Endfaden ”’ of each ovariole, as well as the common one binding the tubes together. The conditions found in Forficula auricularia (Figs. II, II) form an interesting and instructive phase in the line of develop- ment. Here, according to Fabre and Lubbock, the ovary con- sists of very numerous short tubes, perhaps each containing a 238 CLALT ODE, [VoL. XIV. single egg, opening successively into the ovary. This typically illustrates the difference in the position of the myriapod follicle and that of the insect, and shows the possible method of origin of the numerous tubules by progressive localization from the conditions shown in Glomeris (Fig. VIII). Another interest- ing point is the evidence of a cephalic elongation, which, in the case of Forficula, unites with one from the opposite side (Fig. III). Its anterior attachment was not determined. The step from Glomeris to Anurida is short and clear; Anurida still retains the ova developing in intra-ovarian follicles, and a reduction of the germinal epithelium to a mass in the third and fourth abdominal segments is the only change except- ing for the altered position of the outlet. Still further, the arrangement of the ova in strings is possibly an indication of the development of egg tubules similar to those found in the higher Apterygota. Supposing this to be true, it is evident that the morphological value of the egg tube in Machilis and the other thysanurans is not necessarily different from that of the higher Pterygotes. Increase in number simply means arrangement in a greater number of strings, a device for accommodating more germ cells compactly in an individual. Heymons’ study on Blatta strongly seconds this view. This aspect does not support the opinions held in regard to the primary metamerism of the egg tubes in Japyx. If the insect ovary came through any such series, headed by such structure as is shown in Glomeris, it is evident that metamerism, if it existed in yet earlier forms, has been obliterated; and such evidences as are seen in Japyx are either reversions or due to secondary development. Returning once more to the series of text figures (I-VIII), there is an evident lack among the Thysanura of anything cor- responding to the “‘Endfaden.”” With the exception of Anurida, there are none that show a trace of such a structure. This absence may perhaps be accounted for by the following facts. The ovaries of Machilis, Japyx, and Campodea are comparatively simple; in the first two the ovarioles are short and small and, especially in Japyx, spread throughout the body. There is not the mechanical demand for an anterior suspensory ligament No. 2.] ANURIDA MARITIMA. 239 that arises on the increase in number and length of tubes found, for example, in Blatta. In origin the cells of the “ Endfaden”’ arise from the same source as those of the ovary, and it can be considered as simply elongations of certain parts. On this basis it is difficult to see why two forms, preserving as simple an ovarian structure as that found in Anurida and Forficula, both show an anterior elongation. A possible interpretation will lie along another line. The elongation in Forficula is totally distinct from the germinal epithelium, which lies at the free ends of the short tubes. The two parts unite on the middle line (Fig. III). In Anurida the elongations are also distinct, and from their union with the fat body in the thoracic seg- ments evidently serve the purpose of suspensory attachments. Cross-sections of these chords near the ovary show in some cases a distinct lumen in the middle of them. In some cases this persists for some distance, giving the thread the structure of a fine tube. In the embryo this part of the ovary is very striking and bends over distinctly towards the ventral wall of the first abdominal segment (Fig. 58). It appears long before any duct at the hinder end has begun to develop. Its position, structure, and relations in the adult ovary and its early development and peculiar relations in the embryo all strongly suggest a possible connection with the oviducts of the chilognath or symphylid. Change of function would now account for its relations to the second thoracic segment of the adult, but its embryonic relations suggest that it once was con- nected with the first abdominal segment, which bears the collophore. A suggestion might be made in regard to this problematic organ, which has been considered in so many different aspects. Wheeler (90), following Graber ('s9) and Carriére, gives these views at length, and it is only necessary to say that whichever of the three functions it serves, — of gills, sense organs, or glands, —it is undoubtedly a pair of fused appendages. In the Symphyla, which, according to Haase (’g6), are nearly related to the hexapod ancestor, the genital opening lies in the median line between the fourth pair of legs (Haase, 'g9, Fig. 1). A study of the genital openings of Polydesmus, Craspedosoma, and other chilognaths, as given by Fabre ('55), 240 CLAVP OLE. [VoL. XIV. shows that the appendages on the segment bearing the genital opening have been more or less changed. Histologically, the condition of these modified appendages is given as being highly vascular, a description closely agreeing with the structure of the collophore in Anurida. On these grounds it is then possible that the collophore in Anurida is a relic of a former anterior opening for the reproductive organs, and that the cephalic elongation is a trace of the former duct. The exact present function of the collophore has long been a source of much conjecture, and still remains in doubt. . Fernald (90, p. 45) summarizes the views held as to the pos- sible present function of the ventral tube in the Collembola; two writers think it a genital organ, but its equal development in the male and female argues against this. Haase considers it a blood gill, a function he assigns to all the rest of the similar abdominal appendages found in the Symphyla, while it is said also to be a gland for the secretion of an adhesive mixture. Any of these functions may have been acquired since its origi- nal function was lost, but the evidence given above and its position on the fourth body segment — the same as that of symphyloid genital opening — give probability at least to the view here advanced of its primitive function. Kenyon ('95) gives a table of the appendages and their homologies in the various groups of arthropods, in which he considers the fourth segment of the Symphyla as the homologue of the third in hexapods, but upon what basis is not clear. Haase, Wheeler, and Grassi agree in thinking the homology of the fourth with the first abdominal correct. This view is still further supported by the suggestion of Grassi that the symphyloid ancestor of the Insecta had paired genital apertures at the hinder end of the body and also another pair between the third and fourth pairs of legs. That the genital openings among the hexapods are not very fixed in position is evident from the variety of conditions found in the different members of the group. Wheeler ('93) shows that the openings of the male and female differ, and an actual movement takes place in the former from the tenth to the ninth abdominal segment during embryonic life. No. 2. ] ANURIDA MARITIMA. 241 Nutritive Cells. The possession of nutritive cells is a character widespread among the Insecta. In the majority of cases, ova are associated with a definite number of cells that assist to a greater or less degree in yolk formation and in the general growth of the ova. Korschelt ('g6) divides insect ovaries into two classes, according to whether they possess these accessory cells in the form of a nutritive chamber or otherwise. Sometimes they are arranged in separate chambers lying between the eggs, and sometimes in a terminal chamber. Many curious forms are described by different writers. Claus (64), using Coccus and Aspidiotus, found “yolk strings” leading from nutritive cells to the egg, in some cases passing through the chamber of an undeveloped egg, The same structure, a “ Dottergang,” is described by Wielowiejski ('85) in Pyrrhocoris. Sometimes the nutritive cells are arranged in a highly developed follicle and absent in any other form. Korschelt (86) figures Vanessa urticaria as pos- sessing a few such cells, closely applied to one surface of the egg, an arrangement resembling that found in Anurida. Among the Apterygota, Grassi (gs) figures Campodea alone as having anything similar to nutritive cells; here, as shown (Fig. VI), they are interpolated in chambers between the eggs. He expressly states, however, that the nutritive cells are zo¢ homologous with those of the Insecta; why he does not say, unless he wishes to consider the ovary as not the morphological equivalent of the ovariole of the higher Insecta. In the thysanuran (Zomoceras sp.?) nutritive cells were found closely similar to those described for Anurida, but smaller and arranged more distinctly as a fol- licle and present in larger numbers (Fig. 14). In Anurida, as before described, each egg is associated with its string of nutritive cells, varying in number from five to eight. Will (84, ’85) gives the results of his studies, and presents in full his idea of the compound origin of an egg, and the formation by the egg of some of the follicle cells. As he has, however, changed some of his recent statements of the case, these studies will not be mentioned in detail. 242 CLA YVPOLE, [VoL. XIV. Among the myriapods, very little trace can be found of any- thing resembling nutritive cells; in part they are absent, and in part too little histological study has been made of these forms. However, Schmidt ('95) describes in Pauropus a peculiar process that he interprets as the nutrition of eggs by other eggs. Ova have no definite arrangement in the hinder end of the ovary. ~Some cells of the germinal mass grow in size and gain yolk at the expense of the surrounding ova, which cease growing and finally degenerate. In Scolopendrella he describes an even more astonishing process in which he considers the follicle cells to be opposed to the egg cells, and vice versa. Follicle cells have the power of migrating into the egg cells. This migration is followed by one of two alternatives: either the egg absorbs the follicle cells or the follicle cells overpower the egg cell as phagocytes, destroying it, and afterwards wander out to become the follicle cells of a stronger egg cell. A similar case is men- tioned by Weismann in Leptodora, not, however, so phagocytic in character. Yolk is formed after this battle is over, and all changes in the germinal vesicle are subsequent to this process. No mention of any such relation can be found among the diplopods or chilognaths. Stuhlmann ('g6) figures eggs of /zlus sp. 2 and of Glomeris marginata, in neither of which are there any traces of nutritive cells. Passing from the arthropods, Wheeler (’96) describes many forms of “ Nahrzellen,” or accessory cells, in different groups of “worms.” The ovum of Myzostoma is accompanied by two accessory cells, which gradually lose their individuality as the ovum matures. Ophryotrocha, described by Braem ('93) and Korschelt, sheds its ova into the body cavity with one accessory cell. The ovum of Tomopteris is accompanied by seven smaller cells, while in Diopatra it bears two long strings of cells attached laterally, which strings fall off before the ovum is mature. Numerous other cases are given, but these are enough to show the parallelism of development and re- semblance to those found in Anurida. In the latter case the ovum accompanied by a certain number of cells is pushed away from the germinal mass of cells into the ovary, the cavity of which is the homologue in the hexapod of the annelid body No. 2.] ANURIDA MARITIMA. 243 cavity. Here they are pushed forward by those developing behind, and are later inclosed in follicles made from the wall of the ovary. The difference between this follicle and that usually found in the hexapod ovary is in its direct origin from the ovarian wall, not from certain special cells in the germinal epithelium. No further light is thrown by Anurida on the exact part taken by these accessory cells in the development of the ovum. No support is given, however, to Will’s idea of the direct transformation of follicle cells into the yolk (Will, 84). Cer- tain changes occurring in the nutritive cells previous to yolk formation and the disappearance of the material shown just preceding and during the early stages of the growth of yolk supports the view held by Blochmann ('84), Schiitz ('82), and others that the nutritive cells secrete a ferment, the precursor of the yolk; this passes from the cells into the ovum and then the yolk appears. This material is probably formed also in the ovum itself. The persistence of these cells up to the time of maturity of the ovum till all the yolk is formed, their undi- minished size up to this point, and their rapid degeneration afterwards, indicate an active relation between them and the egg. The existence of lines of communication between the acces- sory cells and the ovum, such as was seen by Claus ('64) and Wielowiyski (’85), is evidence of a higher degree of differentia- tion; probably the material passed into the ovum in cases where there is a visible connection is more highly elaborated by the nutritive cells and requires less adaptation by the egg. As far as the origin of these cells is concerned, it is generally agreed that they arise from undifferentiated germ cells; it may be, perhaps, owing to a peripheral position on the germinal epithelium that more nutrition reaches them, or some other less evident cause may prevail. One thing is clear, that an added supply of chromatin is one of the first changes; from an originally small chromosome the mass increases by branch- ing and spreading. Fine threads reach out in every direction as if to offer a larger area for contact with the nuclear plasma. It finally assumes in Anurida a strikingly stellate structure (Rigso10; '11;) 13); 244 CEAVPOLE. [VOL. XIV. Passing now to the early changes in the nutritive cells, a consideration of that peculiar structure known as the yolk nucleus is necessary. The “yolk nucleus” is a term of varied application and, consequently, great indefiniteness. Two distinct classes of structures have been designated by this name: (1) embryonic cells concerned with yolk absorption whose origin is assigned variously to entoderm, mesoderm, superfluous spermatozoa, or the germinal vesicle, and whose fate is said to be absorption or transformation into the midgut epithelium; (2) all those structures appearing in the ovarian egg which can have but one of two possible origins: nucleus or cytoplasm of the cell itself, and whose existence may cease before the yolk appears in the egg or may be continued into embryonic life. It is on the second of these classes that the facts observed in Anurida have a bearing. The peculiar “blue caps” that appear at a certain stage in the nutritive cells can have no other homology than the “yolk nucleus,” the “ Dotterkern”’ or ‘“Nebenkern”’ of some authors. Hubbard ('94) gives a list of groups in which the yolk nucleus has been observed to occur. This list includes all the classes of arthropods, cnidarians, nematodes, Sagitta, lamellibranchs, gasteropods, and all the vertebrate groups. The nucleus is found in eggs associated with nutritive cells and those without. Stuhlmann (’86) described it in Bombus, Vespa, Trogus, Pimpla, and Bauchus, varying in form from a diffuse peripheral mass toa localized spot. He, however, reduces the method of origin in all the Hymenoptera to one type, —that of a small concre- tion appearing close to or in the near vicinity of the germinal vesicle. This mass wanders away, forms a peripheral layer, collects at one pole or is scattered in several diffuse masses. Stuhlmann never satisfied himself of its nuclear origin as Bal- biani (83) had in Geophilus and Will ('s4) in the frog. In all cases it is in very young ova that it appears; it becomes in- visible on the formation of yolk. No mention has been made of the existence of the yolk nucleus in any other of the forms of insects. The appearance in Anurida of this blue cap in the nutritive cells declares the presence of this curious adjunct of No; 2:] ANURIDA MARITIMA. 245 the ovum. It is true that no traces have been seen in the ovum itself, but the position close to the nucleus of the nutri- tive cells at the time just previous to yolk formation and its subsequent disappearance are all indications of its homology with the “yolk nucleus” of the second class. Among the myriapods this body has been extensively ob- served. Balbiani ('83) and Zograff ('90) both report a yolk nucleus present in the small ova of the chilognath Geophilus. Balbiani figures and describes it as originating from the nucleus and in part forming follicle cells and in part the undoubted yolk nucleus, which in this case in its greatest develop- ment has a radiate structure suggesting the aster of cleavage spindles. It disappears while the ova are still very young. Heathcote observes the absence of a complex yolk nucleus in Julus such as was described for Geophilus. Lubbock (61), de- scribing the eggs of Julus, notes the absence of the vitelligenous bodies described for Glomeris, but notes the presence of a small body in eggs of an intermediate size that he compares to the laminated body of spiders’ eggs; evidently it is a yolk nucleus. It is absent in smaller and larger eggs. Lubbock describes vitelligenous bodies as present in the egg follicles of Glomeris, but distinguishes them from yolk nuclei. Stuhlmann (86), however, figures part of the ovary of Glomeris in which yolk nuclei are evidently present, and, from his descriptions and figures, more closely resemble those found in Anurida than any others hitherto described. Kenyon ('95) describes a small body near the nucleus in the young ova of Pauropus which disappears in older stages to be followed by yolk spherules, which is evi- dently the same structure, although not called so by him. Schmidt ('95) describes in Scolopendrella a small body inside of the egg cell staining as deeply as the nucleus, which he calls a migrated follicle cell, but which is quite possibly the yolk nucleus. Stuhlmann ('g6) figures a large yolk nucleus present in the ova of Peripatus edwardsii which, strangely enough, per- sists late in the egg’s history, even after fertilization. It is not necessary to enumerate any more forms of animals in which this structure appears; it can be said to occur among the re- maining invertebrates and the vertebrates, with many variations 246 CLA VYPOLE. [Von. XIV. in its history. Usually it is present only for a short time just before yolk formation. There can be no doubt that the yolk nucleus has an impor- tant part to play in the developing egg, and that its function concerns the formation of yolk, as agreed by Stuhlmann and Schiitz. Balbiani, Sabatier, and Jatta formerly held a view regarding it as a fertilizing element, precursor of the sperm cell, but this has long been abandoned. A question naturally arises as to the origin of this necessary body, for which some name ought to be found excluding the term nucleus with which it has nothing in common, or very little, even if its nuclear origin were demonstrated. Balbiani, working chiefly among arachnids, where the yolk nucleus is largely developed, sug- gested its homology with the centrosome of the spermatozoan and somatic cells. Agreeing with Boveri that the centrosome has no part to play in the female cell, he considers the yolk nucleus as a case of a true hypertrophy of degeneration. It is easily supposable that in the process of degeneration some ferment should be originated useful in yolk formation; hence its preservation under anew form. Its appearance in the ovum just previous to the beginning of growth may indicate the fact that the period after the last division has taken place is when degeneration of the centrosome sets in, accompanied by its useful hypertrophy. Balbiani’s figure of Geophilus shows a strikingly radiate structure in the “yolk nucleus.” The late persistence of this body in the egg of Peripatus cedwardstt is perhaps explicable on these grounds. This species of Peripatus is viviparous, and the appearance of intra-uterine development in this form reduced the amount of yolk needed. This was consequently decreased, but as yet the reaction has not included the formative material; this is still formed, perhaps, in unreduced quantities, and hence remains not transformed into yolk. It has not yet responded to the changed conditions which require a smaller amount of yolk. It is interesting to note that the yolk nucleus and its origin bring up the much-disputed question as to the origin of the centrosome. Balbiani’s arguments demanding the origin of the yolk nucleus from the nucleus naturally carry back the NOw25] ANURIDA MARITIMA. 247 centrosome to the same source. On the other hand, Stuhl- mann and others fail to determine the nuclear origin, their evi- dence pointing to the cytoplasmic nature of the centrosome. As far as Anurida is concerned, there is no clear evidence of a _ nuclear origin. The yolk nucleus always appears in the cyto- plasm close to the nucleus and on one side, but there is no reason to think that it originates more from one than from the other: it has more the appearance of being the result of the joint activities of both. At least, this question cannot be settled without determining the greater one, the origin of the centro- some, if such an homology as has been suggested be accepted. Unsegmented Ovum. The eggs of Anurida when freshly laid are easily recognized by their peculiar light yellow color and smooth, shining surface. They are found fastened together in masses, the number of eggs in a mass varying from a few, five or six, to at most fifteen or twenty; each egg is about 0.27 mm. in diameter. These masses are sometimes arranged irregularly, the eggs being simply fas- tened together in a pile, as shown in Fig. 15, @; or there may be a definite form to the mass, owing to the position of the eggs in two rows in which the eggs alternate with each other (Fig. 15, 0). Very frequently in the central part of an ovum can be recognized a lighter spot; this shows the position of the central mass of protoplasm in which the cleavage nucleus is placed. The egg membrane is closely adherent to the surface of the egg in these early stages, but before cleavage takes place a distinct space has appeared between the membrane and the egg. No further points of differentiation are visible from the outside, but the internal structure is marked. Pl. XXII, Fig. 30, showsa section through an ovum having the external characters described above. The outer egg envelope and the vitelline membrane, which is very thin but distinctly formed by this time, are both omitted in the figure. The egg itself is seen to be formed at this time of a large central mass of protoplasm (Fig. 30, ¢.g.) with many radiating strands (7.), which branch as they pass to the periphery, and in most cases finally connect with a thin protoplasmic layer 248 CLA YPOLE. [Vou. XIV. that surrounds the outer surface of the egg. Between the strands of the meshwork formed by the radiating protoplasm lie the numerous yolk bodies (y.), which vary much in size. In the central mass of protoplasm, either central or slightly to one side, there was usually present a rather large pear-shaped nucleus which proved to be the male pronucleus (m.pr.); after entering the egg it has assumed its permanent central position and is awaiting the return of the female pronucleus from the periphery. In many eggs there was a smaller mass of proto- plasm on or near the outer edge, as is shown in Fig. 30 at fz. In this is found the female pronucleus, the egg nucleus, for the first time recognizable since its disappearance before yolk formation began. Fig. 25 shows an enlarged view of this mass as seen on the periphery. Large yolk bodies (y.) are recog- nized imbedded in a protoplasmic mass that extends to the surface. At fpr. can be seen the extremely small female pro- nucleus returning to the centre of the egg to meet the sperm nucleus that is already there. P.4. shows the small and in- completely separated polar bodies that have been given off by the egg nucleus, thus completing its reduction to the female pronucleus. Fig. 26 shows the two polar bodies at 7.0.1 and p.b.2; as shown, the first polar body is again dividing, giving the typical number of three. Somewhere in the protoplasm is the female pronucleus, but not clearly outlined. The polar bodies never completely separate from the egg and never clearly protrude from the surface of the egg, but remain flattened down and are eventually absorbed into it. No sign has been found of the polar body spindles; the egg nucleus preserves its invisible condition in spite of the most careful searching, the first reappearance being in the form of the female pronucleus and polar bodies, as shown in Fig. 25. With the gradual passage of the female pronucleus towards the centre of the egg there is a withdrawal of the strands of proto- plasm in the same direction, so that the central mass gradually increases in size until at the beginning of cleavage it is plainly visible in a fresh, whole egg as a large, lighter, central spot. In Fig. 27 is shown a part of this mass, and in it at the centre the two pronuclei,— the larger, the male pronucleus, pear-shaped, No. 2. ] ANURIDA MARITIMA. 249 and the smaller, the female pronucleus, a round ellipse. Some of the eggs contain very small central masses; several showed, instead of any central mass, a small one about halfway to the centre, distinctly containing a small nucleus. These facts point to the following interpretation: when the sperm cell enters the egg (just when this occurs was not determined) there is practically no localized protoplasm present. Its en- trance presumably starts the transformation of yolk into proto- plasm. During the passage of the sperm cell to the centre of the egg the protoplasm gradually accumulates, until on its arrival there it is surrounded by a conspicuous amount. Here it remains stationary until the egg nucleus, having been trans- formed in its peripheral position into the female pronucleus, returns to the centre surrounded by a small mass of proto- plasm, the two nuclei eventually uniting to form the cleavage nucleus. Many of the unsegmented eggs apparently undergo a process of degeneration, the process consisting chiefly in the formation of a large number of oil globules, which are protruded between the surface of the egg and the membrane; these form large masses on the surface of the egg. It is probable that such eggs failed in fertilization, and are consequently degenerating. Sections show that the yolk material is undergoing pathologi- cal changes and becoming clear in spots. The external mem- brane adheres more closely to the surface than in the case of the normal egg. There is, moreover, no vitelline membrane present. Ona large number of normal eggs, so large a number as to suggest its constant presence, is a small raised spot, con- spicuous enough to be seen on the whole egg. In section this appears as an irregular protoplasmic mass with apparently no definite structure. It possibly represents the place of entrance of the sperm cell; for a time it remains visible, but is eventu- ally absorbed or otherwise disappears. The vitelline membrane offers no particular points of interest; it is very thin and reacts strongly to a protoplasmic stain; its color is nearly the same as the protoplasm of the outer surface of the egg. 250 CLA VOL. [VoL. XIV. Cleavage and Blastoderm Formation. The cleavage in Anurida, and as far as observed in the rest of the Collembola and Thysanura, forms a striking exception to the method typical of the other Insecta. In Anurida the spherical egg has a cleavage that is slightly unequal but dis- tinctly holoblastic. The first cleavage plane cuts the ovum into two practically equal halves (Figs. 16,17). The second planes appear at slightly different places in the two halves and result in the arrangement of blastomeres so frequently found in annelids (Figs.18,19). Fig. 20 gives a different view of the 4-celled stage from that shown in Fig. 19, and illustrates the slightly unequal cleavage; this is almost a polar view and shows the shifting of the blastomeres. The second planes do not exactly halve the two parts of the egg. From this time on the planes appear regularly, the third being horizontal and the fourth more or less irregular, but in effect vertical, resulting in the 16-celled stage (Fig. 22). From this point on the details become confused by the rapid division and the difficulty of orientation. Holoblastic cleavage continues, however, up to the stage shown in Fig. 24, where a coarse morula stage has been reached. The cleavage planes are still distinct and the different cells stand up dis- tinctly on the surface. After this, however, a change takes place that is clearly visible on the surface; the cell outlines become indistinct and the blastomeres flattened ; until after another division the surface appears almost uniformly thickly scattered with white spots that represent the nuclei and sur- rounding protoplasm. A study of sections shows what has been taking place. Beginning with an egg slightly younger than that one shown in Fig. 16 the first cleavage spindle is distinctly seen (Fig. 28). The spindle does not differ in any way from the usual type. The centrospheres are represented by the darker haloes sur- rounding the ends of the spindle, and scattered through the protoplasm are small yolk granules in the process of transfor- mation into cytoplasm. Fig. 29 shows the reorganization of the nucleus and the amoeboid processes of the migrating masses of protoplasm. The number of chromosomes has not No. 2.] ANURIDA MARITIMA. 251 been definitely determined, owing to their extremely small size, but the indications are that there are eight in each nucleus. Fig. 31 is a section through an 8-celled egg at the line z—-¢ in Fig. 21. It shows the distinct holoblastic cleavage and four of the cells. There is practically no cen- tral cavity, the cells being crowded in together. The nuclei and protoplasmic masses are already preparing for the 16- celled stage; as this division is in vertical planes, the nuclei divide in a horizontal plane and can be seen in this section. In Fig. 32, the 32-celled stage represented in Fig. 23 has been cut horizontally at about the level of the line y-y. This shows distinctly that the typical blastula does not result from the cleavage; some of the cells have been crowded into the interior so that a solid morula results. This condition is still more evident in sections of the typical morula stage shown in Fig. 24. Up to this point the cleavage has been undoubtedly holoblastic and practically equal, for the slight inequality shown in the 4-celled stage becomes more and more obliterated by subsequent divisions. After the morula has been formed a decided change takes place in the internal structure corresponding to the external features already de- scribed. In Fig. 33 there is shown a gradual obliteration of the hitherto distinct blastomeric outlines. The nuclei and protoplasm of the outer blastomeres have migrated to the sur- face, leaving the yolk masses on the inside. In the inner part the nuclei and surrounding protoplasm have entirely left the yolk masses and are evidently moving towards the surface. There has been a cessation of the total cleavage, and now the blastoderm is being formed by the migration of the cells from the blastomeres to the exterior. Consequent on this change the yolk is left behind as an inert mass; and, though at first retaining a separation into masses corresponding to the earlier blastomeres, it gradually assumes a more compact arrange- ment. The cells, if this name can be applied to the migrating masses of protoplasm containing a nucleus, divide as they pass to the exterior, and some remain behind, also undergoing the process of division. Fig. 34 shows the final result of this migration. Here a blastoderm has been formed by migration 252 CLAYPOLE: [VoL. XIV. from the holoblastic egg. The cells have arranged themselves in two definite layers which differ slightly in character. The outer layer is continuous, while the inner is composed of fewer cells at regular and greater distances apart. Some cells remain behind in the yolk. Following this there is a rapid division of the cells forming these two layers, until a stage shown in Fig. 35 is reached. Cell outlines become very indistinct in the blastoderm and the size very much reduced. The protoplasm becomes strongly vesicular. Some of the cells left behind in the yolk cease division at a much earlier stage and remain large, lying in the yolk; others are grouped in masses (Fig. BONE LIE These are the principal steps in the cleavage and formation of the blastoderm as found in Anurida, and they can be seen to widely diverge from the centrolecithal cleavage and conse- quent migration of the cells typically found among insects. Unequal holoblastic cleavage has been described by Lemoine (87) in Smynthurus and retarded holoblastic in Anurophorus. Ryder ('86) while describing some of the later embryological features of Anurida does not consider the early stages, so makes no note of the cleavage. Smynthurus and Anurophorus were not studied in section, so that internal changes were not described. In Anurida, as has been seen, there is a sudden change in the method of development, which results in the final formation of the blastoderm by migration, as in the case of the typical centrolecithal egg. The temporary preservation of the yolk blastomeres suggests the condition found in a centroleci- thal egg after the blastoderm is formed and secondary yolk cleavage has occurred. Before discussing possible interpretations of the facts ob- served in Anurida, a brief sketch of some other somewhat peculiar methods of cleavage will be attempted. There is no other arthropod as yet described in which cleavage takes place in just this way. Many are holoblastic at first and change their method of cleavage during development. Among the crustaceans are found some interesting forms. Korschelt and Heider ('92) classify crustaceans according to cleavage methods, and put in the second group all those forms which start with No. 2.] ANURIDA MARITIMA. 253 holoblastic cleavage and eventually lose it. Brauer’s ('92) figures are given for Branchipus, in which the change takes place in the following way: holoblastic cleavage results in a distinct and regular cleavage cavity. This method continues until a layer of small, narrow, and very long columnar cells is formed, having their nuclei on the periphery and long yolk stems extending in to the centre. Eventually, this inner part forms a fused yolk mass with a single layer of blastoderm cells on the outside not separated from the yolk. Alpheus, Palae- monetes, and Hippa all have a similar cleavage according to Herrick. Ishikawa (’85) states that in a fresh-water form some cells remain behind; these are, he thinks, doubtful in signifi- cance. Among the pantopods there are two methods of cleav- age. Morgan ('91) describes the cleavage in Pallene as at first total; gradually the nuclei become more and more peripheral, and eventually the cleavage planes in the yolk mass are lost. Tanystilum and Phoxichilidium present another variation; holo- blastic cleavage is maintained up to the 16-celled stage with the formation of a central cavity (Figs. IX, X). This is followed by a delamination of the entoderm from the inner ends of the single layer of cells (Fig. X, A, #), filling the blastocoele with entoderm cells. In this way entoderm arises by what might be considered a process of multipolar migration. Among the myriapods certain phases are suggestive. Zo- graff (90) figures Geophilus as having purely centrolecithal cleavage of the nucleus, accompanied by a certain kind of yolk cleavage (Fig. XI, A, B). This gives the outward appearance of holoblastic cleavage that has been claimed as general throughout the group. The central nuclei now migrate out- wards along these yolk cleavage lines, and the blastoderm is formed by migration. Small masses of cells are found at the ends of each cleavage line that eventually spread out and form a complete blastoderm (Fig. XI, D). It is clear from these few cases that change in method of cleavage is a widespread fact among arthropods, but accom- plished in many different ways. Anurida differs from any of the others described. As in Branchipus no blastocoele is formed, the strictly one-layered condition is early lost, and a 254 CLAVPOLE. [ VoL. XIV. condition suggesting Fig. X, B, is reached (compare Fig. 32 and Pl. XXII, Fig. X, B). Later the relations are more like those in Fig. XI, C. This suggests a line of possible explanation for the conditions found in Anurida. These cells found inside the morula, as shown in Fig. 32, ¢c, 6, may originate by a multi- _ polar immigration, which is the primitive method of invagina- Fic. X, A. Fic. X, B. tion. As the second layer of the blastoderm is formed largely by the migration of cells that were earlier inside the morula, it may be considered to arise by an imperfect and incomplete gastrulation. Whether the layer thus formed by migration from the inner part of the morula receives any additions from the outer layer is uncertain. Cells in various stages of divi- sion with the spindle axes directed obliquely to the surface point to such a possibility. The origin of the many cells re- No. 2.] ANURIDA MARITIMA. 255 maining in the yolk is undoubtedly from those cells that were inside the morula. As to the names to be applied to these determinate layers of the blastoderm there is no doubt. With- out hesitation the outer can be called ectoderm and the second mesoderm, while, as will be shown later, the entoderm develops from certain of the cells left in the yolk. It remains dormant, however, until a late stage in embryonic life. Precephalic Organ and Blastodermic Membranes. For a certain time after the blastoderm is fully formed further changes consist chiefly in increase in the number of cells and decrease in their size, the lower layer always main- 256 GLA VPOLE. [VoL. XIV. taining the numerically established ratio of cells (Fig. 35). The next change is that in a certain place the blastoderm cells cease to divide, and assume certain definite characters. This is the beginning of the “‘ precephalic organ,”’ or, as it has been called, the “dorsal organ.’ Figs. 35-39 illustrate the stages by which the organ attains its full size. After the nuclei cease to divide, the cytoplasm begins to increase in amount and becomes highly vesicular in structure, forming a thick layer (Fig. 35). The nuclei increase in size, but do not divide either kinetically or akinetically. These changes -apply only to the ectodermic cells. The mesoderm cells lying below these disappear gradu- ally, partly by migration and partly by disintegration. At first the protoplasm accumulates more rapidly below the ectodermic nuclei, placing these on the periphery, but soon a process of infolding and insinking begins. The nuclei gradually sink lower as the amount of protoplasm increases, until a condition shown in Fig. 37 is reached; this is quickly followed by the stage of sreatest development shown in Fig. 38. Here the precephalic organ (#c.o.) resembles a large gland; on a whole egg it appears as a large, circular, lighter mass that is clearly of some depth. By dissecting it out, the organ is found to have the form of an oblate spheroid, as would be inferred from its form in sections. During the development of the organ by a process of invagina- tion, the surface over which it reaches is much reduced (Figs. 36-38), but the number of cells remains the same. The neces- sary crowding down of the nuclei in this process causes them to remain at different levels, suggesting that the organ is com- posed of several layers. This is, however, simply an appearance, there being only one layer of cells and these ectodermic. The cells always remain distinctly separated from each other, and the protoplasm is very vesicular. The next change is a striking one; the vesicular character of the protoplasm is supplanted by a strongly marked striation that appears first at the outer edge in vertical planes. The final result of this process is seen in Fig. 39, where the nuclei are crowded to the bottom of the organ, which is irregular in shape and apparently beginning to degenerate. The outer ends of the cells have been elongated and drawn out into fine threads, No. 2.] ANURIDA MARITIMA. 257 _ which, after being constricted to a rather narrow neck on the outer surface of the blastoderm, spread out like a fan or, when seen in the uncut egg, like a mushroom. In order that the later stages of this structure may be clear, it is necessary to consider some changes that have taken place in the blastoderm in general. The protoplasm of all the ectoderm cells has been increasing in amount and becoming vesicular, forming a deep protoplasmic layer (Fig. 37, é.c.) over the surface. This pre- paratory stage is followed bya rearrangement of the ectodermic nuclei, so as to form a wavy line (Fig. 38, e.c.); the protoplasm also assumes this form. No cell outlines are distinguishable; the whole blastoderm appears a continuous sheet of protoplasm, containing nuclei at certain definite intervals. After a short time, a very thin membrane separates from the surface. This is formed of thin strands connecting thickened masses (Fig. 40, Pl. XXII, ~.). The latter are found to have come out of the troughs of the folds in the ectoderm, the thin strands from the crests. A thin layer is formed on the precephalic organ. Soon after, a layer of protoplasm is separated from the blasto- derm having a definitely crenated form (Figs. 38, 40, ¢.1, Pl. XXII). The last part of the process is again repeated, and a second crenated membrane is formed (Fig. 39, ¢.2). In the region of the precephalic organ, this last membrane has pecu- liar relations. The elongated ends of the cells spoken of earlier are found to’ be directly connected with the second crenated membrane by thickened ends, which appear as a knob in cross-sections (Fig. 39, &.). These envelopes are developed in a way closely similar to that by which the original egg mem- brane is formed. The superficial protoplasm is at first mark- edly vesicular, and, after becoming homogeneous, is separated as a uniform layer. When the second crenated membrane is nearly formed, the ectodermic nuclei return to their original plane and the two layers again become parallel. At the end of this process there are, surrounding the egg, five membranes. First, the egg membrane formed in the ovary but by the egg (Pl. XXII, Fig. 40, ¢.m.); then the vitelline mem- brane, a thin but distinct envelope (Pl. XXII, Fig. 40, v.). This is followed by 7., the material cast off preparatory to the more 258 CLA YPOLE. [Vou. XIV. complex structures following; it is very thin excepting in places where the knobs are attached, which came from the trough of the fold. The first crenated membrane (c.r) is the next. This is uniform in thickness and is shed over the whole surface, includ- ing the precephalic organ, which is, however, unaffected by the crenations. By the time the last envelope, the second crenated membrane, is formed (Fig. 39, c.2), the dorsal organ is under- going the process of degeneration already described; its cells are elongating, the inner edge is becoming uneven, and the nuclei are shrinking to more solid masses (Fig. 39, fc.o.). The close connection between this organ and the envelope is readily proved by the fact that when the membrane is removed the organ is usually torn away from the embryo and remains attached to the envelope. No suggestion as to the function of the pre- cephalic organ can be made; there was no evidence that it is particularly associated with yolk absorption; its period of great- est development precedes the appearance of the germ band. But its only obvious use begins at about this time. The ultimate fate of the structure is gradual absorption; it becomes smaller and smaller, as is seen at gc.o. in Pl. XXIII, Figs. 41-45. It loses its connection with the envelope, and remains recog- nizable as a darker red staining mass inside the embryo, with a tuft of fine threads outside. On the hatching of the animal it is no longer visible. From these facts it is clear that there is no structure present corresponding in origin and nature to the amnion and serosa of the other Insecta. These are distinctly cellular envelopes, and appear at a later time. The “dorsal organ,” so-called among the higher Insecta, is directly connected with these envelopes. Lemoine ('87) has discussed this organ among the Poduridae, describing its form in Anurophorus and Smynthurus; he also suggests its probable relations to the structures found in the other Insecta. The organ forms a conspicuous part of the embryo in both these genera; in the former it appears early in the development of the blastoderm, but in Smynthurus its appearance is delayed until the formation of the ventral plate, and it persists until hatching. As no sections were cut, the discussion of the relations existing between this organ and the Won 2] ANURIDA MARITIMA. 259 envelopes and their probable connection with similar structures in other hexapods is somewhat vague and unsatisfactory. One point is clear, however: there are membranes in both forms that are attached to the organ, and constitute a structure important to the embryo. Lemoine says there are present at first in Anurophorus an outer thick “chorion,” which is perforated irregularly in numerous places, and an inner very fine vitelline membrane. Later, more membranes are formed, the first of which appears after the formation of the blastoderm; it is described as very fine and not uniform in thickness. The author judges it to be formed of many cells identical in origin with the blastoderm cells, and calls it the amniotic membrane, naming it, however, purely from analogy of form and function. This “amniotic membrane” is connected with the dorsal organ by an ampulla, which Lemoine calls the ‘amniotic ampulla.” Throughout the greater part of development another membrane is also present that he considers a true larval skin, as it forms on the outer parts of the appendages also. Nicolet describes two envelopes for the poduran embryos he studied (Podura, Cyphodeirus, Desoria, Smynthurus), the outer very stiff and the inner fine, probably corresponding to the “chorion ’”’ and vitelline membrane of Lemoine. Oulganine (75) describes two similar structures in Achorutes, Anuropho- rus, and Degeeria. It is evident that these two membranes are found in all these forms, but Lemoine is the only one to describe still more. Leaving out the two preblastodermic envelopes that are similarly described by all authors, one of the inner ones described in Anurophorus and Smynthurus may be considered as resembling the crenated membranes described in Anurida. This “amniotic membrane” of Lemoine is peculiar in its behavior during the life of the embryo. It possesses great powers of expansion and contraction, increasing the size of the egg by one-fourth or one-third at its largest size compared with its smallest. The author goes further and states that in contraction a folding of the surface of the embryo takes place, giving it a roughly four-sided figure with fine wrinkles over the surface. No such powers of rapid contraction were seen in Anurida. 260 CLA POLE, [VoL. XIV. In living specimens the space between the embryo and the membrane is not very large, but it is practically constant; in the early stages the crenations are narrow and deep (Fig. 38, c.1), but later they are wider and shallower (Fig. 39, ¢.2). This is clearly associated with an increase in the size of the embryo, as measurements prove. Very soon after the formation of the membranes the first, the egg membrane, splits, and with it the vitelline membrane. Then the first crenated membrane becomes the outside cover, and a decided increase in size is observable. Growth continues until eventually the wrinkles are expanded so as to make the crenations flat in comparison with earlier stages. The attachment of this inner crenated membrane with the dorsal organ serves as a means of suspension of the embryo in the envelopes; it is thus held in a fixed position. In pre- served specimens, in which some amount of shrinkage of the embryo has taken place, the space round the embryo is consid- erable. The latter hangs eccentrically placed, owing to its attachment to the membrane. There is one other possible use for the crenations in the envelope besides the simple one of allowance for growth. The eggs are subject to considerable variations in pressure and degrees of moisture, owing to the changes in level of the tide. The crenated surface would more readily resist the effects of this change in pressure than an unfolded one. Observations were attempted to determine this point, but nothing definite resulted, and any such sugges- tion must remain an inference. It seems clear that powers of expansion belong to the embry- onic envelopes of at least three of the poduran genera, —Smyn- thurus, Anurophorus, and Anurida. As regards the causes of such changes, there is less known; in Anurida, growth and possibly changes of pressure are the direct agents, while in Anurophorus and Smynthurus contraction and expansion take place regularly without any apparent external or internal cause. Lemoine’s Fig. 16 strongly suggests another interpretation of the crenations found in the embryo; it so much resembles the early crenated stages of Anurida as to make it possibly a corresponding stage, instead of an embryo undergoing excessive contraction. No. 2.] ANURIDA MARITIMA. 261 Leaving the podurans, there are points of interest to be found in connection with the higher groups. Wheeler ('93) discusses a curious structure found in Xiphidium, which he calls the “indusium.” It appears at the same time as the germ band, or a little later, and is ventrally placed on the long oval egg, just in front of the head. It remains unchanged for some time, usually separated from the head, but sometimes connected by a small string of cells. Ultimately by proliferation it forms an envelope, pushing its way between the serosa and the yolk, and finally becomes an inner membrane next to the yolk, and only separated from the embryo by the amnion. Strange to say, this organ forms for itself an amnion, which spreads round the egg and is recognized as the outer “indusium.” The author homologizes this with the poduran “ micropyle,’ and seconds the previous suggestion that the latter is truly homologous with the “dorsal organ,”’ as found in some groups of the Crustacea. Among the Crustacea there are found curious intermediate structures. Bobretsky (74), in his studies of Oniscus, which have been corroborated in part by Nusbaum ('86), discusses the so-called “‘ primitive cumulus” or “dorsal organ.” Its origin as described is similar to that of the precephalic organ in Anurida both in manner and in time. Excepting in the dorsal half of the embryo the two germ layers are distinct. After remaining stationary for a long time the cells increase in number and spread out over the dorsal part of the embryo as acap. This cap is connected with a thin membrane that the author calls a larval skin. At the greatest development of this organ it remains as a saddle-shaped cloak composed of a single layer of cells. In embryos of two species of Idotea found at Woods Holl, a structure similar in its early stages to that of Anurida was found, but it was paired, one small organ being placed on each side of the middle line. This resembled closely Nusbaum’s (87) figures of Mysis. To make a graded series between Anurida, Oniscus, and Xiphidium is easy. In the first the organ is large, active, and functional in very early stages; it later begins to degenerate and assumes certain secondary characters, as, for example, the 262 CLA YPOLE. [Vou. XIV. connection with the membranes. In Oniscus the cells migrate bodily instead of simply elongating, and form a cellular cap instead of a membranous one. In Xiphidium the cellular envelope is completed and entirely encloses the embryo. Wheeler’s suggestion is that the organ he calls the “indusium ”’ had probably lost its original function, and was degenerating and varying in consequence; accidentally acquiring a new value, it was reconstructed for its new use as an embryonic envelope. In Oniscus this process of reconstruction is not yet completed, and in Anurida barely begun. There is an in- teresting suggestion in Wheeler’s ('93) mention of the embryonic sucking disc in Clepsine. A complete series may possibly be made between this disc, the organ as found in Anurida and the phyllopod cervical gland which actually functions as a sucker, and is regarded by Miiller (64) and Grobben ('79) as the homologue of the ‘dorsal organ”’ of the Amphipoda. In this case the power of adhesion that still belongs to the pre- cephalic organ in Anurida is possibly a remnant of its former function. The gradual prolongation of embryonic life causes the young to hatch in a more mature stage, and need for the adhesive disc of the immature larva is lost. Embryo Formation. After the separation of the second membrane the formation of the ventral plate or germ band begins. On surface views it first appears as a narrow band passing round the egg in such a way that it nearly encircles it, the precephalic organ being the separating mass. The head of the embryo lies on one side of it, and by crossing the organ the tail is found at the opposite side (Pl. XXIII, Fig. 41). Almost immediately the outlines of the embryo can be distinguished, the different parts being laid down successively from the head backwards. The mesodermic somites indicating the future segments of the body appear, and almost at once the appendages of the different parts. In as early a stage as that shown in Pl. XXIII, Fig. 41, the begin- nings of the antennae, mandibles, maxillae, and thoracic legs are evident, and Pl. XXIII, Fig. 40, shows an added pair of No. 2.] ANURIDA MARITIMA. 263 appendages between the antennae and the mandibles, as well as the faint outline of the rest of the germ band. Up to the stage figured in Fig. 42 the chief changes are in the clearer definition of the six abdominal segments, the appearance of the median unpaired labrum, and the indication of the procto- deal and stomodeal invaginations. The antennae have become undoubtedly three-jointed, with an indication of a fourth, and the precephalic organ has begun its process of elongation and degeneration (Fig. 42). The embryo still preserves its spheri- cal form, and stretching across between the ends of the appen- dages can be seen the last envelope formed (Figs. 42, 44, m.). Whether this is a true larval skin or is similar to the “ Blasto- dermhauten”’ already discussed is not clear, but it can best be seen after the appendages have appeared. It seems most likely that it is shed just at the beginning of the embryo formation; it passes round the embryo, and is frequently found attached to the ends of the precephalic organ. From this point a radical change of form takes place; a flexure of the embryo begins that results in crowding the mouth-parts together to form a definite head and folding the embryo upon itself. This greatly changes the points of refer- ence in regard to the precephalic organ: the head remains in about the same position, but the tail is drawn much farther away, and the embryo becomes restricted to less than one-half of the circumference of the egg, instead of extending over nearly the whole. At the same time there is a marked lateral flattening, so that the young animal is much narrower meas- ured from side to side than measured dorso-ventrally. Before the final stage of this process is reached, however, certain features of note have appeared. The most striking of these are shown in Figs. 43 and 45. Fig. 43 represents an embryo in which the ventral flexure has just begun, as shown in the side view of Fig. 43, a. The brain lobes, the protocerebrum, have clearly appeared, and their elongation into optic lobes is evident. The labrum, unpaired, and lying on the middle line, is seen just anterior to the stomodaeum. The antennae lie on each side of the future mouth, and are formed of three short, thick, and approximately equal joints. The three pairs of 264 CLA YPOLE. [Vou. XIV. mouth-parts are next in succession. On each side of these has appeared a ridge that passes backward along the embryo, the two folds enclosing the mandibles and maxillae. These folds start from just the region where the small intercalary appendages were seen earlier, but which have now disappeared. _Figs. 43, 46, and 47 show the process by which this change takes place, and leave no doubt that the folds as they finally appear are a development from the intercalary appendages. This sheath-like form of the extra mouth-part explains the well-known peculiar structure of the adult head. The adult mouth has always been described as deeply sunk into the head and appearing as a tube, out of the end of which the points of the mandibles and maxillae protrude. It can be readily seen that the labrum in front and these lateral folds make together a three-sided box in which the mouth-parts, two mandibles, and four maxillae are sheltered. In Fig. 43, where flexure has just begun, the thoracic appen- dages are visibly longer and more distinctively legs. The first abdominal segment bears a large pair of appendages that are ultimately modified to form the collophore, while on three of the succeeding abdominal segments there are also small appen- dages, those on the fourth segment being the largest. This condition is equally evident in Fig. 44, a slightly later stage. In Fig. 45 the conditions are still the same; the collophore is, however, almost hidden by the flexure of the body, and the terminal segment has elongated into two decided folds that surround the proctodaeum. The five single eye-spots have appeared on the sides of the head, and the precephalic organ is much reduced and shows the thread-like elongation of its cells. An interesting question is raised by a consideration of the folds that rise round the mouth. The simple structure of the adult mouth in these forms has been discussed by Fernald ('90) ; he describes it as being a pouch of considerable size, at the inner end of which are attached two pairs of jaws; these are entirely en- closed. He cannot, however, determine the exact homologies of the different parts. Hansen ('93) discusses the homologies of the mouth-parts of the Crustacea and Insecta by studies on Japyx, Campodea, and some of the Collembola. He finds it a No. 2.] ANURIDA MARITIMA. 265 common peculiarity that the mandibles and maxillae are sunk deeply in the head up to the points, as in the case of Anurida. He speaks of a fold of the skin that causes this insinking which is attached to the labrum, and is undoubtedly similar to the fold found in Anurida. Campodea, Japyx, Machilis, and Lep- isma all agree in general details, but the last mentioned is considered by Hansen a transition form between the Thysanura and the Orthoptera. The relations as seen in the anuridan em- bryo are as follows: The unpaired labrum forms the upper part, the front of the pouch, at the back of which work the two pairs of jaws, the mandibles, and the first maxillae, while the second pair of maxillae has been modified to form the back of this pouch. The lateral folds already described make the sides and are developed as shown from the intercalary segment. The question naturally arises as to what homology this addi- tional pair of mouth-parts can have, arising as it does on a dis- tinct segment. Viallanes ('91) and Wheeler ('93) agree in the following structure of the orthopteran head and brain: It con- sists of a protocerebrum, the most anterior segment, forming the mass of the supra-oesophageal ganglion, from which the large optic nerves are developed. A deutocerebrum and trito- cerebrum follow, which together complete the brain and the oesophageal collar. Following this is a series of ganglia cor- responding to the mouth-parts, which eventually fuse to form the suboesophageal ganglion of the adult. These authors find distinct mesoblastic somites in the segments of both the deuto- and tritocerebral lobes, and hence conclude their equivalence in value to any of the succeeding segments. The antennae of insects are enervated from the deutocerebrum, and, as has been demonstrated by Viallanes ('91) and St. Remy ('90), the first pair of crustacean antennae is also connected with this brain lobe, the second pair being enervated by the tritocerebrum. Hansen homologizes the mandibles in the two groups, but does not decide on the antennal homology. It would seem clear from the work already mentioned on the brain that the homology of the first antennae of the Crustacea with that of insects is practically decided. Arguments drawn from the absence or presence of either pair of antennae in the higher 266 CLA YPOLE. [VoL. XIV. Crustacea are not convincing, as there is great variation in the degree of development of these appendages in different groups. In some the first antennae are larger and the second small or absent, and in others the reverse is true. The evidence from the lower forms is more reliable. Ray Lankester enumerates the different appendages found in that archaic type Apus, and indicates that the first antennae are always present while the second are sometimes absent and sometimes present, in the same species, and always missing in some species. As Apus is considered more generalized in its structure than any other crustacean, it is suggestive that the first antennae should be constant and the second the more variable. This immediately suggests an interesting explanation for the added pair of mouth- parts found in Anurida, originating from the tritocerebral seg- ment. On this basis they are a modified form of the second pair of antennae in the crustacean; and hence Anurida, includ- ing, without doubt, its allied forms, possesses an adult structure clearly homologous with the second antennae, the very impor- tant appendages of some crustacean heads. It is interesting in this connection that Hansen considers the musculature of the head of Machilis much more like that of the crustacean than that of the insect. Fig. 48 represents an Anurida just hatched. It can be seen to have many of the characters of the adult form; it is, however, perfectly white, showing none of the black pig- ment characteristic of the adult. The surface of its body is not as wrinkled and folded at this young stage, and the cuticle lacks the finely papillose surface found in the older specimens. The antennae are clearly four-jointed, as described by Ryder ('86); the terminal joint is less pronounced, however, and some- times is not completely separated from the third. Instead of a third joint there exists only a constriction. The collophore is prominent; it originates by the fusion of the two appendages on the first abdominal segment. Young animals at this stage are very active, and may be found in large numbers in the same places as the eggs. Judging from the great variation in the size of the eggs at the time of hatching, there is a great variation in the size of the animal; this must be the case, No. 2.] ANURIDA MARITIMA. 267 because it is not arule that the smallest is the least developed. Quite often the smallest ones have undergone considerable post-embryonic development, while some larger ones are much farther back in the process. Pigmentation and increase in size are the chief external changes that are needed to make the young Anurida resemble the adult. Both of these charac- ters come slowly, though the young are probably all pigmented by the end of the season. They remain small in size, however. Comparing these results with Ryder’s (’86) figures, which are, as far as known, the only published studies of the em- bryonic stages of Anurida, certain differences are observable. There are figured in these the two crenated membranes and the early stages of the germ band, several later embryonic forms, and young and adult animals. The chief difference in the embryos as figured by Ryder and those shown in Pl. XXIII of this investigation lies in the different interpretation of the embryonic head appendages. Ryder recognized but three pairs: one pair of antennae, one pair of mandibles, and one pair of maxillae. He included the second maxillae with the thoracic legs, and did not see the intercalary appendages. In the recently hatched young he describes a structure placed on the anterior part of the fourth abdominal segment which he con- siders represents a rudimentary spring. No evidence of such a structure was seen in the young investigated, and large num- bers were examined. In the embryonic stages the appendages on the fourth abdominal segment are larger than any of the others, excepting those on the first (Fig. 45, a.4); but these, like all the others excepting those on the first, disappear before hatching. The process by which the germ band arises is exceedingly simple. Immediately after the formation of the second cre- nated membrane, or even before, or in some cases before all the entodermic nuclei have sunk to a common level again, the mesoderm cells may be seen migrating to such a position that one meridian passing through the precephalic organ and the centre of the egg would cut the band they form longitudinally into two. The migration eventually leaves the greater part of the egg covered only by ectoderm and the germ band appears 268 CLA YPOLE: [VoL. XIV. girdling the egg. At first one-layered, the mesoderm early shows a further change to two in certain parts which represent the mesoblastic somites. Fig. 49 represents a cross-section through the germ band just after itsformation. In the middle line, under the median ectodermic depression, the mesoderm is a single layer of cells; but on each side there are the early indications of the somites. The two-layered condition arises by migration, and the cavity when present is hollowed out afterwards. Subsequent modifications arise by differentiation from this primitive condition. | Origin and Development of the Entoderm. The place and manner of entoderm formation long remained in doubt, as the appearance of the mid-gut is delayed till very late in embryonic life, and these late stages are difficult to find. However, the following facts and explanation were eventually determined. By the end of cleavage two definite layers are fully established, the ectoderm and mesoderm, as shown in Fig. 35. There are, moreover, certain cells left in the yolk that have never taken part in the formation of either of the two layers. Some of these that are spread singly through the yolk are evidently yolk cells, and function in the transformation of yolk for the nutrition of the embryo (Figs. 35, 37, 39, y.c.). In addition to these, however, there are some cells that remain grouped in clusters, the whole mass evidently arising by division from a single cell or perhaps a few cells. The clusters are _ placed above the centre of the egg, using the precephalic organ as the pole of the reference axis, and very frequently limited in numbers to two (Fig. 35, ez.). One of two things now happens to them: they either migrate from the masses and are scattered through the yolk in small groups of twos or threes, or else they remain unchanged for a considerable length of time. In the former case they are difficult to distinguish from the yolk cells, but their greater transparency, larger vesicular cell bodies, and association in small groups is a sure guide to their identification (Pl. XXII, Fig. 41). The yolk cells early acquire a more deeply staining nucleus, showing the characteristic increase of chro- No. 2.] ANURIDA MARITIMA. 269 matin in cells with a strongly assimilative function. In the second case, when these cells remain permanently associated in one or more clusters, no further change occurs until late in embryonic life. By the subsequent development of the embryo and its changes in form during flexure, the relative positions of the groups are somewhat changed. One large mass may, how- ever, readily be recognized in the region of the proctodaeum, not far from the ectodermal layer. After the embryo has reached a stage corresponding to Fig. 45, or perhaps later, the mass may be seen to be scattering, and certain changes occur in the yolk. Around some cells are large spherical masses of yolk particles contained in extremely vesic- ular protoplasm, in which there is a small central nucleus (Fig. 58, ez.). These are particularly numerous in the regions of the stomodaeum and proctodaeum, and may be clearly seen later to assume a regular arrangement in the yolk, forming two broken lines reaching through the body. It is now clear what these mysterious cells are: they are the entoderm, and are taking up definite positions to form the mesenteron of the young animal. In a newly hatched specimen an interesting relation is shown; Fig. 65 represents part of a frontal section through the body of such an animal. The mesenteron is seen to be composed of large irregular-shaped cells with extremely vesicular protoplasm; the nuclei are irregular in size and stain faintly. At the inner or free edges of the cells masses of yolk are visible, and certain of the cells also contain similar particles. Whether these particles are passing out of the entoderm cells to the enteric cavity or are being ingulfed by them is not clear, but in either case it is evident that the mesenteron when fully formed contains very little food yolk, a condition contrary to the general rule. The entodermic cells are resting on an extremely thin membrane, but there is as yet no sign of muscu- lar walls or other differentiation. The cells are themselves still irregular and almost amoeboid in form (Fig. 65, ¢7.). This is, then, the history of the entoderm in Anurida; it originates without doubt during cleavage, and takes up its posi- tion in the middle of the morula by a process which it is possible to call invagination. When the mesoderm, which also lies within 270 CLA YPOLE. [VoL. XIV. the morula, migrates outward and forms a definite layer below the ectoderm, the entoderm remains in the interior as one or more cell masses and is comparatively unchanged till a late period of embryonic development. Finally the cells separate and increase in size, and ingulfing yolk arrange themselves to form the definitive mesenteron, which contains practically no yolk excepting some in an intracellular condition. Whether the vitellophags are genetically connected with entoderm is not clear, but they very possibly are entoderm cells that early assume their digestive powers. That they do not, however, take part in the formation of the mesenteron is clear from their presence at the time of its formation scattered throughout the yolk, recognizable as shrunken degenerating bodies (Fig. 57, y.c.). At first sight this process is markedly different from that described for other insects; but the differences admit of quite ready explanation. The typical process of entoderm formation in the other Insecta is by proliferation from two formative centres, an oral and anal, that appear at the two ends of an elongate blastopore. This process has been demonstrated for the Coleoptera by Heider ('g9) and Wheeler (89); in the Diptera by Voeltzkow ('s9) and Graber ('s9); in the Hymenoptera by Carriére (90); and in the Orthoptera by Wheeler (93). In several other forms but a single formative centre is described, that one being the anal. From this one or these two centres a continuous band is formed by proliferation that finally incloses the yolk completely. The great difference between the two processes, as described for Anurida and the rest of the Insecta, lies in the different disposition of the yolk; in the former case it is not inclosed in the mesenteron, and in the latter it is. The other variations may be harmonized in the following way: The groups of cells that are usually two in number can be consid- ered to be the equivalent of the two masses in the higher Insecta; the difference in size is marked, the anal mass being the one more likely to persist in a recognizable condition. A similar difference was observed by Wheeler (93) for Xiphidium, where he found the anal centre decidedly larger than the oral. The position of the two masses or, as it may be, one rather scattered mass just below the precephalic organ may be indica- No. 2.] ANURIDA MARITIMA. 27% tive of its future anal position, although at this time the germ band is not yet laid down. By a somewhat early migration the entoderm cells are scattered, and finally assume their definite relations at a very late embryonic period. It is, of course, pos- sible that the entoderm cells even at this stage assist in the transformation of the yolk, but there are certainly separate yolk cells for this duty. . As readily seen, the process of entoderm formation in Anu- rida agrees very closely with the method found among some of the Crustacea. In many members of this group the entoderm is early differentiated from the rest of the cells, but remains sta- tionary for a long time, simply imbedded in the yolk. In some cases its origin is still under discussion; some authors claim that it is composed of vitellophag cells that have been function- ing in the egg from the beginning; in many cases the entoderm only assumes its permanent relations at a late period of devel- opment. Zograff (90), describing the origin of the mesenteric lining in two species of Geophilus, says that it appears from the yolk, and concludes that the process closely resembles that found in Malacostraca and Arthrostraca. Heathcote ('s6) describes it for Julus terrestris in the following way: Certain cells during cleavage remain behind in the yolk and form the entoderm, and in turn give rise to the middle germ layer. After the appear- ance of the ectodermal parts of the alimentary tract the scat- tered entoderm cells arrange themselves to form a central lumen and give rise to the mesenteron. From these few points of comparison it is clear that Anurida constitutes an interesting intermediate form, connecting the processes typical of the crustaceans and myriapods with those of the higher Insecta. It is difficult to say to which of the lower arthropod groups Anurida is the more closely allied, especially as so little work has been done on the myriapods. Certainly the resemblances to the group last mentioned are very striking. The interpretation of the differences found between the higher insects and Anurida would point to a gradual delaying of the entoderm formation to a later embryonic period in those eggs possessing a larger quantity of yolk. This new relation possibly raises again the question as to whether all the cells resulting from cleavage of 272 CLA YPOLE. [Vor. XIV. the central nucleus in centrolecithal eggs pass to the surface to form the blastoderm. Some may remain behind and form the vitellophags and perhaps take other part in development. It is certain in Anurida that vitellophags do not migrate out- wards and then return, but are left in the yolk. Development of the Reproductive Organs. The development of the germ cells was found to be one of the most interesting processes followed in detail. Their appear- ance takes place at a comparatively late period of embryonic life, the earliest stages occurring when the animal has reached the stage shown in Fig. 44.1 At this period the processes rep- resented in Figs. 50 and 51 are seen to take place in the second and third abdominal segments. Both these views represent cross-sections of the mesoblastic somites of one side of these abdominal segments, and show the great variation that occurs in the distinctness with which the cavities of the somites are developed. In Fig. 50 two germ cells (g.c.) are seen, one pass- ing out into the yolk on the free side of the somite, and one as yet imbedded in the splanchnic layer of the mesoderm. These cells are readily recognized by their peculiarly clear transpar- ent cell bodies. In Fig. 51 is shown another stage, when the germ cell is clearly inclosed in the cavity of the somite. Figs. 2, 53, and 58 show the line of development followed in this latter case. The germ cells are distinctly between the walls of the two mesoblastic layers, the splanchnic and the somatic. In Fig. 53 a definite form has already been attained by the germi- nal mass. There is a cephalic elongation and a hinder spherical mass. The surrounding mesoderm has been differentiated into muscles, and connective tissue is beginning to appear. There is, however, a distinct layer of mesoderm separating the germ cells from the yolk, the splanchnic layer; the germinal mass lying in a space appearing to be a true body cavity. In Fig. 58 the mass of cells is much larger, and by the crowding together of the abdominal segments it can be seen that flexure 1 Since the reproductive organs are paired and the process is similar in general principles, descriptions will be made of but one side. The only difference lies in a slight variation in position of the two organs. INO:2.|| ANURIDA MARITIMA. 273 of the embryo has proceeded much farther. The continuous mesoderm sheet separating the germ cells from the yolk mass has been broken; at the most curved part of the mass of germi- nal cells there is direct communication between them and the yolk. Whether this breakage is due to rapid flexure or rapid increase in the number of germ cells, which show evidences of frequent division or to both causes, one thing is clear, that the germ cells are now in close contact with the yolk. Returning now to a consideration of Fig. 50, the fate of germ cells originating in the second way may be seen in the series shown in Figs. 50 and 54-56. The single cell, set free on the outer side of the somite, increases to an irregular mass that lies in part sunk into the mesoderm and in part projecting out towards the yolk. These cells, at first a solid group, migrate outwards and begin to mingle with the yolk (Fig. 54, y.), the migration being most noticeable in the outer cells, those nearest the yolk. In Fig. 55 migration has not gone so far and the greater magnification shows the peculiarly “succulent” char- acter of the cells. In Fig. 56, illustrating the extreme result of the process, the cells have divided into two groups, one (s,¢.c.) remaining near the mesoderm and by repeated divisions increas- ing to a large mass of small cells, another (.¢.c.), which has migrated out and is spreading through the yolk, still maintain- ing, however, a certain relation to the stationary cells. Unfor- tunately, in spite of the most careful search, the latest embryonic forms found do not seem to supply the next step. The final result is seen in the figures in Pl. XXV, Fig. 64 concluding this series. This represents a longitudinal slightly oblique section of a just-hatched animal, showing the reproductive organs of one side of the body. At g.e. is a somewhat irregular mass of cells forming the germinal epithelium, lying in the second and third abdominal segments. Below this, and directly connected with it, is a large irregular sac filled with yellow material, in which are scattered a few large cells. Two lobes of this sac are cut through, and at its lower end, coming from the hinder end of the fifth abdominal segment, is an-ectodermic invagination, the duct of the reproductive organs leading to the exterior. This animal is recognizably a young male, the parts described 274 CLA YPOLE. [VoL. XIV. corresponding to the parts of the adult. The yellow material in the sac-like parts of the organs is yolk (Fig. 64, y.), and the golden yellow globules scattered through the connective tissue is yolk acquiring the characters of fat globules. The whole body is very simple in structure, a few muscle fibres, the ven- tral nerve chain, and a large amount of connective tissue filling up the space being the essential elements; numerous blood corpuscles loaded with yolk present in the small body cavity were not represented in the figure. The reproductive organ is shown at a later stage of development in Fig. 65 in frontal section; the yolk has been absorbed from that part of the sacs near the germinal epithelium, and by rapid proliferation, prob- ably rendered possible by the ample supply of food, very small sperm cells are being formed that will eventually mature as spermatozoa; these fill the sac. In Figs. 59, 60, and 62 the story of the first group of germinal cells originating within the cavity of the somite is continued. Fig. 60 represents a cross-section of an ovary from a recently hatched or at least very young Anurida. The cut does not include the germinal epithelium, but some of the cells that have become detached from it are figured that show char- acters rendering their recognition easy. At z.c. are cells that bear all the distinctive marks of nutritive cells, large nuclei, richly supplied with chromatin, which is irregularly massed together, but not stellate in arrangement. At o. are seen cells that as distinctly possess the characters of ova, large cell body, and small clear nucleus. Scattered among these are gran- ules of true embryonic yolk of irregular sizes. This is even included in some of the ova, as shown in Fig. §9. The ovarian wall is extremely thin; small nuclei occur at intervals that closely resemble the mesodermic nuclei of younger stages. Fig. 61 is a representation of an ovum with its nutritive cells, as found in an animal taken early in the summer, evidently before development had begun for the season. This shows how little, excepting in one respect, the ova and nutritive cells change during the winter. The one respect is in the chromatin of the nutritive cells; in the last figure the definite stellate structure is attained, while in the younger forms the chromatin No. 2.] ANURIDA MARITIMA. 275 is in coarse threads (Fig. 62) or irregular masses (Figs. 59, 60). In the size and external characters of the animal the changes in the interval are marked, but practically no development has taken place in the reproductive cells. The external part of the reproductive organ, the outlet, was found to originate in the late embryonic stages by a median unpaired invagination from the hinder end of the fifth abdominal segment. As shown in the adult, the ectodermic part of these ducts is extremely short, being only the small unpaired part extending from the exterior through to the body space, where it joins the mesodermic part of the organ. This is found completely invaginated in late embryos, and showing in the female the accessory diverticula, the receptaculum seminalis, as a branch of the main duct. When it was found that the yolk was not contained in the mid-gut of the embryo, the question naturally arose as to its final disposal. It has been seen from the above facts that a large part of it is included within the reproductive organs, and serves to hasten very much the maturing of the generative elements. A very large quantity is also found in the blood corpuscles of the newly hatched animal. Fig. 63 shows some of these taken from the same animal as Fig. 59. Gradually during their circulation through the body they must give up their rich supply of food. No complete observations were made on the origin of these cells, but it is probable that they arise from the mesoderm. Already in Fig. 53 small isolated meso- dermic cells can be seen, and many are found later in different parts of the body. Even when loaded with yolk there is no possibility of confusing them with yolk-laden entoderm cells, they are so very much smaller in size (cf. Fig. 63 and Pl. XXII, Fig. 41; 63 is magnified more than 41). Yolk was found in the places already enumerated and also free in the body cavity, lying chiefly under the alimentary canal. Thickly scattered through the meshes of the connective tissue are many yolk spheres, which are eventually transformed into fat globules. When first hatched, the body cavity, as it is called, or, more correctly speaking, the haemocoele, is much obscured by a large amount of connective tissue that originates from the mesoderm. The alimentary canal, reproductive organs, and nervous system all 276 CLA VPOLE. [Vou. XIV. lie more or less imbedded in it, and it is only by later post- embryonic development that the space is finally cleared and becomes as distinct as in the adult. The history and fate of the slight trace of the true coelom, as seen in the female embryo, has not been studied in detail and must remain a point for future investigation. As seen in Fig. 51, there is a distinct cavity in the mesodermic somite, although this is not so clearly marked in all the segments. In Fig. 58 the splanchnic layer of mesoderm forms one side of a spacious cavity, evidently resulting from the fusion of those parts of the somite cavities not cut off in the appendages. This space is a striking feature in animals of this sex and renders them immediately recognizable. It is, however, eventually obliterated; the beginning of this process is shown in the breaking of the splanchnic layer, thus allowing the germinal mass to leave the distinct true coelom. It is curious to find so much more reduction in the coelomic space in the male; it is as noticeably absent from the beginning as it was present in the female. At all times the germ band remains a solid mass; no space such as is shown in the female is ever seen. The loss of the coelomic cavity is without doubt a derived condition, as the ancestors of the insects probably inherited the space more or less completely from their annelid-like progeni- tors. The retention by the female of primitive characters not found in the male is a frequent occurrence, the latter sex being the more subject to modification. The female is the more conservative and adheres more closely to the primitive type. This inclusion of yolk in the reproductive organs is a point of great interest in the development of Anurida. It is a phe- nomenon not frequently observed in any forms, and, as far as known, without a direct parallel among the Insecta. There are certain forms, however, in which resemblances may be noted. Metschnikoff (74), in his classic account of the embryology of Polydesmus and Julus, double-footed myriapods, speaks of the peculiar position of the nutritive yolk in the body of the young embryos. It is present almost exclusively in the body cavity; very little is found in the intestine. This is a fact true also of the daphnids, where the yolk lies in the body cavity between a No. 2.] ANURIDA MARITIMA. Pigg | yolkless alimentary canal and the remaining viscera. Mordivilko (95), discussing the structure and development of some of the aphides, speaks of Metschnikoff’s “‘ secondary yolk ” (Metschni- koff, 66), and shows how it lies in the body cavity surrounding the reproductive organs and causing a wonderfully rapid growth on their part. Among the vertebrates certain forms exist in which a quantity of the embryonic yolk is associated with the germ cells, causing their rapid growth. Petromyzon, the lam- prey, belongs in this category, and, as shown in Fig. 66, much of the yolk is included among the germ cells.} Summary. Summing up the results of this investigation on Anurida the following points are of interest: (1) That the ovary is very simple in character, no arrange- ment corresponding to the ovariole of the higher hexapods being present. (2) A long anterior elongation is present, composed of cells non-germinal in character and serving as a suspensory ligament. Homology with the “‘ Endfaden ” is uncertain. (3) Ova are associated with nutritive cells that show distinct “yolk nuclei” at a certain stage. (4) The germinal vesicle early becomes invisible and the nucleus does not again appear until after the polar bodies are given off. (5) The egg is spherical, cleavage holoblastic at first and slightly unequal. (6) There is a multipolar immigration suggesting gastrulation. (7) Outer and middle germ layers are formed by migration, the entoderm remaining behind in the yolk with yolk cells. (8) A precephalic organ homologous with the dorsal organ of some crustaceans, and the indusium of Xiphidium is developed in the early blastoderm stages. (9) There are at least three cuticles formed during preblasto- dermic stages; two of the three are crenated. 1 This figure is from an unpublished drawing of Dr. W. M. Wheeler, who kindly lent it for this purpose. 278 CLA YPOLE. [Vou. XIV. (10) The embryo appears encircling the egg as a narrow girdle, stopping each side of the precephalic organ. (11) An extra pair of mouth-parts appears, forming in the adult two lateral folds inclosing the mouth-parts. This is homologous with the second pair of crustacean antennae. (12) Yolk is included in the reproductive organs and lies free in the body cavity, but is not found in the mesenteron. (13) Anurida shows characters allying it with crustaceans and myriapods rather than the rest of the Insecta. In consideration of all these points it is clear that Anurida possesses certain characters allying it closely to the lower arthropod groups. The holoblastic cleavage and egg mem- branes ally it to both crustaceans and myriapods, while the structure of the ovary is most like the synthetic type Scolo- pendrella, but more like the chilognath myriapod than the chilopod. In spite of the possession of some generalized characters, it is evident that Anurida is a degenerate type that has been developed by a lengthening of embryonic life and a shortening of adult life. Paedogenesis, the sexual maturing of a larva, is illustrated by this process. The absorption of the embryonic yolk by the reproductive organs and the great maturity of the products even immediately after hatching both point to a tendency to shorten adult life and to omit larval development even to the extent of assuming the larval form for the adult. The decrease in the number of abdominal segments is only another step in the same direction. If the insect may be con- sidered a larval chilognath sexually matured and bearing the three pairs of legs found in the chilognath larva, so can an Anurida be considered a very simple insect embryo matured sexually. Observations have been before advanced to estab- lish the progressive shortening in some forms and gradual elimination of larval forms. Anurida shows additional interesting points. By its curious habitat, chiefly under water, it has lost the need for tracheae, and, consequently, they are so far obliterated as to be absent even in the embryo; its respiration is purely cutaneous. It has been remarked that the amnion and serosa, the cellular envel- No. 2.] ANURIDA MARITIMA. 279 opes of higher tracheates, are connected strictly with terrestrial forms and are one of the necessary adaptations to the exigen- cies of land life. Whether or not the ancestors of Anurida ever possessed such structures and have since lost them in consequence of acquired semi-aquatic life cannot be settled, but it is interesting to speculate on the possibilities of Anurida being a simple form and still retaining a semi-aquatic mode of life and showing a few transitional characters. Notes on Other Points of Interest. Nervous System. —No detailed observations were made on the development of the central nervous system, but a few points of correspondence with other forms were noted. The brain and ventral cord both arise in the same way as that described by Wheeler ('93) for Xiphidium, — by the proliferation from single ectoderm cells until rows of nerve cells arise. Proliferation is in the direction of the dorso-ventral axis of the embryo, and is restricted to certain places in the segments; subsequently, these primitive ganglia are united to form the ventral cord. Ultimately the six abdominal ganglia are fused to form a mass. The brain portion may be readily seen to have the three suc- cessive segments, protocerebrum, deutocerebrum, and trito- cerebrum. The optic lobes form a large part of the young protocerebrum. Above the stomodeal invagination soon arose by proliferation from the hinder end a cord similar in structure to the ectoderm of the invagination that after reaching a con- siderable length remains unchanged, but a prominent character even in late embryonic life. It is entirely missing in the young animal and nothing remains to suggest its former presence. From the method of origin it is concluded that this is a trace of a sympathetic system, this being the history of this system in other insects; but since the adult seems to lack a sympa- thetic system, its degeneration is to be expected. Pauropus, that low, degenerate myriapod, also lacks a sympathetic system. These are mere notes on the general features of the nervous system, and a more complete study will be reserved for a future occasion. 280 CLA YPOLE. [VoL. XIV. Respiratory System.— Anurida, as has long been known, lacks entirely any tracheal system; respiration is carried on wholly by means of the skin. In the embryos no invaginations were seen to represent even the rudiments of such a system. There are, however, at the bases of the legs and at different parts of the abdomen large unicellular glands that may have some relations to tracheal openings or the different glands found in myriapods and other Tracheata. This point, too, remains for further investigation. In conclusion I wish to acknowledge the helpful oversight given to me during this investigation by the Department of Zodlogy of the University of Chicago, where the greater part of the work was done. My thanks are especially due to Dr. W. M. Wheeler, under whose direct supervision the subject was undertaken. I am also indebted to him for many valuable suggestions in the treatment of the subject and in methods and much assistance in reaching literature, and I wish here to express my grateful appreciation of the aid so freely given. Since finishing this article, two contributions have been made to our knowledge of the development of the Apterygota, in both cases of the Thysanura. One article is by Dr. Heinrich Uzel, in the Zoologischer Anzeiger (Bd. XX, Nrs. 528, 529, and 535) for 1897, entitled “ Beitrage zur Entwicklung der Thy- sanuren” (Campodea staphylinus Westw. and Lepisma saccha- vina L.). Another is by Dr. R. Heymons, published in the Zeitschrift fiir wissenschaftliche Zoologie for 1897, on the subject of “ Entwicklungsgeschichtliche Untersuchungen an Lepisma saccharina, L.” There are many points of interest between the observations made by these authors on the Thy- sanura and those given above for Anurida; one or two in particular will be briefly mentioned. Both authors describe the cleavage as distinctly superficial in Lepisma, and Uzel observes the same to be true in the spherical egg of Campodea. This is an interesting point in No. 2.] ANURIDA MARITIMA. 281 consideration of the sizes and shapes of the eggs in the three forms. In Lepisma the egg is a regular oval, and about I mm. in its longest diameter; in Campodea the egg is spherical, and has a diameter of about 0.4 mm., while the egg of Anurida has also a spherical form, but is only about 0.27 mm. in diameter. This increase in size is enough to explain the loss of holoblastic cleavage in the larger forms, considering its imperfect preser- vation in the small anuridan egg. The process of germ band formation as described by Uzel for Campodea agrees very closely with the corresponding one in Anurida. A “dorsal organ” is described as having a posi- tion comparable to that held by the precephalic organ in Anurida. There are in Campodea no embryonic membranes corresponding to the amnion and serosa of the pterygote insect. In Lepisma these structures appear, but the amniotic sac re- mains open for a short distance. In many ways Campodea suggests to Uzel the myriapod type of development. In the more complete consideration of Lepisma by Heymons there are several special points of interest. He finds rudi- mentary appendages upon the tritocerebral segment, which eventually disappear in early embryonic life. In discussing the origin of the mesenteron, several observations agree closely with those made on Anurida. The appearance of this part of the alimentary tract is very much delayed. Not until after hatching is it definitely formed. Unfortunately, certain criti- cal stages were not found; but the author saw in late embry- onic stages small groups of cells taking up a peripheral position on the yolk. These groups increase by rapid division originat- ing from what the author considers yoke cells that have been func- tioning throughout embryonic life in the assimilation of yolk. Hence he says the mesenteron is truly entodermic in origin. There is only one principal point of difference between this view and the one given for Anurida. In the latter case, it is clear that the mesenteron arises from cells originating at the same time as the yolk cells, but remaining latent through the early embryonic stages; the yoke cells themselves degenerate at the close of embryonic life. Possibly there is some such latent source in Lepisma that may have escaped observation. 282 CLAVFOLE: [Vox. XIV. The origin of the germ cells is another question of extreme interest. It was only after prolonged study that the interpre- tations of the facts observed in Anurida, as given above, were formulated. One point observed in Lepisma corroborates in part the conditions described in Anurida. It is clear that, as in Blatta, the germ cells are subject to great changes of posi- tion. According to Heymons they have an ectodermal origin, and appear early in embryonic life. The formation of egg tubes and their connection with each other are all steps accomplished by the process of migration. There is clearly nothing meta- meric in their origin, and any such arrangement must be sec- ondary. At present nothing further can be said on the diverse origins of the germ cells, — from the ectodermic in one case and mesoderm in the other. 1855. 1890. 1889. 1886. ANURIDA MARITIMA. 282 BIBLIOGRAPHY. BALBIANI, E. G. Centrosome et “ Dotterkern.” /ourn: Anat. Phys. Paris. 29 Année, pp. 145-179. 1883. BaARROIS, J. Développement des Podurelles. Assoc. Franc. p. Avance. des Sci.. 7° Sess. 1870. BICKFORD, E. Morphologie und Physiologie der Ameisen-Arbeiter- innen. Zool. Jahrb. Bd. ix, Heft 1. 1895. BLOCHMANN, F. Ueber eine Metamorphosis der Kerne in den Ovarialeiern und iiber den Beginn der Blastodermbildung bei den Ameisen. Verhandl. naturhist. med. Ver. Heidelberg. Bad. iii, pp. 243-246, Pl. I. 1884. BrRAEM, F. Zur Entwicklungsgeschichte von Ophryotrocha puerilis, Meez. Zeit. 7. wiss. Zoot. Ba. tii; pp. 187—223,. Lat. Xa 1893. BRAUER, A. Ueber das Ei von Branchipus grubei von der Bildung zur Ablage. Adt. Acad. Berlin. Anhang 66. 3 Taf. 1892. BoBRETSKY, N. Zur Embryologie des Oniscus murarius. Ze?z¢. f. wiss. Zool. Bd. xxiv, pp. 179-203, 2 Taf., 25 Figs. 1874. Bumpus, H.C. Embryology of the American Lobster. /ourn. of Morph. Vol. v, No. 2, pp. 215-252, Pl. XIV-XIX. 18o1. CARRIERE, J. Die Drusen am ersten Hinterleibesringe der Insect- embryonen. Szol. Centréblt. Vol. xi, pp. 110-127, 3 Figs. 1891. CARRIERE, J. Die Entwicklung der Mauerbiene (Chalcidoma muraria Fabr.) im Ei. Archiv f. mikr. Anat. Bd. xxxv. 1890. Ciaus, C. Beobachtungen uber die Bildung des Insecteneies. Zeit. f. wiss. Zool. Bd. xiv, pp. 42-54, Taf. VI. 1864. DuFour, L. Recherches anatomiques sur les Labidoures ou Perce- oreilles, précédées de quelques Considérations sur l’établissement d’un ordre particulier pour ces insectes. Ann. des Sct. Nat. Ser. 1, Tome xiil. 1828. FABRE, M. Recherches sur l’Anatomie des Organes reproducteurs et sur le Développement des Myriapodes. Azmn. des Sct. Nat. Ser 4, Lomein. 1S5 5: FERNALD, H. T. The Relationships of Arthropods. Stud. Biol. Lab. of Johns Hopkins Univ. Vol. iv, No. 7, pp. 431-513, Pl. XLVIII-L. 1890. GRABER, V. Ueber den Bau und die phylogenetische Bedeu- tung der embryonalen Bauchanhange der Insecta. Bzo/. Centrblt. Bd. ix, pp. 355-363. 1889. GRAssI, B. I progenitori degli Insetti e dei Myriapodi. Morfo- logia delle Scolopendrelle. Jem. d. Reale Accad. d. Sci. da. Torino. Ser. 2, Tome xxxvil. 1886. 284 1888. 1888. 1889. 189g. 1894. 1894. 1885. 1895. 1894. 1884. 1886. CLA YPOLE, [VoL. XIV. Grass!, B. I progenitori dei Myriapodi e degli Insetti. Memoria VII Anatomia comparata dei Tisanuri e considerazioni generali sull’organizzazione degli Insetti. Atte Accad. Linett Mem. (4), Vol. 4, pp. 543-606, Taf. 5. GROBBEN, C. Die Entwicklungsgeschichte der Moina rectirostris. Arb. zool. Inst. Wien. 2 Bd. 1879. GuERIN, M. Iconog. du Regne Animal, Texte Explic. Paris. p. II (not figured). 1829-1838. HAASE, E. Die Vorfahren der Insekten. Adhk. naturf. Gesells. Tris, Dresden. 1886. HAASE, E. Beitrag zur Phylogenie und Ontogenie der Chilopoden. Schles. Zeit. f. Entom. N.F. Heft 8. HAASE, E. Die Abdominalanhange der Insekten mit Beriicksich- tigung der Myriapoden. Morph. Jahrb. Bd. xv, Heft 3, pp. 331 —335, Taf. XIV-XVI. 1889. HANSEN, H. J. Zur Morphologie der Glieder und Mundtheile bei Crustacean und Insekten. Zool. Anz. Nrs. 420 und 421. 1893. HEATHCOTE, F. G. The Early Development of Julus terrestris. Quar. Journ. Micr. Soc. Vol. xxvi, pp. 449-470, Pl. XXIII- XXIV. 1886. HEATHCOTE, F. G. The Postembryonic Development of Julus terrestris. Phzl. Trans. Lond. Vol. clxxix B., pp. 157-179, Pl. 27-30. 1888. HEIDER, K. Die Embryonalentwicklung von Hydrophilus piceus, LL. Jena: 13889. Heymons, R. Die Entwicklung der weiblichen Geschlechtsorgane von Phyllodromia Germanica, L. Zezt. f. wiss. Zool. Bad. lili, pp. 434-536, Taf. XVIII-XX. 1891. HeEymons, R. Ueber die Bildung der Keimblatter der Insekten. Sitzungsber. Akad. Wiss. Berlin. 1894. HUBBARD, J. W. The Yolk Nucleus in Cymatogaster aggregatus, Gibbons. Proc. Am. Phil. Soc. Vol. xxxiii. 1894. ISHIKAWA, CH. Onthe Development of a Fresh Water Macrurous Crustacean, Atyephyra compressa, de Haen. Quar. Journ. Micr. Soc. Nol. xxvion T6G5. Kenyon, F. C. The Morphology and Classification of the Pauro- poda with Notes on the Morphology of the Diplopoda. TZzjfts College Studies. No. 4. 1895. KorotnEFF, A. Zur Entwicklung des Mitteldarmes bei den Arthro- poden. Bzol. Centrblt. Bd. xiv, pp. 433, 434. 1894. KoRSCHELT, E. Ueber die Bildung des Chorions und der Micro- pylen bei den Insekteiern. Verlauf. Mittheil. Zool. Anz. Bd. vii, Nr. 172, pp. 394-398, 420-425. 1884. KORSCHELT, E. Entstehung und Bedeutung der verschiedenen Zellenelemente der Insektovarium. Zezt. f. wiss. Zool. Bd. xlii, p537.. Sess: No. 2.] ANURIDA MARITIMA. 285 1889a. KORSCHELT, E. Die Bildung der Eihullen. MWova Acta Acad. Lzop. Carol. Ba. lit’ “1859, 188gb. KoRSCHELT, E. Beitrage zur Morphologie und Physiologie des Zellkernes. Zool. Jahrb. Bd. iv, pp. 1-154, Taf. I-VI. 1880. 1892. KORSCHELT und HEIDER. Lehrbuch der vergleichenden Entwick- lungsgeschichte der wirbellésen Thiere, Heft 2. Jena, Verlag ‘von Gustav Fischer. 1892. 1876. Leripic. Der Eierstock und die Samentasche der Insekten. Ver. kh. Leop.-Carol. Akad. 1876. 1887. LEMOINE, V. Recherches sur le Développement des Podurelles. Assoc. Franc. p. l'Avanc. des Sci. Paris. 1883. (1887.) 1859. LuBgBock, J. Ovaand Pseudova of Insects. Phil. Trans. Lond. pp. 341-369, Pl. XVI-XVIII. 1859. 1861. LuBsock, J. Notes on the Generative Organs and the Formation of the Egg in the Annulosa. Phzl. Trans. Lond. pp. 595-627, Pl. XVI-XVII. 1861. 1895. McMourricu, J. P. Embryology of the Isopod Crustacea. /ourn. of Morph. Vol. xi, No. 1, pp. 63-139, Pl. V-VIII. 1895. 1866. METSCHNIKOFF, E. Embryologische Studien an Insekten. Zezt. jowtiss.. Zool. Bda..xvi, pp. 437-467; Tak. XXVIlLIZXX XI. 1874. METSCHNIKOFF, E. Embryologie der doppelftissigen Myriapoden. Zeit. f. wiss. Zool. Bd. xxiv, pp. 257-283, Taf. XXIV-XXVII. 1874. 1875. METSCHNIKOFF, E. Embryologische Studien tiber Geophilus. LeU fi WES. 2000») DOW EXV, Pp. 93-322.) ihdkas N= ee 1875. 1895. MorpDIvILKo, A. Zur Anatomie der Pflanzenlause, Aphiden. Zool. Anz. Nr. 484, pp. 345-364. 1895. 1891. MorGan, T. H. Contribution to Embryology and Phylogeny of the Pycnogenids. Studies from the Biol. Lab. of Johns Hopkins Univ., Baltimore: Vol.v. 1891. 1864. MULLER, F. Fir Darwin. 1864. 1886. Nuspaum, J. L’Embryologie d’Oniscus murarius. Zool. Anz. Bd. ix, pp. 454-458. 1886. 1887. Nusspaum, J. L’Embryologie de Mysis chameleo. Arch. de Zool. Expér. Tomev. 1887. 1887. OUDEMANNS, J. T. Bijdrage to de Kennis der Thysanura en Col- lembola. Acad. Proefsch. Amsterdam. 1887. 1875. OULGANINE, W. M. Sur le Développement des Podurelles. Arch. de Zool. Expér. Tomeiv. 1875. 1871. PACKARD, A.S. Embryological Studies on Diplax, Perithemis and the thysanurous Genus Isotoma. Peabody Acad. of Sct. Vol. 1, INO Msi LoyAl. 1890. Vom Ratu, O. Ueber die Fortpflanzung der Diplopoden. Bericht. Nat. Ges. Freiburg. Bd.v, pp. 1-28, Taf. 1. 286 1881. 1886. 1882. 1894. 1895. 1896. 1886. 189go. 18Ql. 1889. 189go. 1889. 1890. 1893. 1896. 1885. 1884. 1885. CLA YPOLE, [VoL. XIV. Ryber, J. A. The Structure, Affinities, and Species of Scolopen- drella. Proc. Acad. Nat. Sct. Philadelphia. 1881. Ryp_Er, J. A. Development of Anurida maritima, Guérin. Amerz- can Naturalist. Vol. xx, pp. 299-302, Pl. XV. 1886. Scuurz, J. Ueber den Dotterkern, dessen Entstehung, Structure, Vorkommen und Bildung. J/naug. Dissert. Bonn. 1882. ScumipT, P. Zur Kenntnis des inneren Bau des Pauropus Huxleyi, Lubb. Zool. Anz. Nr. 448, pp. 189-196, 2 Figs. 1894. ScumipT, P. Beitrage zur Kenntnis der niederen Myriapoden. Zeit. f. wiss. Zool. Bd. lix, Heft 3, pp. 436-510, Taf. XXVI- XXVII, 3 Figs. im text. 1895. SINCLAIR (HEATHCOTE). Insecta. Cambridge Natural History. Vol. v. 1896. STUHLMANN, F. Die Reifung des Arthropodeneies nach Beo- bachtungen an Insekten, Spinnen, Myriapoden und Peripatus. Akadem. Verlagsbuchhandlung von J. C. B. Mohr. Freiburg i. B. 1886. St. Remy, G. Contribution a l’étude de cerveau chez les Arthro- podes tracheates. Arch. de Zool. Expér. Tome v et Suplt. (1887). 18go. VIALLANES, M: H. Sur quelques points de l’histoire du développe- ment embryonnaire de la Mante religieuse. Anz. des Sct. Vat. Ser. 7, Tome xi, pp. 283-328, Pl. XII-XIII. 18o1. VoeLtzkow, A. Entwicklung im Ei von Musca vomitoria. Y i J ae i 1 “ 1 ui ® @ raul. of Morphology Vola 292 CGEAVPOLE: EXPLANATION OF PLATE XXI. Cleavage. Fic. 15. External view of unsegmented eggs, showing grouping. Obj. 4, oc. 2. Fics. 16-24 are surface views of cleavage stages, showing the 2, 4, 8, 16, 32, and coarse morula stages until total cleavage ceases. Drawn from unstained eggs. Obj. 16, oc. 6. Fic. 25. Protoplasmic mass at the surface of egg. /fr., female pronucleus returning to centre; 7.0., polar bodies. Borax carmine. Obj. 7, oc. 6. Fic. 26. Showing division of first polar body. Letters as in Fig. 25. Borax carmine. Obj. 74, oc. 6. Fic. 27. Part of protoplasm from the centre of the unsegmented egg. /r., female pronucleus; .fr., male pronucleus. Borax carmine. Obj. 7s, oc. 6. Fic. 28. First cleavage spindle. Iron haematoxylin and Orange G. Obj. qs; OC. 2. FIG. 29. Reconstruction of nucleus after division into the 2-cell stage. Letters as before. Iron haematoxylin and Orange G. Obj. 4, oc. 4. eS Morphology Vol.AN: “uv ye = ne i. Piha i , an wt . + « % af ar a - = or. fe es a we af sp oa a - m= bs a all 7 Zz ae ee —— = Es te Ee Pat ts [_—_ —— - a = = ’ i ; . 4 Ps - i a ' 7 . ° al 1 e 7 ee NY at a ve - et glee ee y= 2 49 «7 7 eu oi = eS o eo - q : a - a ' = ws - ' a rs ’ + i - t “I : : = A 5 z Ly % _ t 3 | 294 CLA YPOLE. EXPLANATION OF PLATE XXII. Blastoderm Formation. Fic. 30. Section through unsegmented egg. .2., protoplasmic island in which female pronucleus is present; ¢.f., central mass of protoplasm; *., radial proto- plasmic strands; other letters as before. Borax carmine. Obj. 4, oc. 6. Fic. 31. Section through line Z-Z in Fig. 21. 01, blastomere; ¢., egg membrane; v., vitelline membrane. Iron haematoxylin and Orange G. Obj. 8, oc. 6, tube length 153. Fic. 32. 32-cell stage. c.d., central blastomeres. __ Erlich’s haematoxylin. Obj. 4, oc. 6. Fic. 33. Part of egg after holoblastic cleavage has ceased. 4/., blastomeres from which nuclei surrounded by protoplasm are migrating. Borax carmine. Obj. 4, oc. 6. Fic. 34. Early blastoderm. ec., ectoderm ; #e., mesoderm ; y.m., yolk masses showing earlier position of blastomeres ; o.a., oblique division of ectoderm cells. Erlich’s haematoxylin. Obj. 6, oc. 6. : Fic. 35. Blastoderm formation completed. c.0., beginning of the precepha- lic organ; e#., entoderm cells; y.c., yolk cells; the rest as in Fig. 34. Borax carmine. Obj. 8, oc. 6. Fic. 36. Later stage of blastoderm. Letters as in Fig. 35. Borax carmine. Obj. 8, oc. 6. Fic. 37. Still later stage. Letters as in Fig. 35. Borax carmine. Obj. 4, Ocean. Fic. 38. Precephalic organ at its period of greatest development, blastoderm crenated. ¢;., first crenated membrane; remaining letters as before. Borax car- mine. Obj. 4, oc. I. Fic. 39. Precephalic organ elongated. cz., second crenated membrane; 4., knob where elongation is attached to crenated membrane number 2; remaining letters as above. Borax carmine. Obj. 4, oc. I. Fic. 40. Enlarged view of section of membranes and ectoderm of ovum. #., preparatory membrane; other letters as before. Borax carmine. Obj. 7g, oc. 6. Fic. 41. Entoderm cells showing vesicular protoplasm. Borax carmine. Obj. 2s, Oc. 4. 4 » ? coe Pe 1 ( ar ( = : ’ ur a — noe if 4 \ co - oe i 1 - . — ~) ‘ : t f \ aa ' @& ‘ % 7 ? , ana. « - a o y is é , : 7 = _ hip a i) se : ; : ; 7 P= ' : ‘ Oi xt é = x -” \ 1 o. \ iy - ie ah, mW. forphology Vol. >'D eg \ a) 8) a Vy Ji a - —_ 7 <' oa = J - . a - i 296 CLA VPOLE. EXPLANATION OF PLATE XXIII. Surface Views of Embryos. Erlich’s Haematoxylin. Fic. 40. Ventral view of early embryo. The embryo is rolled over and rep- resented as laid out flat. at. antenna, 7.c., intercalary appendage (2d antenna) ; md., mandibles; mx ;., mx2., maxillae; ¢., Ist thoracic legs; fc.o., precephalic organ. Obj. 8, oc. 4, tube length 154. FIG. 41. fg. ¢3, 2d and 3d thoracic legs; a@;., 1st abdominal appendage ; other letters as above. Obj. 8, oc. 4, tube length 154. Fic. 42. 7d., labrum; a@;.-ads., abdominal segments and beginning of append- ages; pd., proctodaeum; remaining letters as in Fig. 41. Obj. 8, oc. 4, tube length 153. Fic. 43. Face view of later embryo, shown in side view in 43a. Flexure just beginning. .f, mouth fold; m.Z., mouth-parts; z#a., thoracic appendages; c/., collophore ; @2.—a4., appendages onabdomen. Obj. 4, oc. 6, tube length 15}. Fic. 43@. Outline side view of embryo represented in Fig. 43, showing begin- ning of flexure. Obj. 16, oc. 4, tube length 15}. Fic. 44. fc.o., precephalic organ elongating ; 7., last membrane shed ; other letters as above. Obj. 8, oc. 4, tube length 15}. Fic. 45. Flexure almost complete. ¢., eyes; remaining letters as before. ay. is larger than any of other abdominal appendages excepting the collophore. Obj. 8, oc. 4, tube length 15}. Fic. 46. Head of embryo showing the beginning of the mouth fold, m.f. Obj. 8, oc. 4, tube length 15}. Fic. 47. Enlarged head of embryo of same stage as Fig. 42. fc., procere- brum; dc., deutocerebrum ; ¢c., tritocerebrum bearing 2.c., the intercalary ap- pendage. Obj. 8, oc. 4, tube length 15}. Fic. 48. Newly hatched young Anxurida maritima. Letters as before. Rei- chert, obj. 3, oc. I. j Litt Anshy: Werner &Winten Frankfurt at. CLAY POLE, iS) \O oa) EXPLANATION OF PLATE XXIV. Development of the Reproductive Cells. Fic. 49. Transection showing early mesoderm formation. Letters as before. Borax carmine. Obj. 7, oc. 2, tube length 15}. Fic. 50. Transection through somite on one side of body in embryo of age shown in Fig. 44, 2d abd. segment. guc., germcells. Delafield’s haematoxylin. Obj. 75, oc. 6, tube length 15}. Fic. 51. A similar section of same age as in Fig. 50. me.s., mesoblastic so- mite, cavity distinct. Borax carmine. Obj. +44, oc. 6, tube length 153. Fic. 52. Longitudinal section through under part of abdomen of later embryo. mc., beginning of muscles; other letters as before. Delafield’s haematoxylin. Obj. #5, oc. 4, tube length 153. Fic. 53. Longisection through abdomen of stage shown in Fig. 45. sp.me., splanchnic layer of mesoderm; 4@/.c., blood corpuscles ; other letters as above. Delafield’s hematoxylin. Obj. +4, oc. 4, tube length 15}. Fic. 54. Longisection through hinder part of abdomen of embryo in corre- sponding stage (slightly oblique). @z., anus; rest of letters as before. Germ cells are migrating intothe yolk. Delafield’s haematoxylin. Obj. #5, oc. 4, tube length 153. Fic. 55. Longisection through embryo. me., two layers of mesoderm, splanch- nic and somatic. Delafield’s haematoxylin. Obj. ;4;, oc. 6, tube length 15}. Fic. 56. Similar section to Fig. 55. s.g.c., stationary germ cells; m.g.c., migrating germ cells. Delafield’s haematoxylin. Obj. 5, oc. 6, tube length 15}. Fic. 57- Section showing migrated germ cells and scattered degenerating yolk nuclei, y.c. Obj. #;, oc. 4, tube length 15}- nm : - = a - ~ La at = 7 7 i 7 = =. - = oe + 7 ~ 7 : , x a9 i gy - E 7 7 E 1% 2 ’ 4h " = — B! i a ah a a 4 - = S ’ ; ‘| ‘ 7 , ; : a . ‘ 2 - - s F ™ ‘Nims i ait = x t hi a = = — = , ? Me ; . PLAX Journal of Morphology Vol.AM ti AD ay : XS Py @ @ ge () A ; e. ) ee, Py Pe j Oot & EF 20% # © yy eo 9 SS its : Ge Moe e a "Sg @o o 90° me ; oe, a oe” ee —— 8 @ 300 CLAVPOEE: EXPLANATION OF PLATE XXvV. Fic. 58. Longisection through abdomen of last stage of embryo. ev., ento- derm. Other letters as before. Borax carmine and Orange G. Obj. 74, oc. 4- tube length 153. Fics. 59, 60, 62. Cross-sections of ovaries of just-hatched Anurida. y., em- bryonic yolk; rest of letters as before. Borax carmine and Orange G. Obj. qs Oc. 4, tube length 15}. Fic. 61. Cross-section of small animal taken early in the summer. Erlich’s haematoxylin. Obj. 75, oc. 4, tube length 15}. Fic. 63. Blood corpuscles from just-hatched animal containing yolk. Borax carmine. Obj. 74, oc. 6, tube length 153. Fic. 64. Slightly oblique longisection of just-hatched male. 7. longitudinal muscles; fg., fat globules; zzz., intestine; 7.c., opening of reproductive organs; n.c., nerve cord; other letters as before. Enrlich’s haematoxylin. Obj. 8, oc. 4, tube length 152. Fic. 65. Longisection through mid-gut of just-hatched male. ez., entoderm ; y. yolk; cg. cavity of gut; s., sperm cells; g.e., germinal epithelium. Borax carmine and Orange G. Reichert, obj. 75, oc. I. Fic. 66. Frontal view of Petromyzon from a drawing by Dr. W. M. Wheeler. This shows the association of yolk with germinal cells. PUXAXV. Tith, Anst x Werner &Winter, Frankfart3M. z ' 7 f FORMATION OF THE GERM ‘LAYERS ‘IN THE AMPHIPOD MICRODEUTOPUS GRYLLO- FPALPA, COSTA: CLARA LANGENBECK. THE interesting results which Dr. McMurrich obtained from a study of the cytogenesis of the isopods led him to suggest to me that I should undertake the study of amphipod development from the same standpoint. This investigation was begun under Dr. McMurrich’s direc- tion in the summer of 1893, at the Marine Biological Labora- tory, Woods Holl, Mass., was continued there during the two following summers, and was completed during my term of the Biological Fellowship at Bryn Mawr College, in the winter of 1895-96. I wish here to express my thanks to Dr. Mc- Murrich, as well as to Dr. Whitman, Director of the Marine Biological Laboratory, and to Dr. Morgan, Professor of Zoology, Bryn Mawr College, for their kind interest and assistance. Up to the present time observations which have been re- corded upon the segmentation stages of the amphipod ovum have been made only upon the living egg. For a historical sketch of the literature I refer the reader to the Monograph upon the Amphipods, by Della Valle ('93). The only paper upon the subject which has appeared since then was published in December of the same year by Bergh ('93), who calls atten- tion to the interesting rotation of the embryo upon the egg, and points out the necessity of studying the whole cleared egg before sectioning it. The paper does not go into detail, being intended rather as a suggestion for future work than as an exposition of amphipod development. My observations were made upon the egg of a small marine amphipod, Mzcrodeutopus gryllotalpa Costa, which lives in shallow wateramong decaying seaweed. This species is widely distributed, being found on the coast of New England as well 302 LANGENBECK. [VoL. XIV. as on the European coast from Norway to the Mediterranean.! It is most plentiful and accessible, and, as I was able to collect a complete series of embryos, it proved a very favorable species upon which to work. For a description of the animal I refer the reader to Della Valle (93), who places it in family IV, Corophiidae. To this same family belong also Amphitoé and Sunamphitoé, whose development has been described by Mlle. Rossiiskaya (91). Judging from the resemblance of her figures to those I have of Microdeutopus, the modes of development of these species must be almost identical; our interpretations, however, differ widely. The Corophiidae are placed in the sub-order Crevettina together with the Gammaridae and the Orchestiidae (Leunis), families whose development has received the most attention. Microdeutopus itself was studied by Della Valle. In his introduction to the embryology he says he studied not only the eggs of Orchestia and Gammarus, which he figures and describes, but also amphipods of other families, especially Microdeutopus gryllotalpa, as control observations. Della Valle is convinced from his studies that, on the whole, there is no essential difference in the development of these groups. There are many points in his description which I am not able to bring into harmony with what I found for Microdeutopus, but I shall defer the discussion of these points until I have given my own results. Methods. Microdeutopus lives in shallow water among decaying sea- weed. By taking small portions of the seaweed at a time and squeezing them the animals came out of hiding and could be easily caught. They were then placed in glass dishes with fresh salt water, and kept in captivity for several days. It is very difficult to catch the animals with eggs in the early stages, though why this should be so I cannot tell. Quantities of females with the eggs twenty-four hours old could be found 1 Prof. Sidney I. Smith, of Yale University, who very kindly identified the amphipod, made the statement about its distribution in a letter to Dr. McMurrich, to whom I am indebted for the information. No: 2:5 ZICRODEVUTOPOS (GRVELOTALPA COSTA. 303 each day; but, although the seaweed was carefully searched, the number of eggs in the segmentation stages found in the material brought in on one day was out of all proportion to the number of eggs twenty-four hours old found in the material brought in the next day from the same place. The animals do not hide in the mud during the.early period of development of their eggs, because the bottom of the pool in which they were col- lected was composed of a black refuse, which gave off so much marsh gas that the animals could not live in it. By carefully watching those kept in captivity, I observed that when there was a moulted amphipod shell floating at the surface of the water, an animal which had just deposited its eggs was almost always to be found in the dish, and in this way the early stages were obtained. The females were caught and firmly held with a forceps while the eggs were removed from the brood pouch with a dis- secting needle. The eggs were then killed in a modification of Kleinenberg’s picro-sulphuric solution, in which sea water was substituted for the ordinary distilled water. This solution gave better results than the ordinary Kleinenberg killing fluid, _which distends the egg. Corrosive sublimate, as suggested by Della Valle, also distends the egg and injures the protoplasmic structure. The living egg contains a fluid substance which exudes into the space between the surface of the egg and the chorion as soon as the egg is killed. This fluid substance coagulates and is stained by haematoxylin, the stain, however, being extracted by the acid alcohol before the protoplasm is decolorized. I could find no killing fluid which would prevent this exudation. Hot corrosive sublimate, Perenyi’s, Flemming’s, and Kleinenberg’s fluids, alcoholic picro-sulphuric acid, and hot water all affected the egg, in this respect, in the same way. With the modified Kleinenberg solution the eggs shrink con- siderably, but the parts are not distorted, and good, clear nuclear figures were always obtained. The protoplasm showed no ab- normal vacuolization, such as occurred when corrosive subli- mate was used. The chorion closely invests the fresh egg, but in the killed specimen there is a large space between it and the surface of 304 LANGENBECK. [VoL. XIV. the egg. It was, therefore, found advisable to dissect off the chorion, since it collapses when the egg is placed in oil of cloves, and the resulting folds in it are easily mistaken for cleavage furrows. The eggs were overstained in Kleinenberg’s or Delafield’s haematoxylin, washed out with acid alcohol, dehydrated, cleared in oil of cloves, and mounted under a cover slip supported by wax feet. By pushing the cover slip from side to side the eggs could be rolled into any desired position while they were being studied. Mlle. Rossiiskaya attempted to work upon the whole egg viewed as a transparent object, but says that she met with no success. I found that, unless a strong condensing lens is used, it is, as Mlle. Rossiiskaya ('88) says, almost impossible to distinguish the cellular structure, but with the condensing lens the cells can be distinctly and clearly seen. These same eggs which had been studied zz toto were then imbedded in paraffin and sectioned. It was not possible to cut the seg- mentation and early blastoderm stages thinner than 10 y, be- cause the protoplasm at this time is so distributed that it does not seem to offer sufficient support to the yolk. Sections of the later stages were cut 5 u thick, and preferably stained upon the slide. Eggs of the third day and after, when the yolk was partly digested, were stained with a %% aqueous solution of haematoxylin and washed out in iron alum, according to the usual iron-alum method. This stain could not be used for the earlier stages, because the undigested yolk becomes very black and totally obscures the structure of the egg. I tried to orient the eggs for sectioning according to Patten’s method. They were so small, however, that the least amount of celloidin which would hold them to the paper formed a coat over the eggs so that the paraffin did not penetrate. Knowing the relative position of the dorsal organ with respect to the rest of the embryo, from a study of the cleared egg, it was not difficult to orient sections. Now2.))) JICRODEUTOPUS GAYELOTALPA COSTA. 305 Segmentation. A complete account of the cell genesis of the amphipod egg has never been published. The segmentation has been followed only on the living egg. The most complete account was pub- lished by Van Beneden and Bessels in 1869, who carry their observations up to the time when the blastoderm begins to appear upon the surface of the egg. Other authors merely state that the segmentation is total; that the third cleavage plane divides the egg unequally; that after the 32-cell stage the segmentation becomes irregular; that just before the blasto- derm appears on the surface of the egg the difference in size between the micromeres and macromeres is lost; and that the protoplasm rises to the surface and the cells migrate toward the micromere pole to form the blastoderm. In the present work the segmentation was followed on the living egg as far as the 8o-cell stage, and eggs in all stages of development were also studied as transparent objects. The drawings were all made from stained and cleared eggs, which were afterwards imbedded in paraffin, cut as described above, and studied in section. I have never seen the process of fertilization in Microdeuto- pus, but I have caught many pairs of a closely allied amphi- pod in the act of copulation. In these forms the male rests upon the dorsum of the female, clasping her with the large chelae. He probably assists the female to slough. Some- times the sloughing takes place soon after the animals have come together, and sometimes I have seen them united for days before the female sloughed. Shortly before she sheds her shell the male leaves her, and as soon as it is shed he returns, and the animals unite in the same way that they did before the sloughing took place. They remain together a short time and then separate, and shortly after the male has left for the second time the eggs are extruded. I do not know how nearly the process of fertilization in Microdeutopus agrees with what I have observed for the other amphipod, but a female of Microdeutopus which had not sloughed, but whose eggs were just in a condition to be extruded, was isolated. After a time she sloughed, and the eggs were extruded in the 306 LANGENBECK. [VoL. XIV. normal way; but the eggs, although apparently quite normal, evidently were not fertilized, because they did not segment. I concluded, therefore, that in Microdeutopus, as in the other amphipod, fertilization takes place between the time of slough- ing and the time of extrusion of the eggs. It was also ob- served that when a moulted amphipod shell was found floating at the surface of the water a female which had lately extruded her eggs was almost always in the dish. When the eggs are first extruded into the brood pouch they are of a bright opaque green. The chorion closely invests the egg, but no other membrane could be seen either in the fresh specimen or in the sections. The eggs seem to be covered with some sticky substance, which causes those coming from a single ovary to cling to one another; but the groups of eggs from the two ovaries are separate. This substance is subse- quently either absorbed or loses its sticky properties, because after the first cleavage the eggs separate readily as soon as they are removed from the brood pouch. The protoplasm is found at the center of the egg. It is irregular in outline, sending out long pseudopodia-like prolongations, which ramify throughout the egg, very much as Dr. McMurrich ('93) has shown to be the case in the egg of Jaera. No protoplasmic layer could be seen around the periphery of the egg, however; if it is there, it must be very thin. Fig. 19, which represents an egg passing from the 2-cell into the 4-cell stage, shows the manner in which the protoplasm ramifies throughout the yolk mass. The nucleus is found in the center of the protoplasmic area. The segmentation in the early stages is total, but not equal; later it is superficial. The protoplasm loses its control over the inner ends of the blastomeres as it moves nearer the sur- face in the succeeding divisions, and the inner ends of the blastomeres fuse, so that the blastocoel, which is at first present, is obliterated. 2-cell stage. — About three hours after the eggs have been extruded into the brood pouch, the protoplasm in the center of the egg divides, the nuclear spindle lying in the long axis. After the two halves of the central protoplasm have separated a furrow appears at the surface of the egg and gradually deepens, No2z:) J77CRODEUTOPUS GRYLLOTALPA COSTA. 307 dividing the egg into two equal parts. The two blastomeres then flatten against each other, and the living egg presents the same form as it did before cleavage. I have never seen the blastomeres unequal in size at this stage, as Van Beneden and Bessels and Della Valle describe for the gammarids. 4-cell stage. — One hour elapses before the completion of the second division. Fig. 19 represents an optical section of an egg which was killed half an hour after the first division. The proto- plasm has almost divided and the second cleavage furrow has begun to appear. This cleavage plane makes an acute angle with the first plane, giving rise to two small and two large blasto- meres, the smaller blastomeres being even less than one-half the size of the larger ones, as shown in Fig. 1. When the egg comes to rest the two large cells flatten against each other, pushing the smaller ones apart in such a way that one lies above and the other below the plane of the equator, this plane being sup- posed to pass through the long axis of the egg and at right angles to the first and second cleavage plane; z.¢., in Fig. 1 it lies in the plane of the paper. No rotation of the blastomeres, as described by Wagner for Melita ('91), has ever been observed; the blastomeres always flatten against one another without changing their position. I always find, at this stage, two cells smaller than the other two, and not three of the same size and one somewhat smaller, as in the gammarids described by Van Beneden and Bessels ('69) and Della Valle ('93). Sometimes one of the two smaller blastomeres is larger than the other small one, but never as large as the large ones. For convenience in describing the later stages, I shall name the larger cells AS and CD, the small cell above the equator EF, and the remaining one GAH. S-cell stage. — At the end of the fifth hour an equatorial furrow divides the egg into four micromeres and four macro- meres (Fig. 2). The four micromeres bear to each other the same relation in size as the four macromeres, and the larger micromeres are smaller than the smaller macromeres. When the egg comes to rest the two large macromeres flatten against each other, and likewise the two large micromeres, while the two small macromeres and the two small micromeres are forced 308 LANGENBECK. [VoL. XIV. apart. The larger micromeres stand just above the larger macromeres, and the same holds true for the smaller micro- meres and macromeres. In the figures the macromeres and their descendants will be designated by the large letters, and the micromeres and their descendants by the small letters. The macromeres AB, CD, and EF and their descendants are drawn in red ink, while GH and the micromeres are in black. From this time the macromeres always divide before the micromeres, and the larger macromeres before the smaller ones. The subsequent cleavage planes which divide the two larger macromeres are alternately meridional and equatorial, while the planes dividing HF and the micromeres are always meridi- onal. This may be due to mechanical causes; since these blastomeres, lying upon the larger ones, are somewhat flat- tened, as shown in Fig. 25, the spindle would find more room in the horizontal than in the vertical plane. 16-cell stage. — After the sixth hour two vertical cleavages atmeht aneles to each other @ive rise tow, 2 .C, Dawa, Ff, a, 6; \6, die; J, & 2k (Pigs. 3,\4)) > When! the’ egs comes to rest A and C flatten against each other, but B and D are forced apart by G and A (Fig. 3). It is to be noticed that the zone of small cells lies obliquely over the oval egg. Mlle. Wagner ('91) finds the same oblique zone of small cells in Melita, but the obliquity was, in that form, brought about by a rotation of the cells in the 4-cell stage. Her account agrees in part with the results of Van Beneden and Bessels (69), whose Figs. 10, 11, and 12 show exactly the same oblique arrangement. In the text Van Beneden and Bessels make no mention of this oblique zone of cells, but in their Fig. 9, which represents the 8-cell stage, the cells are arranged symmetrically with refer- ence to the long axis of the egg, while in their Fig. 10, where the macromeres are just beginning to pass into the 16-cell stage, the oblique position of the micromeres is manifest; therefore, it would seem that a rotation occurred just at this time. In Microdeutopus the obliquity is occasioned by the angle which the second cleavage plane makes with the first (Fig. 19). Now2.)) A77CRODEUTOPRCS (GRYLEOTALPA COSTA. 309 22-24-cell stage. — Figs. 5-8 show four views of an egg of the 22-cell stage, Fig. 5 representing the macromere pole of the egg. In this stage A, Bb, C, D, G, and # have given rise to At, A’, B, B*, etc., by an equatorial cleavage (Fig. 8), while £ and Fare still in the act of dividing and the micromeres show no trace of division. jo-cell stage. — Figs. 22-24 show three views of an egg of thirty cells passing into the 42-cell stage. The cells E and & have sgiven Tse: to 22) 227A, and) 7777(fic. 22) ihe cells'a and c, 6 and d@ have all divided, forming a’-a%, cc’, and 6°62, a*—d’, while g@ and h*, x and y are the descendants of g and Z%. Instead of the division being by an equatorial plane in these cells, as it is in the macromeres, and as Van Beneden and Bessels ('69), Rossiiskaya ('90), Della Valle (93), and Ulianin (81) found it for the micromeres of other amphipods, it is vertical and at right angles to the last plane of division of these cells, which also was vertical (Figs. 3, 4). That the divi- sion of the micromeres at this time is vertical and not equa- torial in Microdeutopus is shown by the spindles in g and / (Fig.11). I havealso seen spindles in 6 and d@ lying in the equa- torial plane and at right angles to those shown in g and 4; z.2., in the direction of the arrows in the cells 4 and d (Fig. 11). Figs: 11,02 show the protoplasm in A, .A% 52 Bb C+, C42. D divided and the yolk deeply constricted; the nuclei of G, ff in the aster, and of g, / in the diaster stage. In Figs. 24, 25 G’, 77/2 are beginning to divide; in. G?; 27 the process is further advanced, and g and % have divided completely. The daughter cells of g and ZI shall name g*, h*, x, and y, the x, y cells being those lying next to 6°-6%, d*-d’. These are always present before G*, H/ have divided, although e and 7, the cells corresponding to g and 4, upon the other side, show no trace of division until a much later period (about the 72-cell stage). It is interesting to note that the cells of the A/ and ef groups divide later than those of the GH and gh groups. It may be that the ZF cell corresponds to the smaller cell which Della Valle ('93) found to result from the second division in Orchestia. He describes this cell as lagging behind the others in development. 310 LANGENBECK. [VoL. XIV. At this stage x and y appear smaller and at a lower level in surface view, as though they were being pushed inward (Fig. 13). Sections just after this time (Figs. 9, 10) show forty-three cells arranged around a blastocoel, within which, at one end, lie two cells x’, y’ (Fig. 9). The nucleus in one of these cells may still be seen (Fig. 9); in the other it is no longer present. I have also sections of one egg (Fig. 20) showing forty-one cells arranged around a blastocoel, and two of the cells have spindles whose equatorial planes are parallel to the surface of the egg. (Only one of these is shown in the figure.) The question now arises, Do the two cells which are, after this stage, ALWAYS found in the center of the egg, go in by a division parallel to the sur- face, or are they x and y which have been pushed in during the later division of the other cells? Unfortunately, I was not able to tell whether the two cells dividing inwards (Fig. 20) were the cells g, # or not. Although I have cut a number of eggs passing from the 32-cell into the 42-cell stage, I have never seen spindles directed radially when x and y were present. I hoped by tracing the cells into the next division to determine whether x and y were pushed in; if these two-cells in that place were wanting, they might be accounted for in this way. I did not succeed, however, because the descendants of G4, A, and G*, H® take such different positions that it is impossible to be sure of the following generation. For instance, in one egg the arrangement was Ge He G* He G® G> H*® He as shown in Fig. 23. In another Ge Ge He He G*® G> He H* while in a third it was G* FH G* H# G> Hf? G*® H® Nor 24) MICRODECTOP OS GRYELOTALPA COSTA. 311 so that in the next stage I was not able to decide from their posi- tion whether g?, 4* had divided again or whether certain of the other cells had divided. I did find, however, one egg with about sixty-four cells, in which the two cells next to 4°-0%, d*—d* were as large as g and / are before dividing. The arrangement of the other cells is diagrammatically sketched in the cut. It was neces- sary to make a diagram, because all the cells could not be seen in the same field of view. In this one egg I did not find the two cells in the center. I cut sections to see if I could find spindles going in, but the critical section was completely broken. I know there were no cells in the interior for the reason that when they are present they can always be distinguished in optical section in the whole cleared egg. After this time the two cells in the interior are found in various stages of disintegration. As late as the 112-cell stage, after the blastoderm has appeared on the surface of the egg, two deeply stained patches can still be seen in the interior. Shortly after this stage they disappear altogether, and no other cells are seen in the yolk until the egg is about forty-eight hours old (Fig. 39). Weismann and Ischikawa (87) have described three secondary polar bodies in Bythotrephes longi- manus, which are carried into the interior of the egg during the early segmentation stages. These polar bodies subsequently disintegrate, though as late as the 32-cell stage remnants of them could still be detected in the axial space between the blastomeres. Dr. Mead (95) also has found that the polar bodies of Amphitrite are taken into the axial cells, where they are absorbed. These results of Weismann and Ischikawa and of Mead led me to suppose that the two cells found in the interior of the Microdeutopus egg were polar bodies. I there- fore made a careful study of the eggs before the 32-cell stage. Since I found no cells which had not been derived from one of the two blastomeres of the 2-cell stage, I conclude that the cells in the interior cannot be polar bodies. In the literature which 312 LANGENBECK. [VoL. XIV. I have seen I have found nothing to which I might compare these two cells. Dr. McMurrich, however, found two cells in the interior of an advanced isopod egg; but as they were seen only once, and no traces of disintegrating nuclei were found in the later stages, Dr. McMurrich supposed it to be an abnor- mality. It may be, however, as Dr. McMurrich suggested to me, that the two cells found in the interior of the isopod egg are comparable to those which I have described for Microdeu- topus. Dr. Conklin has kindly permitted me to state that he, too, finds that two cells (the tip cells in one arm of the cross) in Crepidula are lost in the later stages. However, they are not, as I understand, absorbed, but thrown out. It would be interesting to know if these cells in Crepidula could be com- pared to those in Microdeutopus. Comparisons have been made between the amphipod and molluscs before (Ulianin 'g1), with how much right is still to be decided. But certainly the loss at an early stage of two blastomeres is very remarkable, and has as yet not been described for other forms. 44-cell stage. — The blastomeres £%, F* divide, giving rise to £*, F and E”, #2, The cleavage is parallel to the planes which divided £ and F before. Fig. 21 shows an egg passing from the 44-cell stage into the 46-cell stage. The blastomeres E~ and F” are dividing for the last time before they rise to the surface of the egg. 73-cell stage. — Figs. 14-16 represent three views of an egg of seventy-three cells. In this ege 2°5 £21) hor) 8%, and F® have all been divided by vertical planes at right angles to the last, and the micromere f has also divided in the same way. The protoplasm in these cells has come up to the surface, and the nuclei have become very large and clear. This is the first appearance of the blastoderm. The sixteen large macromeres have divided into thirty-two by an equatorial cleavage. aa’, cc’, and 6’-6+, d*-d* have not changed, and there are seventeen cells derived from the gi and GH groups. The descendants of A and C lie next to each other and border upon the EF group, while the B and D groups are forced apart by the GH group. Sections (Fig. 26) show the protoplasm quite near the surface at this time, the cell boundaries breaking down at their Nov2.) AICRODEUVUTOPUS, GRYELOTALPA COSTA. 313 inner ends, and the blastocoel becoming obliterated. The sec- tion shows one of the two cells which have wandered in. Its nucleus appears as a dark, homogeneous mass, showing signs of disintegration. 100-cell stage. — The thirty-two large cells all divide once more, by vertical cleavages, into sixty-four. I should state here that, although I have always spoken of the descendants of the four large macromeres of the 16-cell stage as large, I have done so merely to distinguish them from the descendants of the small macromeres. Somewhat before the 73-cell stage, owing to their more rapid cell division, the difference in size has disappeared, and in the living egg, as stated by Mlles. Pereyas- lawzewa ('88), Rossiiskaya ('88), and Wagner (91), the macro- meres and micromeres cannot be distinguished. In the cleared egg they are recognized by the different appearance of their nuclei and the smaller amount of protoplasm contained in the micromeres. It will be recalled that in the 16-cell stage A and C were flattened against each other, while 6 and PD were forced apart by G and H. Because of the obliquity of the second cleavage furrow and the flattening of the larger macromeres against each other, the group of small cells lies obliquely on the oval egg, as shown in the diagram. Since, as_ stated before, no change in the position of the cells takes place, their descendants have exactly the same position as the cells themselves originally had. So the AB group lies over the pole near- est the EF group, while the CD group lies over the opposite pole, and the A and C groups lie near- est each other and border the ZF group. At this time the protoplasm of all the blastomeres rises to the surface (Fig. 17). The protoplasm of the macromeres becomes more con- centrated, while that of the micromeres spreads over the yolk, thus making such a thin layer that only the dark nuclei remain visible (Fig. 18). Since the concentration of the 314 LANGENBECK. [Vou. XIV. protoplasm of the macromeres is toward the £F group, the cells of the AB group, as can be seen by the diagram, would come to lie over the lower pole, whereas the cells of the CD group would be drawn away from the upper pole and lie on the side of the egg, and the whole ventral plate so formed would lie eccentrically over the oval egg. Figs. 17 and 18 show an egg at this stage. The eleven cells at EF are the descendants of the ZF group, the twelfth cell of this group lying beneath the surface, as was shown by sections. The end cells of the CD group can be seen in Fig. 18 on the side of the egg, while the end cells of the AB group cover the lower pole. The cells of the EF group will form the head region and the dorsal organ. Bessels ('70) is inclined to believe that the dorsal organ arises exactly at the same point where the blastoderm first appears on the surface of the egg, but, according to my observations, it does not appear exactly at this point, but a little lower down, as the cells of the ZF group, which are the first blastodermic cells to appear, spread during their growth. In sections of an egg at this stage cells are seen underneath the surface in the ZF region, and I think these are the descendants of ef, because the sections of the egg represented in Fig. 17 show five cells beneath the surface in this region. One of these is, I think, the twelfth cell of the ZF group, as only eleven were seen on the surface. It is only in this way that the ef group can be accounted for. It will be remembered that only four cells were derived from this group. Another reason which led me to this conclusion is that all the other micromere cells are overgrown as the embryo develops and come to lie in the lower layer. Summary. The first cleavage plane appears three hours after the deposi- tion of the egg. The three succeeding divisions, vertical, equa- torial, and vertical, occur at intervals of an hour each, and after this the large macromeres divide synchronously and regularly, a vertical cleavage alternating with an equatorial cleavage; but the micromeres no longer divide synchronously with the macromeres. NOxzZziW i ChRODE OCTOPUS GRYELLOTALPA COSTA, 315 The £F group and the micromeres divide only vertically. The ventral plate is formed by the descendants of the large macromeres and of the £F group, and has an oblique position upon the egg, owing to the obliquity of the second cleavage plane. After the 42-cell stage two cells are found in the interior of the egg in different stages of disintegration. Formation of the Embryo. In the last section I have described the ventral plate as being formed by the macromeres on the lower pole of the egg. I am well aware that in this I stand alone, all previous writers describing the descendants of the micromeres as the first which rise to the surface. Further, they describe the embryo as forming over the micromere pole, and the macromeres as gradually added to the outer layer during the growth of the ventral plate over the egg. Della Valle, who studied Microdeu- topus as a control observation, agrees in this point with what has heretofore been published. He describes the blastoderm as arising on the micromere pole in Orchestia, and makes no ex- ception in the case of Microdeutopus. The results which Mlle. Wagner obtained for Melita agree most nearly with what I found for Microdeutopus. According to her account, when the cells emerge from the yolk they lie on the sides as well as on the oral pole of the egg, and later grow over the dorsal face. This corresponds almost exactly to what I have found. The figures 4, 5, and 6 of Sunamphitoé, by Mlle. Rossiiskaya, are almost identical to those I have for sections of eggs in the stage figured on Pl. XXVI, Fig. 17. Her figures show that the blastoderm probably arises in the same way as in Microdeutopus. The EF group lies above the plane of the equator on the side of the micromeres, and the cells of this group are the first which appear on the surface to form the blastoderm. Previous investigators may have been led by this to suppose that the ventral plate forms on the micromere pole, but, having traced the development cell by cell on the stained and cleared egg, I 316 LANGENBECK. [Vou. XIV. am convinced that the ventral plate ts formed from the descend- ants of the macromeres and over the macromere pole. The pole upon which the ventral plate is formed will become, as in all other Crustacea, the ventral side of the embryo. The cells after reaching the surface rapidly increase in number, ‘and as the embryo grows backward over the egg it shifts its position, so that its long axis finally corresponds with the long axis of the egg. In Fig. 29 we can still see that one side of the ventral plate is a little nearer the posterior pole than the other side, the shifting at this time not having been completed. Fig. 35 shows an egg in which, for some reason, the shifting has not taken place, so that at this late stage the embryo is still oblique upon the egg. In Fig. 33, a stage which is a little later than that shown in Fig. 29, the ventral plate has grown almost over the posterior pole (Fig. 32), and the axis of the embryo lies parallel to the long axis of the egg. The rotation of the embryo upon the egg has been described by Bergh (93) for Gammarus pulex, in which the embryo lies at first parallel to the shorter axis of the egg, and rotates during development through an angle of 90°. Bergh said he could not explain why it arose in that position. Della Valle also figures the embryo as lying at first obliquely over the egg, then parallel to the short axis, and, lastly, parallel to the long axis. I could not make out, however, whether he sup- posed a rotation to take place, or whether he considered the short axis to be drawn out at the expense of the long axis. In Microdeutopus we have seen that the cause of the oblique position of the embryo upon the egg is due to the obliquity of the second cleavage plane. In the stages represented in Figs. 33 and 34 the outlines of the cells are quite sharply marked off from the yolk. Their arrangement in definite lines is partly due to the fact that the blastodermic cells appear on the surface of the egg arranged in definite rows, on account of the regular cleavage of the two large macromeres. Yet this cause cannot hold good for the sides of the embryo, for in Fig. 28, the side view of an egg shown in Fig. 29, the spindles lie at all sorts of angles to each other, and the cells have at this time no definite arrangement, Nooza) Ma(CRODEUTOPUS (GRVELOTALPA COSTA. 317 although later they fall into line. In the isopods and in Mysis teloblasts give rise to regular rows of cells in the postnaupliar region. In Microdeutopus, however, I could find neither ecto- dermal nor mesodermal teloblasts. Bergh supposed that the regular arrangement of ectoderm cells, which is found in the amphipods, arose from some indistinguishable teloblasts, but I think this assumption is not necessary for Microdeutopus, since the regular arrangement is found also in the naupliar region (Figs. 27 and 31), where no teloblastic growth occurs. The dark patches (d@) in Figs. 28, 29, 32, and 33 represent cells below the surface, which, I think, have been overgrown by the ventral plate as it extends over the egg. In the isopods, as described by Dr. McMurrich, and in Mysis, as described by Bergh, the cells scattered over the dorsal pole are added to the ectodermal layer of the ventral plate as it grows over the egg, and with these facts in mind I searched carefully to see if this was the case with Microdeutopus. Further and careful exami- nation only strengthened the view that in Microdeutopus the dorsal cells are overgrown and so form part of the lower layer. At a little later stage (Fig. 34) the head region has increased in extent, a few ectodermal cells are beginning to differentiate at the sides, and the edge of the ventral plate is even more sharply marked off from the yolk than in the preceding stages. In the next stage (Fig. 30) the patch of dark cells has become very characteristic in appearance. The dorsal organ (d.0.) has begun to differentiate, and the outline of the ventral plate is no longer distinct. It appears as though a layer of protoplasm had spread over the yolk, and through this layer the nuclei are found irregularly scattered. This appearance is due, I think, to the lack of definite cell walls, for I have never been able to distinguish them in Microdeutopus. In the earlier stages the cells seem to be “raumlich centriert,’ as Flemming expresses it; therefore, in eggs of stages represented in Figs. 29, 33, and 34, the cell boundaries can be distinguished, but in the later stages, where the cells become closely packed, cell outlines are lost. In eggs after the stage represented in Fig. 34, it seems as though the cells at the edge of the ventral plate have no longer the power to assume the spherical form, so making 31 8 LANGENBECK. [VoL. XIV. the outline of the cell distinct, but their protoplasm spreads out over the surface of the yolk, as seen in section (Fig. 47, ppl... As the cells in this region multiply, the protoplasm of one cell fuses with that of the surrounding cells (Fig. 47, v.), so that it appears as though a layer of protoplasm, through which the nuclei are scattered, was spreading over the surface of the egg, as shown in Fig. 30. The large cell (Fig. 30, @') near the region of the differentiated cells lies below the two small nuclei which are imbedded in the protoplasmic layer. The d! cell is almost always seen in this position, even before the ventral plate has reached that point (Fig. 34). In this case, therefore, it is unmistakably one of the dorsal cells which has been overgrown. As the embryo develops, the cells which are spreading dor- sally arrange themselves in definite rows corresponding to the rows of cells of the ventral plate. During the second day the yolk is completely overgrown by the ventral plate, the edges of which meet at the dorsal organ. At this stage the dorsal organ has moved to the center of the dorsal pole of the egg, and is composed of large triangular cells, whose apices are at the surface of the egg; the antennules are clearly de- fined, and the appendages could be seen just beginning to form. Fig. 31 represents an egg of the second day. The appendages have appeared asa series of ridges on the ventral surface, gradu- ally shading off dorsally, and it will be observed that the series extends over the posterior pole and dorsal surface of the egg, reaching almost to the dorsal organ (Fig. 36). The appendages are formed by the pinching off of from eight to ten parallel rows | of cells, and while being pinched off each ridge includes some of the cells of the lower layer from which the mesoderm of the appendage develops. This mode of origin of the limbs and their musculature recalls what has been figured by Dr. Bumpus (91) for Homarus. The embryo now elongates, the area of growth being chiefly in the dorsal region, posterior to the dorsal organ, as shown in Figs. 36-43. By comparing these figures it may be seen that the distance between the dorsal organ and the last appendage increases in extent, in consequence of which a fold appears on Nor2))) 227CRODEUTOPUS (GRYLLOTALPA COSTA. 319 the ventral surface (Fig. 38, add.) in the region of the first abdominal appendage. Figs. 40-42 are three views of the same egg. At this stage the appendages no longer extend over the dorsal surface (cf. Figs. 36 and 40), although they still cover the lower pole of the egg (Fig. 41). The anlagen of the seven abdominal appendages are pushed inwards, and finally all lie in the abdominal fold (Fig. 43), whereas the space between the dorsal organ and the last appendage extends over the whole lower hemisphere of the egg. The mode of formation of the abdominal fold in Microdeuto- pus differs widely from that in Orchestia, judging from the comparison of Figs. 36-43 for Microdeutopus with those of Orchestia, as figured by Della Valle (93). Entoderm. The entoderm in Microdeutopus arises as a true invagination at the hind end of the embryo. During the second day, when the ventral plate has completely overgrown the egg, the cells immediately behind the dorsal organ invaginate. These cells migrate into the interior of the egg, and there arrange them- selves to form the liver tubes and the greater part of the digest- ive tract. The entodermal invagination is shown in optical section in Fig. 37 (ez. zv.), and Fig. 53 represents a trans- verse section passing through the center of the entodermal sac, while in Fig. 54, which represents a small portion of a sagittal section, the relative position of the entodermal invagination and the dorsal organ are shown. In this egg the cells of the dorsal organ have the characteristic bottle shape which is peculiar to them, but the ends of the cells still contain granu- lar protoplasm; later they are filled with some clear, unstain- able substance. . A section of an egg a little older (Fig. 55) shows the cells of the entodermal sac migrating into the interior as an irregu- lar mass. These cells distribute themselves throughout the yolk area, the greater number, however, remaining in the region at which they entered. These are the first cells which appear in the center of the egg, excepting the two blastomeres 320 LANGENBECK. [VoL. XIV. described above, which were absorbed. In eggs at about the stage represented in Fig. 33 I have seen cells in the anterior part of the head region which were apparently migrating off into the yolk area, as the one shown in Fig. 49, but these lay very near the surface, and in the stages shortly before the entodermal invagination had appeared cells were never seen in the yolk area. I conclude, therefore, that these were probably dorsal cells which were overgrown, and had not taken their final posi- tion at the time when the egg was killed. Cells which appear to be similar to these were described by Dr. Pereyaslawzewa ('88), who considers them to be entoderm cells derived from the ventral plate and migrating into the yolk area. Ido not be- lieve, however, that in Microdeutopus they take any part in the formation of the entoderm. Ulianin (81) finds a number of cells scattered through the yolk area which he supposes are derived from the dorsal organ, as at first they are found in that region. He leaves the origin of these cells an open question, however, as he was not able to secure a complete series of embryos, and, therefore, could not trace them to their source. The whole entoderm, according to Ulianin, is derived from these cells, and the lower layer cells of the ventral plate form mesoderm only. Dr. Pereyas- lawzewa ('88) believes that the entoderm is derived from two sources, from the ventral plate and from the dorsal invagina- tion, which she considers to be the dorsal organ, as the follow- ing passage states: “ A mesure que l’organe dorsal se développe, l’ectoderme avoisinant s’épaissit visiblement et garde pour longtemps cette configuration, vu qu'il ne détache aucun organe nouveau. Ce rdle passif qui lui est propre, me permet de le comparer a la plaque dorsal chez les Insectes. La dis- semblance consiste en ce que chez ces derniers la formation de la plaque précéde celle du tube, tandisque chez les Crusta- cés nous remarquons le contraire. D’aprés les recherches de M. Korotneff sur le développement de Gryllotalpa les cellules qui dérivent en grande nombre de la plaque dorsal s’introdui- sent dans les masses nutritives et apres les avoir élaboré de maniére a les préparer pour l’assimilation, qui aura lieu dans les cellules de l’intestine, elles se détruisent completement. No. 2.] MICRODEUTOPUS GRYLLOTALPA COSTA. 321 Il est indubitable que chez les Gammarus et de plus chez les Orchesties l’organ dorsal, ainsi que l’ectoderme avoisinant, détachent de cellules; leur nombre n’est pas grand, elles sont tout a fait liberées et s’enfoncent dans le vitellus nutritif. Or, tandisque les cellules en question se logent dans les masses vitellines, les cellules entodérmiques, d’une parfaite ressemblance avec les premiéres, sont aussi en voie d’y chevaucher; leur résidence simultanée dans le vitellus ne nous permet d’affir- mer aucunement que les cellules issues de la plaque dorsale s’atrophient; aucune de mes préparations ne le prouve pas.” I think the plaque of cells from which, Dr. Pereyaslawzewa writes, no organ is derived is the dorsal organ, and what she calls the dorsal organ is the entodermal invagination. At a later stage a cavity is found in the dorsal organ in Microdeuto- pus which agrees, in this respect, with what Korotneff found in Gryllotalpa. The characteristic appearance of the dorsal organ cells led me to recognize the plaque of cells as the dorsal organ. Dr. Pereyaslawzewa, it would seem, occupies the middle ground between Ulianin (81), who supposes the entoderm is derived from the dorsal organ alone, and Bergh, who derives all the entoderm from the ventral plate. Bergh does not hold that the entoderm is formed at many points on the ventral plate, as Dr. Pereyaslawzewa holds, to judge from her figures, but he supposes that “vielmehr entsteht dasselbe durch Einwuch- erung von Blastodermzellen an einer bestimmten Stelle die also dem Blastoporus entsprechen diirfte.” In his figures Bergh marks the cells in the second layer of the ventral plate, in the naupliar region, ez. My own results agree with those of Ulianin, who believes that a// the cells in the second layer of the ventral plate give rise to mesoderm, and that oly the cells carried in by the dorsal invagination form the entoderm. It would seem, then, that as far as the entoderm is concerned the amphipod agrees most nearly with what Bobretzsky has found to be the case in Palaemon, according to the statement of Heider. B22 LANGENBECK. [VoL. XIV. Mesoderm. At the time when the entodermal invagination takes place the mesoderm is completely laid down and the appendages have begun to pinch off, each ridge containing mesoderm cells, as described above and figufed in Figs. 36, 37, and 55. To judge from what takes place in the great majority of other Crustacea heretofore described, we should naturally ex- pect to find the mesoderm arising from the region where the anterior lip of the entodermal invagination will be formed. In Microdeutopus, then, the extreme posterior end of the ventral plate would be the region where we should look for a prolifera- tion of mesoderm cells, since that is the region of the ento- dermal invagination. However, the posterior end of the ventral plate is exactly the region where the smallest number of lower layer cells are found; in fact, except for the few dorsal cells which were overgrown, that region is composed of only one cellular layer, whereas the head region at the time is composed of two or even three layers, and patches of cells are found irregularly scattered under the ventral plate, decreasing in size and number as they approach the posterior end. When the protoplasm rises to the surface of the egg to form the blastoderm the four cells of the ef group were found under the cells of the EF group. The dorsal cells, which are the descendants of the remaining micromeres, are over- grown by the ventral plate, and, subsequently, form part of the lower layer of cells. In Fig. 18 only the dark nuclei of the dorsal cells can be seen, their protoplasm being spread over a large surface, making such a thin layer that it cannot be dis- tinguished. As the ventral plate grows over the egg the proto- plasm of the dorsal cells concentrates around their nuclei, and the cells present the stellate appearance shown in Figs. 27 and 34. Fig. 46 shows a section of the head region of an embryo at the stage shown in Fig. 30. A large cell (d) is seen just below the edge of the ventral plate. The beginning of the yolk area is shown at Y, and this area was greater in extent in the next section, showing that the cell above the large stel- late one is at the edge of the ventral plate. Fig. 48 also shows No: 24) (-A7IERODE OCTOPUS, GRYELOTALPA, COSTA. 323 a large, clear nucleus (@) below the outer edge of the ventral plate, while in Fig. 47 the protoplasm (f/.) of a cell also at the edge of the ventral plate has spread out over the surface of the yolk for some distance, anda large stellate cell (¢) is seen beneath it. Ido not think that these cells arise by division from the ventral plate, as Della Valle ('93) supposes. I draw this con- clusion because there is no cell above the one shown in Fig. 36 from which it could divide, and especially because these cells at this stage are very different from those of the ventral plate, and their nuclei are similar to those of the dorsal cells. It may be that they are analogous to the vitellophags of the isopods, which arise from the PD cell, described by Dr. Mc- Murrich.‘ The dorsal cells of Microdeutopus and the vitello- phags of Jaera resemble each other in three points: in that they appear during the segmentation stage, in that their nuclei have a characteristic appearance, and in that they are over- grown by the ventral plate. The vitellophags in Jaera also give rise to mesoderm. The dorsal cells in Microdeutopus, however, take no part in the digestion of the yolk. After the ventral plate has completely inclosed the egg the dorsal cells have lost their characteristic appearance, and they cannot be distinguished from the other cells of the second layer. I have still to add that, to judge from the appearance of embryos like the ones shown in Figs. 29, 32, and 33, the cells of the GH group also are overgrown by the descendants of the AB and CD groups. In the head region of the embryo, in the stages shown in Figs. 29 and 33, numerous cells are found in the lower layer,} whereas only a few large stellate cells (overgrown dorsal cells) are found under the ventral plate in the postnaupliar region. At a little later stage (Fig. 30) there are more cells in the lower layer of the postnaupliar region than could be accounted for by the division of the stellate cells. They are found in patches irregularly scattered throughout the region of the ventral plate. As I had a very complete series of embryos, I do not believe that 1 The mesoderm cells are not represented in Figs. 27 and 31, because they were so numerous at these stages that they could not be well represented, and because the regular arrangement of the ectoderm would have been obscured. 324 LANGENBECK. [VoL. XIV. a plug of mesoderm cells arises at any one point, and that this stage had escaped my notice, but rather that in Microdeutopus the mesoderm is formed at many points in the ventral plate. I have a section of a cell dividing obliquely inwards in the anterior end of the head region, and Fig. 51 shows another case in which there are two cells in the aster stage. The equatorial plate of each spindle makes an acute angle with the tangent to the surface of the egg at that point. Had divided, the greater part of one daughter cell would have lain in the second layer. In Fig. 52 we see a cell () which may have been derived from a cell like %, or it may be one of the cells of the outer layer drawn under the surface; from the appearance of the nucleus I am rather inclined to the latter view. I never have seen spindles whose axes were parallel to the radius of the egg in cells at the surface, although I have seen radially directed spindles in the second layer (Fig. 50). Cells which appear as though they were drawing in or had arisen by oblique division were found at any point on the ventral plate. I there- fore conclude that part of the mesoderm in Microdeutopus is derived from the ventral plate. When the dorsal pole of the egg has been completely over- grown by the ventral plate the ventral portion of the blasto- derm, posterior to the head region, is composed of two layers of cells. With the digestion of the yolk by the entoderm cells, which begins during the third day, all the cells of the embryo rapidly increase in number. Cell boundaries are en- tirely lost at this stage, the protoplasm of the cells fusing and making it impossible to distinguish where one layer ends and the other begins. Since the nuclei of both ectoderm and mesoderm appear exactly alike, I could not tell whether the ectoderm was only one layer deep and the mesoderm many layered, or whether the ectoderm consisted of more than one layer. Towards the end of the third day numerous spindles are found in all the layers making any angle with the tangent to the surface. In sections of the fourth day (Figs. 59-64) the muscle cells show their characteristic striations. Bergh (93), in his paper on Gammarus, suggests that the cells of the muscle plates are derived from teloblasts, as he Nov2:|) J77CRODEUTOPUS GRYLLOTALPA COSTA. 325 found to be the case in Mysis. In Microdeutopus I find no evidence of a teloblastic growth. The mesoderm cells seen in optical section in the stained and cleared egg are irregularly scattered under the ventral plate. Morphologically, the isopods and amphipods are very closely allied, and it is, therefore, all the more interesting to note how similar are their modes of development. If we compare a dia- gram of Jaera, after the germ layers have been laid down, with one of Microdeutopus, somewhat before the entodermal invagi- nation has taken place, the similarity will at once become apparent (see diagrams). Heider (91), in writing upon the formation of the isopod embryo, states that the dorsal cells, as the embryo develops, become pushed together, and these cells later undergo degene- ration. On page 352 he adds: “ Es ist die Méglichkeit, die wir oben andeuteten nicht ausgeschlossen, dass in dem Dorsalorgan bloss die Involutionsform des Nahrungsdotter bedeckenden Blastodermtheils vorliegt. Die Involution wurde sich dann bei den Amphipoden-Typus durch Einstiilpung, bei den Onis- cus-Typus durch Amputation einleiten.” If such is the case, then in both forms we have a ventral plate covering the ven- tral pole, at the posterior end of which the entoderm arises. Behind the entoderm lie the vitellophags or the dorsal cells in the amphipods, the similarity between these has been pointed out, and beyond the vitellophags the dorsal cells of the isopods or the dorsal organ. of the amphipods, according to Heider. 326 LANGENBECK. [Vou. XIV. The great difference lies in the origin of the mesoderm, and more work will have to be done upon other amphipods before this can be decided. It, however, is a great question to my mind if the dorsal cells in the isopods and the dorsal organ of the amphipods can be homologized. Heider himself merely suggests the possibility, as his conclusion was drawn solely from the work of Bobretzsky ('74). If we compare embryos of the later stages we find that in both cases the embryo is folded dorsally (see diagram and Figs. 2.0 Beg JL8 J-d.g a. 36, 37). In the amphipod, however, when the cells which will form the dorsum of the animal develop, the least resist- ance to the pressure exerted through their growth seems to be on the ventral pole, and, therefore, the embryo folds over ventrally (Figs. 38 and 42), whereas in the isopods the least resistance is in the dorsal region. Might not the different mode of folding in the two cases be due to the fact that by the time the cells which will form the dorsum of the animal de- velop the ventral pole in the isopods is further differentiated than is the case in the amphipod, and, therefore, offers greater resistance to the pressure exerted upon it by the growing region? Formation of the Liver Tubes and the Intestine. As has been described above, the entoderm arises as a true invagination. After the closure of the blastopore the cells migrate into the yolk area as an irregular mass, and by amoe- boid motion migrate to both sides of the body in much the No. 2:| MICRODEUTOPUS GRYLLOTALPA COSTA. 327 same way that Bobretzsky ('74) describes for Oniscus. These become the liver tubes. Fig. 51 represents a section of an egg which is in about the stage shown in Fig. 38. The section passes somewhat obliquely through the region marked a-a. In the region of the dorsal organ the cells are arranging them- selves to form a tube on the left side of the body. The sections (Figs. 57, 58) of a stage between Figs. 38 and 42 pass through the lines a—a and 6-3, represented in Fig. 42. In the sec- tion lying nearest the dorsal organ (Fig. 58), where the greater mass of invaginated cells was found, the tubes are almost com- plete, and a mass of cells is still seen in the center of the egg. Only the dorsal walls are formed in the section nearer the anterior end, although in this case there are as many cells found in the part of a tube as there are in the whole tube of section (Fig. 58). This, however, is not always the case. The tubes are always complete in the region of the dorsal organ before they are complete in any other region of the body. Although no cell walls could be seen, the entoderm cells can be dis- tinguished readily from those of the ectoderm and mesoderm by their nucleiy and in one egg there was a little yolk space between the cells of the body wall and the liver tubes. In other eggs the tubes have been seen in various stages of for- mation; for instance, in one section, where there was a break in the wall of the tube, like the one shown on the right-hand side of Fig. 58, three cells were lying in the gap, but they had not sent out protoplasmic prolongations at the time to complete the tube. Ina section of an egg somewhat older (Pl. XXVII, Fig. 45), the liver tubes are complete in the region of the dorsal organ, and a few cells are found in the center of the yolk mass. Only in one instance have I seen an entoderm cell dividing. It was in the ring of the liver tube, and the plane of cleavage was parallel to the radius of the tube. I feel con- fident, however, that the cells must increase in number by division, because, as I shall show, most of the digestive tract is formed from entoderm cells. By this time the elongation and consequent folding of the embryo, described above, has been completed. The area between the dorsal organ and the last appendage now extends 328 LANGENBECK. [VoOL. XIV. over the whole dorsal region of the abdomen. The blastopore, evidently, is carried over the posterior pole of the egg, and finally lies at or near the extreme tip of the abdomen (¢f. Figs. 36-43). In consequence, the proctodaeum, which invaginates just posterior to the last appendage, forms either in the blasto- poric area or just anterior to it, as is the case in the decapods. In the decapods, however, the stomodaeal and _ proctodaeal invaginations form the greater part of the intestine, while in the amphipods both stomodaeum and proctodaeum are very short. Figs. 59-64 represent sections of an embryo of the fourth day cut parallel to the line +r (Pl. XXVII, Fig. 44). Owing to the folding of the embryo, the sections pass through stomodaeum and proctodaeum, cutting them transversely, while the dorsal sections cut the digestive tube horizontally. In Fig. 59 we see both stomodaeum and proctodaeum. The cells are closely packed and columnar. In the next section (Fig. 60) the stomodaeum is still seen as a round tube, but the proc- todaeum has broken through. Another section showed that the stomodaeum has also broken through. On examining Fig. 61 it will be seen that the part of the digestive tract immedi- ately adjoining the stomodaeum and proctodaeum is not formed at this time. Six large nuclei are seen bordering the yolk area in the thoracic region, whereas only three long, spindle- shaped cells could be seen bordering the yolk of the abdominal region. Two sections beyond this (Fig. 62) show the anterior ends of the liver tubes. The cells have large vacuoles, and inclose the whole yolk area. In the next section (Fig. 63) the liver tubes are pushed somewhat apart, and the irregular mass of cells between them represents the digestive tract just begin- ning to form in this region. In Fig. 64, where the section passes through the dorsal organ, the digestive tract is com- pletely formed of large, vacuolated cells, with large, clear nuclei looking exactly like those of the liver tubes. Another section shows the digestive tract shading off into the large yolk areas of the thoracic and abdominal regions. All the yolk in these sections is inclosed in the liver tubes and the digestive tract. I have never seen any outside of them being digested by special vitellophags as Dr. Pereyaslawzewa ('88) NOn2. |) /AZIECRODEUTOPOCS GRYELOTALPA COSTA. 329 figures in Gammarus, or as Dr. McMurrich (95) describes for the isopods. We have seen how the liver tubes formed first in the region of the dorsal organ, where the greater mass of the invaginated cells lay; now we find the digestive tract completely formed in that region, whereas, anteriorly and posteriorly, it has not begun to form. The cells also present exactly the same ap- pearance as those of the liver tubes; therefore, I conclude that the whole digestive tract, except the short anterior and pos- terior portions, is formed from the invaginated cells. If cells from the ventral plate or from the stomodaeum or proctodaeum were carried in to form the digestive tract, then I should ex- pect to see the ends formed before the central portion. 69. 93. 93. "70. "74. 91. 85. 85. "91. 95. 95. 88. 86. "77. 88. Eph 81. ful. LANGENBECK. [VoL. XIV. LITERATURE. BENEDEN, E. vAN. Recherches sur l’Embryologie des Crustacés. Bull, ’ Acad. Roy. Belgique. Tomes xxviii, xxix. 1869-70. BENEDEN, E. VAN, et BESSELS. Mémoire sur la Formation du Blasto- derme chez les Amphipodes, les Lérnéens et les Copépodes. Mém. cour l’Acad. Roy. Belgique. Tome xxxiv. 1869. BERGH, R. S. Zur Bildungsgeschichte des Keimstreifens von Mysis. Zool. Jahrb., Abt. f. Morph. Ba.vi. 1893. BerGH, R. S. Beitrage zur Embryologie der Crustaceen. Zool. Sahrb., Abt. f. Morph. Bd. vii. 1893. BESSELS, E. Einige Worte tiber die Entwicklungsgeschichte und den morphologischen Werth des kegelf6rmigen Organs der Amphi- poden. /Jenaische Zett. Bd.v. 1870. BoBrRETzsky, N. Zur Embryologie des Oniscus murarius. Zez¢. f. wiss. Zool. Bd. xxii. 1874. Bumpus, H. C. The Embryology of the American Lobster. /ourn. of Morph. Vol.v. 1891. IscHIKAWA, C. On the Development of a Fresh-water Macrurous Crustacean. Quar. Journ. Micr. Sci. Vol. xxv. 1885. KorROTNEFF, A. Die Embryologie der Gryllotalpa. Zezt. f. wiss. Zool. Bd. xii. 1885. KORSCHELT und HEIDER. Lehrbuch der Entwicklungsgeschichte der wirbellosen Thiere. Jena. 1890. McMorricu, J. P. Embryology of the Isopod Crustacea. /ourn. of Morph. Vol. xi. 1895. MeEAp, A. D. Some Observations on the Maturation and Fecundation in Chaetopterus pergamentaceus Cuvier. /ourn. of Morph., 1895. PEREYASLAWZEWA, S. Le Développement de Gammarus poecilurus Rthk. and Le Développement de Caprella ferox Chrnw. Bzdl. Soc. Nat. Mos. N.S. Tome ii. 1888. REICHENBACH, H. Studien zur Entwicklungsgeschichte des Fluss- krebses. Abhandl. der Senckenberg. Naturf. Gesell. Bd. xiv. 1886. REICHENBACH, H. Die Embryonalanlage und erste Entwicklung des Flusskrebses. Zeztt. f. wiss. Zool. Bd. xxix. 1877. RossiskAyA, M. Le Développement d’Orchestia littorea Spence Bate. Bull. Soc. Nat. Mos. N.S. Tome ii. 1888. RosstiskKAyA, M. Développement de la Sunamphitoé valida Czer. et del’Amphitoé picta Rthk. Bull. Soc. Nat. Mos. N.S. Tomev. 1891. ULIANIN, W. Zur Entwicklungsgeschichte der Amphipoden. Zezz. Jf. wiss. Zool. Bd. xxxv. 1881. WAGNER, C. Développement de la Melita palmata. Bull. Soc. Wat. Mos. N.S. Tomev. 1891. No: 2,] ’°87. WEISMANN und IscHIKAWA. perchen bei thierischen Eiern, Freiburg. MICRODEUTOPUS GRYLLOTALPA COSTA. 331 1887. Ueber die Bildung der Richtungskér- Ber. der Nat. Gesell. Bad:in. ‘92. WELDON, W. F. R. The Formation of the Germ Layers in Crangon vulgaris. Quar. Journ. Micr. Sci. Vol. xxxiii, 1892. LETTERING USED THROUGHOUT THE PLATES. abdomen. en.inv. abdominal fold. Vb. antennae. 1a. appendage. m. blastocoel. ppl. cephalothorax. dorsal cell. pr. dorsal organ. st. ectoderm, x and y. entoderm. Ve entodermal invagination. liver tube. last appendage. mesoderm. protoplasm of cells at the edge of the ventral plate. proctodaeum. stomodaeum. degenerating cells. yolk area. oo" Fic. FIG. FIG. Fic. Fic. Fic. FIG. ise: Fic. OI Anew ws 9. LANGENBECK. EXPLANATION OF PLATE XXVI. 4-cell stage. 8-cell stage from micromere pole. 16-cell stage from macromere pole. 16-cell stage from micromere pole. 22-24-cell stage from macromere pole. 22~-24-cell stage looking down upon GZ cells. 22~-24-cell stage seen from CJD side. 22~24-cell stage looking down upon ZF cells. Section of an egg of forty-five cells passing through the two blasto- meres found in the blastocoel. ides, 11}, blastoc FIG Fic. Fic. Fic. Fic. Fic. Fie. oel. 5 iti. 12. cP 14. rg 16. Te Section of the same egg shown in Fig. 7 passing through the 28-40-cell stage looking down upon the g% cells. 28—40-cell stage looking down upon the G/ cells. 40-cell stage showing x and y cells. 73-cell stage looking down upon the ZF group. 73-cell stage from micromere pole. 73-cell stage looking down upon the GH group. 1o02-cell stage from the macromere or ventral pole after the blasto- derm has risen to the surface of the egg. FIG. Fic. Fic. 18. 19. 20. Micromere or dorsal pole of the egg shown in Fig. 17. Optical section of an egg passing from the 2 into the 4-cell stage. Section of an egg of forty-three cells, showing one cell with the spindle at right angles to the surface of the egg. Fic. Fic. FIG. FIG. Fic. Fic. 21. 22. a5. 24. 25. 26. 44—-46-cell stage looking down on #F group. 30-42-cell stage looking down on #F group. 30-42-cell stage looking down on GH group. 30-42-cell stage seen from micromere pole. Section of an egg of the 8-cell stage. Section of an egg of 72-cell stage. iF Bp Journal of Morphology Vol.XIv: ai Langenbeck del. bik Anstc Werner aWinter, Frankfort 200, FIG. Fic. Fic. Fic. Fic. FIG. Fie. Fic. 31. Fic SP) 27. 28. 29. 30. a0. ae 33: 34: . LANGENBECK. EXPLANATION OF PLATE XXVII. Dorsal view of an egg of about twenty-four hours. Side view of the egg shown in Fig. 27. Ventral view of the egg shown in Fig. 27. Side view of an egg of about thirty-six hours. Anterior pole of an egg of about thirty hours. Posterior pole of the egg shown in Fig. 31. Ventral view of the egg shown in Fig. 31. Side view of an egg of a stage between those shown in Figs. 30 and Ventral view of an abnormal egg of about the same stage as that shown in Fig. 33. FIG. FIG. Fic. Fic. Fic. Fic. Fic. Fic. Fic. Fic. Dorsal view of an egg of the second day. Side view of the egg shown in Fig. 36. Side view of an egg about the end of the second day. Ventral view of the egg shown in Fig. 38. Dorsal view of an egg of about the beginning of the third day. Ventral view of the egg shown in Fig. 4o. Side view of the egg shown in Fig. 4o. Side view of an egg at the end of the third day. Side view of an egg of the fourth day. Section of an egg of a stage shown in Fig. 43. —— T° = ss Morphology. Vol. Abd P aba. f abd abd. P pada se abd Fabia. Langenbeck dei. Lith. Anstv. Werner &Winter: Praak flr 33 6 LANGENBECK. EXPLANATION OF PLATE XXVIII. Fic. 46. Transverse section of the head region of an embryo shown in Fic. 47. Transverse section of the same egg about the middle of the ventral Fic. 48. Transverse section of the same egg nearer the posterior pole. Fic. 49. Transverse section of the head region of an egg of the first day. Fic. 50. Transverse section of an egg of the first day. Fic. 51. Part of a section of an egg of the first day. Fic. 52. Part of a section of an egg of the first day. Fic. 53. Transverse section passing through the center of the entodermal invagination. Fic. 54. Small portion of a sagittal section, showing the relative position of the dorsal organ and the entodermal invagination. Fic. 55. Sagittal section of an egg of the second day. Fic. 56. Oblique section of an egg of the stage represented in Fig. 38 passing through the region a-a. Fic. 57. Section of an egg of a stage between 38-42 passing through a-a, Fig. 42. Fic. 58. Section of the same egg passing through the line 4-d. Fic. 59. Section of an egg of the stage shown in Fig. 44, passing through the stomodaeum and proctodaeum and cut parallel to the line x—x, Fig. 44. Fic. 60. Another section of the egg shown in Fig. 59. Fic. 61. A section dorsal to the one shown in Fig. 60. Fic. 62. Thoracic portion of the second section beyond the one shown in Fig. 61. Fic. 63. Thoracic portion of the next section to the one shown in Fig. 62. Fic. 64. Thoracic section beyond the section shown in Fig. 63. Sib. Anst-e Werner & Winter Frankfar?at, Langenbeck det. THE REGENERATION OF THE NERVOUS SYSTEM OF PLANARIA TORVA AND THE ANATOMY OF THE NERVOUS SYSTEM OF DOUBLE- HEADED FORMS. SIMON FLEXNER. DurRInG the summer of 1895, through the courtesy of Prof. C. O. Whitman, I enjoyed the pleasure of spending two months in the Marine Biological Station at Woods Holl. At the suggestion of Dr. Jacques Loeb, who extended to me the privileges of the Physiological Laboratory, I began the study which forms the subject of this paper. The following year I obtained additional material, which was sent to me at Balti- more, where the investigation was continued and brought to its present state of completion. My thanks are due to both these gentlemen for many acts of kindness, and to the latter for much valuable advice. The drawings which accompany this paper I owe to the kindness of Dr. Alice Hamilton. The researches of Loeb! upon heteromorphosis have shown that it is possible, through the use of several different pro- cedures, to bring about a substitution of an organ of one physiological value for that of another, and even to cause to be developed in one part organs which normally belong to widely removed localities. The light which the observations have thrown on the underlying forces and the manner of the formation of organs is considerable ; the study of the histologi- cal details of the process of organ-building in such cases has been little pursued. That heteromorphosis can be produced experimentally in the planarian has been proven by Van Duyne,? whose studies also 1 Untersuchungen zur physiologischen Morphologie der Thiere, Wiirzburg, 1891 and 1892. 2 Ueber Heteromorphose bei Planarien, Archiv f. die ges. Physiologie, LXIV, 1896, 569. 338 FLEXNER. [VoL. XIV. indicate the ease and variety of the development in these forms of duplicate parts. A knowledge of the histology of the re- generative phenomena in the nervous system is of interest, therefore, not alone in itself for its bearing on the question of the growth of highly specialized organs, but as affording a basis for a closer study of the phenomena of heteromorphosis in these animals. The methods employed in this study consisted in (A) de- capitation of the worms ; (4) decapitation to which was added an incision in the longitudinal axis, passing through the entire thickness of the animal, and extending about one third of its length; (C) complete longitudinal division with and without decapitation. At different periods in the process of regenera- tion the worms were killed with HgCl,, formalin 5%, alcohol or osmic acid (Flemming’s and Hermann’s solutions), hardened and sectioned in paraffin. A number of staining agents were employed. The decapitation was performed on the extended animals, the precaution having been taken to remove the entire cephalic extremity and contained nervous ganglia. When partial longitudinal incision was also carried out, the object of which procedure was to produce double-headed forms, it was found necessary, in order to prevent healing, to separate the incised halves every twelve hours or oftener for the first two or three days. After complete longitudinal division, each half immediately becomes rolled up in the form of a spiral, which gradually unwinds as regeneration proceeds. The rapidity of the regeneration of new and duplicate parts varies in several ways. The shortest periods were noted where a single extremity was replaced (head or tail); a longer period is required for the completion of two parts of the same sort (double heads); and the longest are observed in the regenera- tion of the longitudinally divided halves. Temperature also plays an important part. At the quite uniform and moderately low temperature of Woods Holl, the growth takes place far less rapidly than at the much more elevated temperatures which prevail in Baltimore during the No. 2.] DOUBLE-HEADED FORMS. 339 months of July and August. As this study was begun at Woods Holl in 1895 during the corresponding months and completed upon material forwarded from Woods Holl to Baltimore in the summer of 1896, this comparison was easily possible. The especial purpose of this inquiry was to ascertain the manner in which the nervous (cephalic) ganglia and nervous cord were regenerated under the several conditions mentioned, and also what the new relations were which arose in artificially produced monsters. A few words regarding the anatomy of the normal parts may properly precede a detailed account of our study. The nervous system of the planarian with which I have worked consists of a cephalic and a trunk portion (Fig. 1). The cephalic extremity consists of two bulbous enlargements, lying near the lateral edges of the animal, on a line with the eye-spots. These are connected by a commissure which is somewhat smaller in dimensions than the ganglia, although it does not differ in structure from these parts. Passing out- wards towards the caudal extremity are two cords, one pro- ceeding from each ganglion, which run ina parallel direction quite to the end of the animal. These cords are connected by semicircular commissural processes which come off at somewhat irregular intervals. From the cephalic extremity, as well as from the nervous cords, nerves pass outwards to the periphery of the body. There is no essential difference in the microscopical struc- ture of the ganglionic masses and the nervous cords. Each consists of a bundle of very fine non-medullated nerve fibers in close proximity to nerve cells. These cells — ganglionic cells —are more numerous in the cephalic ganglia. But cells similar in type and appearance exist in the cords, and may even be found, although much more sparsely, in the nerves themselves. In the ganglia they exist within the bundles of nerve fibers, grouped in the center, scattered in the periphery but most thickly placed in the outermost zone. In the cords they are found quite regularly disposed among the fibers. The process of regeneration was studied in all stages, begin- ning twelve hours after decapitation, until the complete restora- a 340 | FLEXNER. [VoL. XIV. tion was effected. At the end of the first twelve hours and about equally at the conclusion of eighteen hours, active cell proliferation in the divided end is going on. The evi- dences for this are found in the rich mitosis encountered as well as in the accumulation of small, immature cells at the injured extremity. The most active division is found in the tissues immediately adjacent to the epidermal elements close to the superficial epithelial cells and to a far less extent in the cells at a distance (Fig. 2). The surface epithelial epidermal elements show no evidence whatever of mitosis; while close to the site of operation degenerative changes, consisting of fragmentation of cell nuclei (karyorhexis), are to be made out. Although especial attention was directed to the ganglion cells in the nerve cords at this time, mitotic figures were never demonstrated in them. The new cells accumulate about the cut end and quickly cover over the defect. At the end of twelve hours they have pushed forward to the extremity, have become continuous with the intact epidermal cells, and have already formed a covering for the denuded structures (Fig. 3). Whether an embryonic form of epidermis or not, considerable masses of multi- nucleated protoplasm (syncytium) form the outermost layer of new cells. At the end of twenty-four hours cell division in the regen- erating end is quite over. Long and painstaking search is required to discover a single karyokinetic figure. But the cellular accumulation is now considerable. The new cells not only cover in the defect and are evident to the naked eye as a projecting white point, but they surround and inclose the divided nerve cords. The changes noted at the end of forty- eight hours consist in a somewhat more orderly arrangement of these cells about the cord and the invasion by them toa small extent of the bundle of fibrils themselves (Fig. 4). The completed ganglion is found at about the sixtieth hour (Woods Holl temperature), although at this time the cephalic extremity does not appear to have attained its maximum size and the regenerated epidermis is still unpigmented. It may happen that in the ganglia at this time there is an undue rich- Noy.) ' DOUBLE-HEADED FORMS. 341 ness of ganglionic cells (Fig. 5), while in specimens four days old no difference from the normal can be detected (Fig. 6). More detailed changes are as follows: At the end of twenty-four hours the new cells have increased greatly and have quite filled up the cut surface, covering over the alimentary tube, etc. The cells have increased in size ; they are oval or oblong in shape, and exhibit vesicular nuclei. They inclose the cut ends of the nervous cords and lie often in parallel rows, the long axis of the cells being placed in the horizontal plane. The most considerable group of cells is at the cut extremity and fibers are already visible between these cells. It cannot be positively stated that these cells have not migrated inwards among the old fibers and that these latter are the objects seen between them. But on the other hand, certain cells which lie to either side and just above the cords show similar fibers. It would be difficult to account for the pres- ence of these upon the supposition that they were pre-existent ; it appears more probable that they are newly formed. This conclusion is impressed upon one the more as these fibers are less distinctly linear and wavy than those in the cords them- selves. Many of them are indeed short and granular, and some may not be fibers at all, but perhaps a sort of granular intercellular substance. This material is, however, not present in other places between cells; and it seems limited to the region of the old nerve cords. It is conceivable that the granules are derived from young and immature fibers altered by the fixing and hardening process. These cells present the characters of neuroblasts, and are doubtless destined to compose the reproduced cephalic nervous structures (Fig. 7). The histology of the growth of the nervous ganglia in the double-headed is not distinct from that of the simple forms. The gross anatomical result is, however, quite different. Although ganglia, two in number, connected by a commissure and closely resembling the normal structures, come to be devel- oped, each is in association, not with a pair of nerve cords, but with a single cord (Fig. 8). The eye-spots bear about the same relation to the ganglia as they do in animals with single heads. 342 FPLEXNER. [VOL. XIV. The regeneration of longitudinally divided animals goes on with far less rapidity ; finally, however, the result is quite as perfect as in the other instances. What takes place at once after section is, as already stated, a curling of the halves, the unraveling of which may require two weeks or even longer. As the restoration of the removed parts precedes this unwind- ing the degree of regeneration may be inferred from the extent of the return to the normal form. The manner of the production of an anatomically complete individual is of much interest... Confining our description to the nervous system it is evident that the new segment could be produced in the growing half in one of two ways. Either a new and independent formation of the removed parts which become united with the remnant of the old system takes place, or the segment of the old system furnishes the starting- point of the regenerating half. The cellular changes following median division are similar to those which succeed to simple decapitation, in so far as the exposed segment of the nervous mechanism becomes sur- rounded with cells which are gradually differentiated into ganglion cells and fibers. This increase in cells which, under- going successive differentiation into neurones, gradually extends the nervous elements into the growing half, gives rise in succession to the cephalic ganglion, including commissure and nerve cord, thus projecting, as it were, the old system into the new part. There is, then, not a formation de novo of the removed segment, but a continuous outgrowth from the intact half (Fig. 9); the new cells do not arise by division of the old nerve (ganglion) cells, but come from other sources. The phenomena attending regeneration of the central nervous system have been studied in both vertebrates and in- vertebrates, and with somewhat varying results by different observers. Most investigators agree in having found that in vertebrates the capacity for reproduction of removed or injured parts is very slight or even 2/1 While Coén, von Kahlden, Sanarelli, and Friedmann found that defects in the cerebrum and cerebellum were replaced by connective tissue, and 1 See Barfurth, Regeneration, Merkel-Bonnet’s Ergebnisse, i, 1891, 132. No. 2.] DOUBLE-HEADED FORMS. 343 Schiefferdecker failed to observe regeneration in the spinal cord of mammals, Danilewski! claims to have demonstrated in a frog in which one cerebral hemisphere had been removed nine months before, a newly formed cerebral mass containing young nerve cells. To the proof furnished by Mingazzini? that in tunicates complete restitution of the brain, etc., takes place, may be added the observations of Friedlander on the manner of the reproduction of the central nervous organs in certain worms. According to this author, the restoration takes place by an outgrowth from the remaining normal portion. It is, however, not clear from Friedlander’s® study just what the origin of the nerve cells may have been, nor what is the nature and origin of the leucocytes which give rise to a compact, cellular new tissue. My studies of the manner of regeneration of the central nervous organs of the planarian are capable of a different inter- pretation. There is no evidence of an outgrowth proceeding from the intact and non-degenerated nerve cords, nor is there any indication of a proliferation of the nerve cells existing among their component fibrils. The new organ results from a new growth of cells which originate independently of the old nervous system and in close proximity to the epidermal cover- ing. They are not the fully formed and completely differenti- ated epidermal cells ; for in these there were never found active changes which could be regarded as regenerative in character. It seems much more probable —although complete proof can- not at present be adduced — that the actively dividing elements are of the nature of Arsazz cells, which, while destined to give rise to the surface epithelium, are still capable of being transformed into neuroblasts. A simple outgrowth from the several nervous cords can be excluded. For, besides the fibers which compose the ganglia, 1 See Barfurth, Regeneration, Merkel-Bonnet’s Ergebnisse, iii, 1893, 171. 2 Ueber die Regeneration bei den Tunicaten, Bol. della Societa di Naturaliste in Napoli, 1891, ser. 1, vol. v, p. 76. Ref. Merkel-Bonnet, Ergebnisse, i, 1891, 122. 3 Ueber die Regeneration herausgeschnittener Teile des Centralnervensystems von Regenwiirmern, Zeitschr. f. wissenscls Zoologie, 1x, 1895, 249. 344 FLEXNER. a more considerable and important constituent is furnished by the nerve cells. First appearing as indifferent cells, entirely without orderly arrangement, they quickly undergo changes through which they become disposed in a regular manner, agreeing in position with the original cells which make up the parts, and after suffering some further alterations in form and staining capacity, may even be seen to develop fibers, which it is fair to assume are nervous in character. There occurs, also, at first an over-production of new cells, a surplus which in the end has disappeared. It would appear as if the new cells somewhat distant from the nerve trunks do not develop fibers, nor do they assume so orderly an arrangement ; and, moreover, they are the individuals which are finally lost. If this view of the regeneration of the nervous ganglia is correct, it must be admitted that a metaplasia of cells destined to become a simple external protective organ into the most highly differentiated structures of the body is, in these forms, possible. Barfurth! has pointed out the similarity in the regeneration of the epithelium of the skin and that of the cen- tral nervous organs of amphibian larvae, and attributes this to their common origin from the ectoderm. This conception, moreover, contains less that is startling in view of the obser- vations of Wolff and Erik Miiller? on the regeneration of the crystalline lens in tritons from the epithelium of the iris. JouHNs HopkKINS UNIVERSITY, BALTIMORE. 1 Regeneration, Merkel-Bonnet’s Ergebnisse, i, 1891, 132. 2 Archiv f. mikroskop. Anatomie ad. Entwickelungsgeschichte, xvii, 1896, 23. » 346 FLEXNER. EXPLANATION OF PLATE XXVIII 4. Fic. 1. Semi-diagrammatic representation of the nervous system of Planaria torva. A.A. nervous ganglia; &.4. nervous cords. Ganglion cells indicated in gray. Fic. 2. Karyokinesis in cells adjacent to the site of injury. The greatest activity in and increase of cells is near the ectodermal covering. Two fields are shown, A. near, &. somewhat removed from the injury. 12 to 18 hours after decapitation. Fic. 3. Newcells at the site of injury. Pushing forward of the cells to cover the defect, the new elements becoming continuous with the ectodermal cells. Large, thin, protoplasmic masses (syncytium) cover the extreme edge. A.A. cells pushing forward to epidermis; 2.4. syn¢ytium; C.C. masses of newly formed cells. 12 hours after decapitation. Fic. 4. A nerve cord (4.) surrounded by new cells. The thickest group covers the cut end, and individual cells of the same type may be seen pushing their way among the fibers. Indistinct fibrillae appear between the cells above the cord. 48 hours after decapitation. Fic. 5. Completion of the ganglion. Increased richness of cells on each side of the cord and normal radial arrangement around the bulbous end. Differ- entiated nerve cells show a lighter tone than the remainder. After 60 hours. Fic. 6. Completely regenerated nervous system. 4.d. ganglia; 2. commis- SUIe; 9G: C. nervous cords. After 4 days. Fic. 7. Showing neuroblasts next to and beyond the sectioned nerve cord. Only the cells in close proximity to the original cord exhibit processes. Fic. 8. Complete double-headed form. A.A. nerve cords; £&.28. ganglia. The new ganglia on each side are united by commissures, and to each pair a single cord is attached. Eye-spots shown. Fic. 9. Almost completely regenerated longitudinally divided half. 4.4. ganglia; B.B. cords. The cord &.1 not quite perfect. After 13 days. ra iS = = a? . = Y = ee 7 os ae , ~~ = es -_ = Al i Lhe — = 4 : s = a fournal of Morphology Vol. XIV. 1 8] tf & a) 4 € Re < a? - DEVELOPMENT OF THE VENTRAL ABDOMINAL WALLS, IN: MAN! FRANKLIN P. MALL. AFTER the neural plate is formed within the amniotic cavity, the tail end of the embryo remains attached to the chorion by means of the allantoic stalk (Bauchstiel). The amnion surrounds the embryo only on its dorsal side, as has been shown recently by the study of a number of young human embryos. The ven- tral side of the embryo, however, hangs free in the coelomic cavity, as the body walls have not been formed by the exten- sion of the amnion. The extension of the amnion proceeds hand in hand with the flexion of the embryo. It first extends over the face of the embryo, then tucks under the tail end, and at the same time it encroaches from both sides of the body. In covering the head it first crosses the mouth and then the heart, this movement being accelerated by the growth of the amnion over the head end of the body from left to right.2 While this is taking place, the stem of the umbilical vesicle elongates to produce the general condition, as represented in embryos four weeks old. Soon, however, the amnion covers the whole allantoic stalk, including within its folds the stem of the umbilical vesicle, thus forming the true umbilical cord. At this stage the intestine begins to enter the cord. After we have reached the stage in which the umbilical cord is formed we find that in transverse sections of human embryos, as well as in embryos of other mammals of the same stage, the . ventral abdominal walls are composed wholly of a membrane of connective tissue, without any ribs, muscles, or blood vessels as they are found in the adult. 1 The present paper is to be considered a continuation of two papers published recently; one upon the development of the human coelom (/ourn. of Morph., vol. xii), and the other upon the development of the human intestine (His’s Archiv, Supplement Band, 1897). 2 Mall, Journ. of Morph., vol. xii, p. 430, Figs. 27, 28. 347 348 MALE. [VoL. XIV. It is now generally believed, and to a great extent proved, that the ribs and muscles wander into the ventral walls of the embryo from the sclerosomes and myomeres on the dorsal side of the embryo. It is easy to demonstrate the growth of the ribs from the dorsal to the ventral side of the embryo, but it is not so easy to demonstrate the growth of many muscles from the muscle plates to their final position in mammalian embryos. With the growth of the ribs and the wandering of the muscles from the myotomes into the ventral wall of the embryo we have associated with them their nerves and blood vessels, which ultimately form the intercostal nerves and blood vessels respec- tively. The segmental arteries fuse at their tips early in their development immediately below the rectus abdominis to form the internal mammary and deep epigastric arteries. This chain of anastomosis is formed in a manner with that seen in the formation of the vertebral artery, as shown by His, Froriep, and Hochstetter. The primitive internal mammary and deep epi- gastric arteries, together with the rectus, at first lie immediately below the mammary line in pigs’ embryos, and all three, artery, muscle, and line, wander together towards the ventral middle line of the body. No doubt the same process takes place in the human embryo, but the mammary line lacks prominence here and cannot be used as a landmark. As the segmental nerves appear, each 1s tmmediately connected with its corresponding myotome, and all of the muscles arising from a myotome are always innervated by branches of the nerve which originally belonged to tt. This generalization is difficult to prove in all of its details, but all of the work in embryology, as well as a tabulation of our knowledge of motor nerve distribution, indicates that it must be true. The simpler muscles of the back, as well as the inter- costal, are innervated by their corresponding nerves. The muscles of the eye, of mastication, and of the face can be correspondingly grouped. The trapezius, latissimus dorsi, serrates anticis, and rectus abdominis wander great distances and carry with them their original nerves. Moreover, it would be practically impossible to explain why each muscle has its proper nerve supply, which is so evenly distributed in it, were No. 2.] VENTRAL ABDOMINAL WALLS IN MAN. 349 not this original relation between muscle and nerve retained. With this view we have no grave mechanical difficulties to overcome in conceiving how it is possible for the simple periph- eral nervous system of the embryo to be evolved into that of the adult. It is simply that each segmental nerve grows to its myotome, or corresponding group of tissue or branchial arch, and with the further growth of muscles from myotomes the original nervous connections are retained. The required free growth of a nerve is then extremely short, as the myotomes receive them as soon as they leave the spinal cord, and then as the different muscles or parts of muscles arise from each myo- tome the nerve bundles split to correspond. The primitive muscle, then, is from its earliest appearance connected with the cord, and, as its muscle fibers appear and increase in number, this first connection guides the new nerve fibers to their destination. The studies in comparative anatomy by Gegenbaur and by Huxley ? all lead to the same general conclusion that nerve and muscle are associated with each other in their earliest stages of development. This idea has been the foundation for a number of studies in comparative anatomy, and the more it is investigated the stronger the foundation becomes, as may be seen in the critical review by Kollmann.? In a discussion of Testut’s “ Anomalies musculaires chez l’Homme,” Gegenbaur 4 shows clearly that the study of variations of muscles would have great meaning if their nerve supply were included, and that all the reports of muscle anomalies for a period of more than one hundred years cannot be used at present, due to this omission. The great value of the comparative study of the muscles in con- nection with their nerve supply has recently been shown again by Ruge.® His study shows that comparison of muscles can be made satisfactorily only when their nerve supply is included. All this indicates that the history of a muscle is indicated by its nerve, and that in studying the development of a muscle our 1 Gegenbaur, Grundriss d. vergleich. Anatomie. 2 Huxley, Comparative Anatomy of Vertebrates. 3 Kollmann, His’s Archiv, 1891, p. 76. * Gegenbaur, Morph. Jahré., Bd. x, p. 331. 5 Ruge, Morph. Jahro., Bd. xix, p. 376. 350 MALL. [Vou. XIV. main guide is its nerve. This has been plainly indicated recently by Nussbaum,! and in my studies in embryology and anatomy I cannot find a single instance to contradict this idea. The same generalization, no doubt, applies also to the sensory nerves, which are distributed in a segmental way to the integu- ment. Their first distribution would then be to the regions immediately over the spinal ganglia, and as the skin shifts in its development it carries with it its original nervous supply. That the nerves cannot be distributed to any great extent without some guide is further demonstrated by all experiments on regeneration, which show that they have great powers of growth if they are guided in their distribution by a canal or by the connective tissue sheath of some degenerated nerve. Although it is impossible to prove at present that all of the skeletal muscles arise from muscle plates or their corresponding coelomic diverticula into the branchial arches, the generalization that they do arise from the mesoderm segments is based upon sufficient observation to make it highly probable. In sharks the coelom extends into the branchial arches and into the myotomes ; in man the cavity in the muscle plates is never con- nected with the coelomic cavity, while in the branchial arches no such cavity exists. Yet we do not hesitate to assert that the condition found in sharks is the primitive, while that in man is due to secondary changes. Of course the problem is more difficult when the fate of the plates is to be studied. In the sharks, where they are definite, it is easy to show that all of the skeletal muscles of the body arise from them, while in man it is extremely difficult to show from what myotome any given muscle arises. The first difficulty encountered is that in the head (with the exception of in the occipital region) there is no grouping of the mesoderm in the branchial arches, while in the extremities the myotomes lose their sharp outline and appear to blend with the surrounding mesenchym. This fact has been sufficiently emphasized by Paterson? a number of years ago, and recently has been used by Fischel? and by Har- 1 Nussbaum, Verhandl. d. Anat. Ges., 1894-18096. 2 Paterson, Quar. Journ. of Micro. Sci., N.S., vol. xxviii, p. 109. 8 Fischel, Alorph. Jahro., Bd. xxiii, p. 544. No. 2.] VENTRAL ABDOMINAL WALLS IN MAN. 351 rison! in combating the notion that all muscles arise directly from the myotomes. Harrison studied the development of the fin muscles in the trout, and finds that, with the exception of those in the pectoral fin, they arise directly through buds from the myotomes. In this one fin the muscle bud is small, and is so closely blended with the mesenchym that it is impossible to show whether or not the muscles of this fin arise from the muscle bud or mesenchym or from both. It appears to me that if all the body muscles of sharks and all of them in the trout arise directly from the myotomes, with the exception of those of the pectoral fin, it is better for the present to ascribe the muscles of this fin to the myotomes rather than to mesenchym, when mesenchym and buds are so completely blended. Not only does Harrison? deny that all the skeletal muscles arise from the myotomes, but he also attempts to prove that the segments of a muscle, like the rectus, do not indicate that it has arisen from a corresponding number of muscle plates. He has shown that in teleosts the rectus is first split from the muscle plates as a band of tissue, and then undergoes secondary segmentation. After the muscle is laid down its front end remains connected with the myotomes until it has segmented, and hence these segments of the rectus correspond with the myotomes, while behind they do not. That the segments in the posterior end are formed after the muscle is separated from the myotomes indicates that there are mechanical causes which favor segmentation, but it is not proved that these segments do not correspond to the myotomes until it is shown that the nerve supply of the two are different. Even if it were shown that the succession of segments in the rectus did not correspond fully with the segmental nerves it would not prove anything special other than the history of this muscle. We must only recall the serratus anticus, which is composed of beautiful segments, all of which are of secondary formation. In its development the muscle wanders down from the neck, attaches itself to the scapula, and then successively to the ribs. During all this process of growth it retains its original nerve connec- 1 Harrison, Arch. f. mikr. Anat., Bd. xlvi, p. 500. 2 Harrison, Johns Hopkins University Circular, No. 3, 1894. 352 MALL. [VoL. XIV. tion, which shows its origin; that it is segmented is due to the ribs, a mechanical element. While in the sharks it is comparatively easy to follow the development of the eye muscles with their respective nerve connections, it is impossible at present to follow them in higher animals. The same applies to the muscles arising from the hyoid arch and innervated by the seventh nerve. It is a remark- able fact, however, as emphasized by Rabl, that just those muscles which belong to the hyoid arch are innervated in man by the seventh nerve. Even where we can see no sharp line whatever in the embryo, there is one which has been indicated to us by the study of sharks. Rabl? states, after speaking of the muscles of mastication: “ Die iibrige Gesichtsmuskulatur dagegen wird vom Facialis innerviert und sie ist, wie Gegenbaur und Ruge auf vergleichend-anatomischem Wege gezeigt haben, aus einer Differenzierung des Platysma hervorgegangen; das Platysma selbst aber gehort genetisch dem Hyoidbogen an und wird daher, wie seine Differenzierungsprodukte, vom Facialis versorgt. Ebenso scharf sondern sich auch die beiden Inner- vationsgebiete in der Paukenhohle. Hammer und Amboss entwickeln sich aus dem ersten, der Steigbiigel, wie ich ausein- andergesetzt habe, aus dem zweiten Kiemenbogen; der Muskel des Steigbiigels vom Facialis innerviert. Der Musc. tensor tymp. gehort mit dem Tensor veli palatini genetisch und anatomisch zu einer Gruppe (Schwalbe); der Musc. stape- dius bildet in ahnlicher Weise aller Wahrscheinlichkeit nach mit dem Stylohyoideus und hinteren Biventerbauch eine Gruppe.’ The short muscles of the trunk are innervated by single nerves, and in them the embryonic condition is retained. But in the case of the shifting of a muscle its nerve supply is not so simple as in case of the diaphragm. In this extreme example the muscle wanders down with the development of the septum 1 Rabl, Avzat. Anz., Bd. ii, p. 226. 1 There is a very extensive literature to show that the muscles of the limbs arise from the myotomes. The systematic work of Mollier (Anat. Hefte, No. viii) is one of the best of this group of papers. In my discussion above I have only brought forth some general objections to the views of Paterson and the much stronger and better arguments of Harrison. No.2.] VENTRAL ABDOMINAL WALLS IN MAN. 353 transversum,' and its nerve supply indicates that the muscle must have arisen from the fourth or fifth cervical myotome or both. After the septum transversum has reached its final location, to form the diaphragm, muscles arising from the lower dorsal myotomes wander into its periphery and these are inner- vated by the lower intercostal nerves. In the same way the sterno-mastoid and trapezius grow over from the head to the shoulder, while the latissimus dorsi and serratus anticus grow from the shoulder to the ribs and pelvis. I have already spoken of the serratus anticus above, and wish to add a few words about the latissimus dorsi. It undoubtedly arises from the seventh and eighth cervical myotomes, is soon separated, and free to move. It attaches itself first to the humerus and then gradually spreads out over the back, attaching itself first to the vertebrae, then to the ribs, and finally to the ilium. As the growth takes place in this order the shoulder end is the older, and the pelvic end the younger, thus accounting for the nerve distribution as shown recently by Nussbaum.” In spreading, its fasciculi cross and encircle branches from the intercostal and lumbar nerves, thus making them perforate this muscle. Other muscles that shift, like the deltoid and pectoralis major, are likewise perforated but not innervated by nerves foreign to them. Sometimes muscles which remain segmental cover metameres beyond their nervous supply. Quain’s? Azatomy, in discussing the quadratus lumborum and complexus muscles, asserts that this indicatesa reduction of the number of nerves, while from the above discussion it appears to me that it is not a reduction of nerves but an extension of the muscle beyond its original bounds. Likewise a complete absence of an important muscle (trape- zius), or a group of muscles (facial), is not necessarily to be accounted for by a degeneration of the nerve. Anything which arrests the development of the muscle in its very earliest stage will accomplish absence of the nerve, while a nerve destroyed at so early a stage might be replaced by a neighboring nerve through anastomoses. 1 Mall, Journ. of Morph., vol. xii, p. 395, Figs. 30, 41. 2 Nussbaum, Verhandl. d. Anat. Ges., 1894, p. 179; 1895, p. 26; 1896, p. 64. 3 Quain’s Anatomy, vol. ii, p. 354. 354 MALL. [Vou. XIV. One of the most complicating elements in the study of the development of a muscle is the formation of nerve plexuses. In those instances in which there is but little cross shifting in the movement of a muscle, its nerve remains single or nearly so. But when there is considerable cross shifting very early in the development we have the formation of a plexus. Furbringer 1 has shown from comparative anatomical studies that there is a plexus formation in the nerves supplying the limbs which shift in development, and that the plexus formation indicates that the limb is wandering. In the development of the extremities in man it has been clearly pointed out by the method of recon- struction that the arm bud makes a rapid excursion from the head backward in the early embryo.? In so doing it appears to drag the nerves together, thus forming a plexus. The upper intercostal nerves do not shift and, therefore, do not form a plexus, while the lower intercostal nerves passing to the anterior abdominal wall shift in their development, and thus favor the plexus formation. ; In this shifting and mixing of nerves the portions of the muscle plates which they supply are also hopelessly mixed, and this I think accounts, in part at least, for the fact that the muscle buds are no longer defined in the arm and leg by buds. But after the muscles are fairly well outlined they move on to their destination without any more mixing of nerve fibers further than additions which may grow along the existing trunks from the spinal cord. The differentiation of the muscles in the extremities takes place after the plexus nearest the spinal cord is formed, thus permitting of a second group of nerve anastomoses in the extremity. This process in the fore- arm, for instance, accounts for the double nerve supply of some of the muscles in it. Physiologists have studied the distribution of nerves in muscles much more carefully than anatomists, and we have to thank Mays? for very careful description of nerve plexuses in 1 Furbringer, Morph. Jahrb., Bd. v, p. 324. 2 His, Abhandl. d. k. s. Ges. d. Wiss., Bd. xiv, p. 341, Taf. II; and Mall, Journ. of Morph., vol. v, p. 459, Pl. XXX. 8 Mays, Zeit. f. Biol., vol. xx, p. 449. No. 2.] VENTRAL ABDOMINAL WALLS IN MAN. 355 muscles. In a segmental muscle like the frog’s rectus abdomi- nis, irritation of the nerve supplying one segment not only makes this contract but the rest of the muscle also, showing that there must be an anastomosis between the nerves supply- ing the different segments.! Therefore, the irritation of the nerve passing to a single segment sets the whole muscle to contracting. The heart is affected in a similar way, for irri- tation of the minutest sympathetic twig has the same effect upon the heart beat as the irritation of its main trunk.2, And the same is true regarding the influence of the splanchnic nerve upon the muscle walls of the vena portae.? In addition to the physiological verification of these nerve plexuses we have ample confirmation of them by Nussbaum, and very recently again by von Bardeleben and Frohse# for a number of muscles in man. So every bundle of nerves which leaves the spinal cord to pass to a muscle has a chance to mix its fibers once, twice, or three times, as the case may be, before it reaches the muscle fibers, and a nerve stimulus may pass to a given muscle ina number of ways and from a number of sources. The plexus within the muscle will, no doubt, prove to be a valuable object for investigation, but it does not concern us much when we consider the origin of a given muscle. I have given it for the sake of completion and have discussed the subject somewhat extensively in order to show a number of difficulties in my way, as well as my standpoint in the present problem. Development of the Rectus. — In the earliest human embryos which have been studied it is observed that the allantoic stalk and the umbilical vesicle are located much nearer the head than in the adult. The relation of these organs to the body of the embryo is shown in the accompanying figure, which is taken from an embryo not over two weeks old.® It’ will be noticed 1 Mays, Ueber die Nervatur d. Musculus rectus abdominis d. Frosches, Heidel- berg, 1886. 2 Johansson, Archiv f. Physiologie, 1897, p. 103. 8 Mall, Archiv f. Physiologie, 1892, p. 423; and Johns Hopkins Hospital Reports, vol. i, p. 148. 4 von Bardeleben and Frohse, Verhandl. d. Anat. Ges., 1897, p. 38. 5 The anatomy of this embryo is fully described by me in the Journ. of Morph., vol. xii, p. 417, and in His’s Archiv, Supplement Band, 1897. 356 MALL. [VoL. XIV. from the figure that the allantoic stalk and the umbilical vesicle must shift their position through a distance of at least twelve segments in order to gain the position they will hold in older embryos. The possibility of this shifting is all easily under- stood when we consider how soft the tissues are at this time, and the lack of any framework to hold the different parts of the embryo together. Later on, when nerve bundles and skeleton appear, the shifting still takes place, but by no means to as great an extent, and its course is now marked by the arrangement of the nerves as well as other tissues. At this stage, however, the most fixed points appear to be the myotomes, and from them I have made all of my measurements. For reasons given in the earlier descriptions of this embryo I have ascribed three of the muscle plates to the occipital region, making the first dorsal myotome immediately over the neuren- teric canal. In later stages each myotome lies immediately over a spinal ganglion, and in referring to the myotomes I give them the numbers corresponding to the spinal nerves. This causes less confusion than by numbering them from the auditory vesicle backward, for by so doing we do not convey any idea of adult anatomy. In embryos slightly older than the one figured above, the spinal ganglia and vertebral column are outlined, thus aiding us easily to locate the position of any of the organs. With the descent of the heart the communication of the umbilical vesicle with the midgut is rapidly cut off, and the stems connecting them, together with the allantoic stalk are then encircled with the amnion. All these intermediate stages are sufficiently well shown in His’s A¢/as, and I need not describe them. At the end of the fourth week the umbilical cord is fully formed, as is shown by a number of specimens in my possession, two of which are especially valuable because there is no doubt whatever as to their being normal. One is the specimen I have studied a great deal, which was obtained from a criminal abor- tion, and the other was gotten from a murdered woman and sent me by the coroner’s physician of Chicago. The first specimen (No. II) is somewhat older than the second (No. LXXVI), and for that reason is more valuable. No. 2.] VENTRAL ABDOMINAL WALLS IN MAN. 357 Fig. 2 is a profile of this embryo! drawn from the specimen before it was cut, with the outlines of the myotomes of the body added from a reconstruction. It shows the relation of the cord to the rest of the body and the extent of the myotomes have grown towards it. In the body itself the myotomes have distinct buds which are growing into the membrana reuniens, and each of these is intimately associated with its nerve. Where buds from the myotomes enter the extremities, their ventral tips appear to be fully blended to correspond with the blending of the nerves which enter the limbs. The extent of the growth of the myotome into the membrana reuniens is shown in Fig. 3. It shows the marked bud from the myotome. Earlier stages need not be given, and a later stage has already been described and pictured by Kollmann. There is, however, a time when the buds from certain of the dorsal myotomes blend to form the rectus, and I have not been able to obtain a good specimen to demonstrate this. A stage somewhat older than one pictured by Kollmann ? will no doubt give the rectus arising from the myotomes. ) co ow )) 5 w wo 5 o 5 | set 7 as r= Journal of Morphology, Vol. XTV. Plate XXX V. G pee eee | ee ee Se oe eS ee ee et en a Journal of Morphology, Vol. XIV. Plate XXX VI. 59 64 By clesthymer del. 3 s ~ Journal of Morphology, Vol. XTV. Plate XXX VII. eo de) 2 = 5 or Q ve) To ° 2 ° — © S D Ne) . bo lo) ~ 2 cae | Se) OO YS © a Eycleshun er del. THE COCOONS AND EGGS OF ALLOLOBOPHORA FOETIDA. KATHARINE FOOT. INVESTIGATORS (I) (3) (11) (20) who have observed the copu- lation of earthworms mention a slimy substance secreted by the two worms during union, which encircles the pair around those segments that are in contact. In March, 1893, while examining a pair of worms surrounded by this slime, its form led me to suspect that two cocoons are formed during copula- tion. Within a few days this surmise was strengthened by finding a freshly deposited cocoon (Fig. 3) encased in a slime- covering entirely similar to that which surrounds the copulating worms. In the following summer I was able to witness the deposition of the cocoons and to find many worms with cocoons in the early stages of formation. Fig. 1 represents about fifty of the anterior segments of two copulating worms. The anatomical features have been so frequently described that it is necessary here only to call attention to the covering of transparent slime that completely surrounds the two worms from the 8th to the 33d segments. In the figure I have represented the average limits of this sub- stance. These limits vary in the 200 or more worms examined ; but I am inclined to think the variation is due largely to the efforts of the worms to escape during the process of capturing and killing them. They were quickly seized and dropped into boiling water, the entire process occupying only one or two seconds, but that would be sufficient time to allow the worms to change the position of several segments in relation to the slime surrounding them. Fig. 2 represents this slime-covering after the worms have been disturbed and allowed to withdraw from it. If placed in water, it will regain its original shape and will be found to be tube-like, sometimes showing the impress of the seg- 482 FOOT. [Vou. XIV. ments of the worms, and so elastic that after pressure it springs back to its original shape. At this stage, and when encasing the cocoon (Figs. 3 and 4), its form is so tube-like that I shall designate it the slime-tube. It can be injected from both ends ; in some cases the colored fluid will flow freely through the entire tube, and again it will fill only one- half, and the other half can be injected with another color. I am inclined to think this semi-independence of the two tubes is the usual condition; for after deposition each cocoon is encased by its own tube (Figs. 3 and 4). There is, however, an intimate connection between the two; for when the copulating worms are disturbed, in many cases one worm will escape first, leaving both tubes around the other worm. When the slime-tube is torn, as seen in Fig. 2, the torn portion can be peeled off from the rest as though it were a separate layer. The entire layer can be pulled off back- wards with a pair of forceps, — can be turned inside out without tearing. Within the slime-tube flows the seminal fluid, containing free spermatozoa and spermatophores. I designate as sperma- tophores the branched bundles of spermatozoa which are aggre- gated at the distal ends of the slime-tube (text Fig. 2). Dur- ing the early stages of the formation of the cocoon, these com- pletely cover the dorsal surface and sides of segments 9 to 11 of each worm and are packed between the two worms at this region. The contents of a partly formed cocoon, when shaken in water, break up into several of these formations, often as many as six, many of them as large as that represented in text Fig. 2. I have never found any eggs in the slime-tube before the cocoons were partly formed, this fact indicating that the eggs are deposited towards the end of copulation. As these worms copulate at a greater or less distance beneath the surface of the earth, at least one function of the slime- tube must be to protect and confine the seminal fluid and spermatophores. If this interpretation be correct, we should find this slime-tube encasing all freshly deposited cocoons of worms which copulate beneath the surface of the earth. I have found this to be the case for several species, and No. 3-] ALLOLOBOPHORA FOETIDA. 483 Vejdovsky (19)! has figured for Lumobricus rubellus a struc- ture which unquestionably answers to the slime-tube of A//o- lobophora foetida, though he does not suggest any connec- tion between this “schleimartiger Fortsatz’’ and the copulating worms. He states also that in all the species of the Lumbri- cidae studied by him, he finds a like structure in connection with the freshly deposited cocoons. Both cocoons are formed while the worms are united, and when they separate each deposits a cocoon, encased by a moiety of the slime-tube. During five summers devoted to collecting and preserving this material, I have seen many cocoons deposited, and in some cases have found the worms copulating so near the surface that by carefully removing a little of the earth, I could watch them for hours, and finally secure and open both cocoons. The examination, however, has necessarily been superficial ; for if enough earth is removed to allow an examination with a lens, the worms soon become rest- less and slowly move away below the surface. In one case they remained undisturbed from g A. M. until 2.52 p. M., and I was then able to secure one of the cocoons, though I was not quick enough to catch the second worm. The worms separate quickly, each drawing back from the other into the hole in which the posterior part of its body has remained during copu- lation. In some cases they leave the two cocoons attached to each other by the slime-tube; but I am convinced that this is not the usual process, but is due to the worms being disturbed ; for in those few cases where I was able to watch without dis- turbing them, only one cocoon was deposited at the spot where the worms had copulated, the second being deposited at some distance from the first. Each cocoon appears to be formed around both worms, encircling the clitellum of one worm and three or more of the anterior segments of the other worm (Fig. 1). As the worms withdraw backwards out of the cocoons, the eight free anterior segments of each must be withdrawn first, leaving a cocoon around the clitellum of each worm; the worm finally leaving this cocoon at the end 1 His figure differs from my Fig. 3 mainly in the fact that the ends of his cocoon are reversed in relation to the slime-tube. 484 FOOT. [Vou. XIV. represented in Fig. 3 by the thread-like extension. Owing to the rapidity with which the worms separate, I have not been able to satisfy myself definitely on several points relating to the formation and deposition of the cocoons. In Fig. 1 are seen four denser, cord-like portions of the slime-tube, circling the anterior and posterior edges of the clitellum of each worm, and binding closely against it three (9-11) of the anterior segments of the other worm. These cord-like portions seem to have contracted until they press into the bodies of the worms like a thread tightly wound around them, and they are so tough that by slipping a needle under any one of them, both worms can be lifted from the table in spite of their efforts to separate and escape. Possibly they are differentiations of the slime-tube; possibly they are of the same secretion that forms the cocoon, though they are present before the slzghtest indication of a cocoon can be detected. They narrow the lumen of the slime-tube (Fig. 2) in planes finally occupied by the ends of the cocoons, suggesting that they may later aid in closing the cocoons. When the cocoon is first deposited, its case is perfectly white, less opaque than the albumen within it, and nearly as soft as the slime-tube. It does not acquire the slightest tinge of yellow until some minutes after deposition. Exposed to the air, it very rapidly changes color, becoming first a delicate pinkish straw-color and assuming later the more distinctly yellow tone. As it acquires this yellow tinge, it becomes more resistant, and finally attains its hard chitinous character. An immature cocoon changes color much less rapidly. After opening such a one and preserving the eggs, the case of the cocoon remained white for more than an hour, and after five hours had acquired only a delicate yellow tinge. This appears to indicate that the secretion which forms finally the chitinous constituent of the case is not formed at the earlier stages of the construction of the cocoon. The slime-tube of the freshly deposited cocoon is transparent, adhesive, and elastic, adhering so closely to the soft white cocoon that it seems part of it and it is difficult to separate the two. As the cocoon becomes yellow and attains its hard No. 3.] ALLOLOBOPHORA FOETIDA. 485 chitinous consistency, the slime-tube loses its slime-like char- acter and elasticity, can be easily torn and separated from the cocoon, and finally entirely disintegrates. Fig. 4 shows a slime-tube around a cocoon, probably containing eggs in the pro- nuclear stages. The tube was found torn away from one end of the cocoon, and its tube-like character is shown by the fact that parts are telescoped into each other. Sometimes the tubes can be turned completely inside out, like the finger of a glove. The time required for the disappearance of the slime- tube depends largely upon the character of the earth in which it is deposited. I have been able to preserve it many hours, and again it will disintegrate in two or three hours. Some- times a remnant will be found adhering to a cocoon containing eggs in the second and third cleavage stages ; but as a rule, there is only a part of the slime-tube left around those cocoons containing eggs in the first cleavage stages. The persistence of the slime-tube until the cocoon has acquired its chitinous character suggests that it may have a protective value for the freshly deposited cocoon. As a rule, in cocoons opened just after deposition, the maturation spindle is at the metaphase and either at the center of the egg or at the periphery (Fig. 5). In these cocoons only an occasional egg shows a spermatozoén just penetrating the periphery. Of the many cocoons either seen deposited or found while still white and soft, I have preserved the eggs from thirty-one, and only two out of this number are exceptions to the above rule. In one of these exceptional cases all the eggs of the cocoon contained a first cleavage spindle. In the other, one of two freshly deposited cocoons found side by side contained odcytes of the second order. This was not due to delay in opening the cocoon, for these eggs were found in the cocoon first opened, while the second contained the usual odcytes of the first order (Fig. 5). I have preserved many freshly deposited cocoons with a view to testing the rate of development, but this uncertainty as to 1 It is impossible to confound the first maturation spindle with the first cleavage spindle; for in normal eggs the latter is always accompanied by pronounced polar rings. 486 FOOT. [VoL. XIV. the stage reached by the eggs in fresh cocoons makes these experiments of little value. After preserving a fresh cocoon for 14 hours in the compost at a temperature of 21 C., the slime-tube was still comparatively fresh and the cocoon con- tained odcytes, second order, with sperm attraction-sphere and rod. Another cocoon, similarly preserved for two hours, contained eggs showing exactly the same stage of development. The eggs in another cocoon, similarly preserved for three hours, had reached the pronuclear stages. In cocoons opened about ten minutes after deposition, only an occasional egg has remained unfertilized, the rest showing the head of the sperma- tozoa just penetrating the egg, or having passed its periphery.” The total number of normal eggs? in 100 cocoons was 399, or about four to a cocoon, which may serve for a rough estimate of the number of normal eggs in each cocoon. At the height of the breeding season, however, the average of normal eggs is greater ; for towards the end of the breeding season (after September 1) a cocoon is often found to contain only one normal egg. In the above-mentioned thirty-one freshly deposited cocoons, the average of normal eggs is about the same ; thus the causes which produce the many structurally disintegrated eggs found in each cocoon must be sought in conditions prior to the deposition of the cocoons. I have opened 453 cocoons and preserved about 1900 eggs, in varying stages of develop- ment prior to the first cleavage. It appears to be the rule that the normal eggs in each of these cocoons have reached very nearly, if not quite, the same stage of development. For example, in a cocoon containing 19 normal eggs, 18 are odcytes, 2d order, the spindle having reached the telophase (z.e., the chromosomes appearing as small vesicles) while the sperm rod has reached the same stage. Only one of these nineteen eggs is an oocyte, 2d order, with the spindle still at the metaphase. Again, among 228 eggs taken from 50 cocoons containing 1 For figures of egg at this stage, see Foot (5), Fig. 3; (6), Fig. to. 2 For figures representing the last stage, see Foot (5), Fig. 1; (6), Fig. 9; (8), Fig. 2. 8 I use the term “normal” here, merely to designate those eggs that do not show any marked disintegration of the cytoplasmic or other structures. No. 3.] ALLOLOBOPHORA FOETIDA. 487 eggs in the pronuclear stages, I find only eight eggs with a cleavage spindle and one with the first cleavage completed. The most marked cases of unequally developed eggs in the same cocoon are those in which one or more of the eggs has reached the metaphase of the first cleavage spindle, while the rest are oocytes, 2d order, — the sperm being still at the rod stage} I have found only two sich cocoons, and in one of these the retarded eggs show varying stages of structural disintegration. Vejdovsky, in his classic work, « Entwicklungsgeschichtliche Untersuchungen ”’ (19), describes the freshly deposited cocoons of the Lumbricidae as “ ziemlich weich,”’ whereas the cocoons of Rhynchelmis, which he has seex deposited, he designates as “ganz weich”’ (p. 36). This, added to the fact that among his figures of eggs of Al/olobophora foctida are none showing a first maturation spindle or a fertilization cone, convinces me that the youngest eggs of A//olobophora foetida found by Vejdovsky are those represented in his Figs. 8 and 9, Pl. XIII, v1z., oocytes, 2d order (fertilized eggs, with the first polar body formed, metaphase of second maturation spindle, and male attraction- sphere), though he has omitted the sperm rod. His figures of Lumbricus rubellus, however, show somewhat earlier stages (Taf. XIII, Figs. 1-4). In Figs. 2-4 he represents a structure undoubtedly answering to the cones of Allolobophora foetida, though he saw no sperm thread in connection with it, and his description (p. 68) of the probable method of fertilization is not supported by the facts, as seen in Al/olobophora foetida. In Pl. XIII, Fig. 10, Vejdovsky has figured what he interprets as an unripe egg ; but I am compelled to question this interpretation and for the following reasons: only in the ovaries have I found unripe eggs, — eggs in the germinal vesicle stage, — never in the cocoons, not even in those seen deposited. This forces me to doubt the possibility of such eggs being found in cocoons containing eggs in the cleavage stages.2 As stated above, 1 Foot (6), Fig. ro. 2 Vejdovsky (19), p. 40: “Dass das Ei der weiteren Entwicklung nicht fahig ist, beweist der Umstand, dass ich es stundenlang ohne jede Veranderung beobach- tete, wahrend die iibrigen Eier desselben Cocons in der Furchung begriffen waren.” 488 FOOT. [VoL. XIV. greatly retarded eggs disintegrate beyond any structural recogni- tion. Thus each cocoon contains eggs at about the same stage of development. I am convinced that Vejdovsky’s Fig. 10 represents an unfertilized egg with the female pronucleus formed. I have found many such in relatively fresh cocoons, z.e., those retain- ing part or all of a disintegrating slime-tube. Sometimes only one such egg will be contained in a cocoon with several others having one or more male pronuclei, and again a larger propor- tion of unfertilized eggs will be found, while in a few cases not even one egg will have been fertilized. The fertilized and normal eggs a/ways show most pronounced polar rings,’ whereas in the unfertilized eggs the polar rings often show various abnor- mal conditions. In some eggs only one is formed ; in others the polar ring substance is still confined to the periphery of the egg (not having aggregated at either pole); in others, again, it is scattered throughout the cytoplasm. The only other figure of which I am aware that represents an unsegmented egg taken from the cocoon, is that by E. B. Wilson (21), Fig. 1. As, however, he shows neither polar rings nor pronuclei, the figure undoubtedly represents a disintegrating egg. The fixa- tive recommended by Wilson himself (Perenyi’s), if applied to normal eggs, has never failed to show pronuclei and polar rings. The latter structures are so pronounced that they can be seen in the living egg under a dissecting microscope. Breeding Season. — The breeding season may be said to begin with the warm days of spring and to close when the nights become cold in the fall. Thus, of the five years I have devoted to collecting material in Evanston and Woods Holl, no two have begun or closed at exactly the same time. Ass the breed- ing worm shows a most marked clitellar region, it is a very simple matter to decide when the season has closed. Worms that, a few days before, showed pronounced clitella, will have that region only faintly marked, or quite indistinguishable from the rest of the segments. Experience has taught me that when a large proportion of worms in a compost heap show these indications of having stopped breeding, it is a waste of time to 1 For figure representing this stage, see Foot (5), Fig. 7. No. 3.] ALLOLOBOPHORA FOETIDA. 489 collect the material, — z.e., to select from these worms those that still possess a marked clitellum. They may continue to deposit cocoons for some days, but as a rule the eggs in these cocoons are either entirely disintegrated structurally, or show abnormal features. At Woods Holl, the close of the season has varied from September I to October 1. It has been possible to collect worms with the clitellar region still marked, and to find fresh cocoons as late as October 20; but few of those cocoons have contained normal eggs. The breeding season closes much earlier in very old compost heaps, — those receiv- ing no warmth from fresh manure. As early as August 15, in such a compost heap containing thousands of worms, it required a search of three hours to find fifty breeding worms. I can support Wilson’s statement (21) that “egg-laying seems in special cases to continue throughout the year . . . but only in decomposing compost heaps, where the temperature is main- tained at a tolerably high point” (p. 394). In 1893-94 I found breeding worms and cocoons in December, January, February, and March, but only in compost heaps that were covered with fresh manure. Method of Obtaining the Fresh Cocoons+—TI select from a compost heap about a hundred full-grown breeding worms, z.z., those having the clitellar region most pronounced, and place them in a one-gallon earthen pot, filled with the compost in which they have been found. To prevent the worms from escaping, it is well to tie over each pot a cloth, with a hole in the center. In order to maintain the average of normal eggs in the cocoons, it is advisable to change the compost about once a week, and to collect a new supply of worms every two or three weeks. If the compost is kept dry, the worms copulate and deposit their cocoons near the bottom of the pot; but if it is kept moist, they come very near the surface. I have fed them with various vegetables and fruits, but have found that the best method is to renew the compost, and to maintain the proper 1 T use the term “fresh cocoons ” to designate those still surrounded by the slime-tube and containing eggs no further developed than the first cleavage spindle. 2 These facts support Vejdovsky’s observations on this point (19), p. 37- 490 FOOT. [VoL. XIV. degree of moisture and temperature. It has proved best to keep them as nearly as possible in a temperature of 21 C., avoiding extremes of heat or cold. Cocoons are deposited at all hours of the day, as fresh ones are found at all hours, vary- ing from 4 A.M.to 6 p.m. They are undoubtedly deposited also at night, as I have found cocoons containing 2-, 3-, 4-, and 6-celled stages at 5 A.M. In midsummer, the early morning hours have proved most favorable for finding the fresh cocoons; but when the nights are cold, better success has been obtained later in the day. At the height of the breeding season from 2 to 10 fresh cocoons have been found daily in a pot containing 100 worms, and the number of cocoons found in these pots leads me to surmise that the worms deposit cocoons very often. Technique. —The cocoon is placed in a small watch-glass under distilled water, and separated from its slime-tube by grasping the long end of the slime-tube with a pair of small, toothed forceps, and with a fine needle tearing it from the cocoon sufficiently to allow the latter to be pushed out. The cord- like projection of the cocoon is then held by the forceps, and with a sharp pair of scissors about one-sixth of the cocoon is cut off at its opposite (blunt) end, the albumen with the eggs being then readily pushed out by pressing the cocoon with the needle. The amount of the cocoon that can safely be cut off is easily determined, for the position of the eggs is readily observed under the dissecting microscope. When the cocoon is first deposited, it is difficult to separate it from its slime- tube, for the former is then so soft that the two appear almost fused. At this stage it is sometimes advisable not to attempt to separate them, but to cut through both with sharp scissors, removing the albumen by pressure. The eggs are set free from the albumen by teasing the latter with very fine needles. The albumen of the freshly deposited cocoons is so adhesive and elastic that it is very difficult to separate it from the eggs without injury to the latter. It gradually loses these proper- ties, however, until by the time the cocoon contains odécytes, 2d order, the albumen can be readily cut or torn by the needles, allowing the eggs easily to be set free. To avoid losing the Se ee No. 3.] ALLOLOBOPHORA FOETIDA. 491 eggs, it is necessary to draw only one egg at a time into the pipette, and for this purpose it is advisable to make extremely small pipettes. Those that have proved the most serviceable have a final aperture of two-thirds of a mm. and are 6 cm. in length. The advantage of the latter is that it allows the hand to rest on the stage of the dissecting microscope. The fixative recommended by Vejdovsky (19), chromo-acetic, has proved the best for whole mounts, and allows the entire egg to be studied under a very high magnification. Alum cochineal has proved the most satisfactory stain for these whole mounts. In order to study these eggs with a Zeiss 2 mm. immer., it is necessary to mount them with great care, as this lens has a working distance of about .2 mm. and many of these eggs measure .15 mm. I found it necessary to make glass feet by cutting square cover glasses of the proper thickness into nar- row strips, about 15 mm. X 3 mm. It is safer to use four of these, inserting half their length under the cover, in order to be able to push it in any direction a sufficient distance com- pletely to revolve the eggs. It is essential to ascertain the diameter of the egg, the thickness of each foot, and the thick- ness of the cover glass. A working distance of .23 mm. can safely be allowed, as it is not essential to focus entirely through an egg. Ifa first cleavage stage measures .16 mm. one can safely use feet measuring .17 mm. and a cover.06 mm. These very thin covers can be obtained only by special order. The feet are now in the market ; but as their thickness is variable, it is not safe to use them without remeasurement. They can, however, be cut in the laboratory as quickly as those of card- board. This method is given in detail to avert any blunder similar to one made by myself. In the fall of 1893 I was obliged to remount all the material collected the previous sum- mer, as the mounts were made with the regulation paper feet. It was only after much experimenting with both feet and cov- ers, that the above method was reached. Thin layers of isin- glass were first utilized as covers, but they have too much spring to admit of being safely pushed over the feet, and are thus apt to crush the eggs. Any number of eggs can be 492 FOOT. [Vor XIV. safely oriented on the slide by using a single hair inserted in the regulation needle holder. The general distribution of this material and the possibility of studying the whole egg under a high magnification make it of especial value for class work, and I publish the above detailed description of obtaining and mounting the whole eggs, in response to requests from those who wish to use them for demonstration in the class room. For the present, however, I reserve the publication of my methods of imbedding and sectioning. As only a few hundred eggs can be collected each year, the time required for obtaining all the successive steps in the development is necessarily prolonged and uncertain, and I shall fill later such gaps as I am now obliged to leave. In the present paper I shall briefly state a few of my earlier observations. OBSERVATION ON THE LiIvinG Eac. (Zeiss 0b. 10 to hom. tmmer. 2 mm. 140 ap.) The egg must be removed from the albumen in order to study it with high powers. Distilled water (a few drops), with as much of the albumen dissolved in it as is consistent with preserving the necessary degree of transparency, has proved the least injurious medium for examination. In this the eggs have grown and developed normally from 30 to 60 minutes. The normal condition has been tested by removing the eggs from this medium, at definite periods of time, killing, imbed- ding, and sectioning them for comparative study. The following phenomena have been seen in the living egg: the maturation spindle in the unfertilized egg, and an indication of the rays of its attraction-sphere ; the fertilization of the egg, z.e., the appearance of the cone ; the constricting off of the first polar- body, and the subsequent disappearance of the cone ; the con- stricting off of the second polar-body, and the appearance of the polar rings. Not all these phenomena, however, are seen in any one egg; for the artificial conditions in which the egg must be studied soon arrest normal development. The first No. 3.] ALLOLOBOPHORA FOETIDA. 493 appearance of the cone is indicated by a lighter area at the periphery, this projecting beyond the periphery and the pro- jection continuing for some minutes after the cone is com- plete. The cone remains sharply differentiated from the rest of the cytoplasm during the constricting off of the first polar- body. The spindle is indicated by a lighter area clearly differ- entiated from the rest of the egg. The time occupied by the formation of the first polar-body has varied from 22 to 45 min- utes, and I am inclined to think that the artificial conditions hasten development; for in the above-mentioned cases (p. 484) where fresh cocoons were kept in the compost 14 and 2 hours, the eggs were still odcytes, 2d order. The changes in the shape of the egg during the process agree with those described by Vejdovsky ( (19), p. 51, Fig. 2) for Rhynchelmis, except that as the constricting progresses, the periphery of the egg at that point flattens, and finally becomes concave, the polar- body resting in the center of the concavity. At this stage, the membrane over the polar-body is so taut that the latter is often somewhat flattened by its pressure, suggesting that this pressure of the polar-body against the periphery of the egg may be at least partly responsible for the above-mentioned concavity. The phenomenon as seen in the living egg accords entirely with my observations on fixed material. A small part of the cytoplasm appears to be pushed out by the spindle, the latter first moving to the periphery and then projecting beyond it until its equator has reached the periphery of the egg, when the process of constriction takes place. The time occupied by the formation of the second polar-body has varied from 20 to 60 minutes, the two phenomena (the formation of the first and second polar-bodies) being similar. In each case a lighter area first appears at the periphery, the egg slowly elon- gates from this point, and the process terminates in the comple- tion of the polar-bodies.!_ Preserved material shows that the first polar-body divides by mitosis. Vejdovsky (19) has figured this in Taf. XIII, Fig. 8. Before, during, and after the formation 1 One expression of a pathological condition of the egg is a constricting off at the periphery of small portions of its cytoplasm, and care is necessary to avoid confounding these with the polar-bodies. 494 FOOT. [VoL. XIV. of the polar-bodies, the egg turns slightly at indefinite inter- vals. These movements are not oscillatory, as sometimes three consecutive turns will occur in the same direction, revolving the egg one quarter its diameter. Later they appear to change the relative position of the egg and first polar-body, thus removing the first polar-body from the path of the second. Pathological changes in the egg, which are induced sooner or later by the artificial examination medium, seem to appear first in the cytoplasm. An egg studied during the formation of the second polar-body (60 minutes) and for three hours thereafter, showed marked pathological changes in the cyto- plasm. The polar rings did not develop, and the cytoplasm finally appeared as a mass of large vacuoles. During the period of these changes in the cytoplasm, the pronuclei con- tinued to develop ; for after killing, staining, and mounting the egg, the pronuclei were found to have reached their maxi- mum size, and were in contact in the center of the egg, neither showing pathological features. The cytoplasm, however, showed the same pathological condition seen in the living egg. As I am at work on a paper which will give the results of a comparative study of the living and fixed cytoplasm in these eggs, I shall omit here any description of the living, normal cytoplasm. A large number of living eggs have been measured with a view to testing the shrinkage or swelling produced by various fixatives. The living odcytes, Ist order, have varied in size from 100p to 132m, the odcytes, 2d order, from 114m to 140p, the ripeegg from 128 to 144, the pronuclear stages from 1364 to 152. These figures differ slightly from those given in my preliminary note (5), owing to the fact that the measure- ments were taken from fixed material. The eggs are not spherical at all stages; but these details will be illustrated later in a series of photomicrographs. Spermatozoa. — Free spermatozoa can be procured by removing the slime-tube from copulating worms, drawing out part of its contents with a fine pipette, and drying on slides a thin layer of the seminal fluid thus obtained. They can be procured from a freshly deposited cocoon by drying on slides No. 3.] ALLOLOBOPHORA FOETIDA. 495 a thin layer of the albumen of the cocoon. In this case, it is well to preserve and mount the eggs in the cocoon, in order to test the stage of development reached by the spermatozoa, by ascertaining whether the eggs are at the exact stage to be fer- tilized ; for, as stated above, I have found freshly deposited cocoons containing eggs past the fertilization stage. Com- parative measurements of the spermatozoa in the slime-tube, in the freshly deposited cocoon, and in the cocoon containing fer- tilized eggs, show only a slight difference in size. I wish here to correct a statement made in my preliminary note (5). I stated there that the full-grown spermatozoa taken from the freshly deposited cocoon were about 2% times the length of those in the immature cocoon, and that they did not respond to differential staining. Further investigation has proved that the structures from which these measurements were taken are not normal spermatozoa, and as at the time I had not learned to control the investigation by preserving some of the eggs, I am convinced that these measurements were taken from spermatozoa in cocoons containing fertilized eggs. These spermatozoa of abnormal growth vary greatly in size, and are relatively far more numerous in the cocoons containing ferti- lized eggs. In some cases they appear to be developed by an abnormal growth of the head alone, while in others, though all parts select the same stain, the spine, head, and tail can be clearly identified. When stained with a chromatin and plasma stain, they, as a rule, select the latter; for example, with Biondi-Ehrlich all parts select the red, whereas the heads of the normal spermatozoa select the green. The large speci- mens suggest the giant spermatozoa of authors, though in this case they appear to be merely hypertrophied spermatozoa (text Fig. 1).1 In the cocoons there are relatively few sperma- tozoa in which the spine, head, middle-piece, and tail respond to differential staining as do those in the slime-tube. Their 1 In cocoons containing fertilized eggs, besides the individual spermatozoa, there appear to be attenuated masses of degenerating spermatozoa. Just such masses can be seen in certain spermathecae in which the spermatozoa show abnormal features, and in some cases from two to four heads are fused, making a relatively thick mass, whereas the tails are separated and can be counted, indicat- ing just how many heads are fused. 496 FOOT. [Vou. XIV. number is very small as compared to the masses in the slime- tube and spermathecae, and for that reason IJ at first (errone- ously) interpreted them as retarded individuals. I have since found that the head and tail of those in the fertilized egg respond to differential staining. As many as nine spermatozoa have been found in one egg ; but in such cases they do not all have an attraction-sphere. It A B Fic. 1.— A, hypertrophied spermatozoén; B&, normal spermatozoén. Camera. X about 687. appears as though the cytoplasm of the egg finally responded no longer to the stimulus. In many cases the tails can be identified within the fertilization cone. In two cases two spermatozoa are in the same cone without producing any apparent disturbance in its form. SPERMATHECAE. Authors agree in ascribing to the spermathecae the function of collecting the seminal fluid during copulation and storing it until it is needed for the cocoon, which has been supposed to / No. 3.] ALLOLOBOPHORA FOETIDA. A97 be formed by each worm subsequent to copulation.!. The fact that both cocoons are formed during copulation seems to indi- cate that another function must be found for the spermathecae; and yet these, when crushed on a slide, stained and mounted, are found to be packed with spermatozoa. If one taken from a breeding worm is placed on a dry slide and pressed with a needle, its contents flow out in the form of a thick, viscid, cloudy fluid. Adding a few drops of water to this fluid and examining it under the microscope, shows it to be a mass of extremely active spermatozoa, the tails moving very rapidly and propelling the heads, which remain straight. When these spermatozoa are mounted and stained, comparative measure- ments indicate that they have reached the same stage of development as those in the spermatophores and seminal fluid of the slime-tube. Careful measurements have been taken of the various parts of several hundred spermatozoa found in the seminal vesicles, spermathecae, spermatophores, seminal fluid of the slime-tube and freshly deposited cocoon,” with the hope of determining whether the spermatozoa in the cocoon are from the spermathecae, but the differences in size were too slight to be of any value as a determining factor. An examination of the spermathecae of worms having just deposited their cocoons seemed to promise the only decisive answer. Thus far I have been able to examine the spermathecae of only three worms 1 In studying the literature I have been able to find only one report that sug- gests the cocoons being formed during copulation, vz., Vogt u. Yung. (Verglei- chende Anatomie, pp. 481, 482). “ Wahrend der Begattung legen sich die beiden Wiirmer mit ihrer Bauchseite in entgegengesetzter Richtung derart an einander, dass der Kopf des einen dem Schwanze des anderen zugekehrt ist und dass die Geschlechtsoffnungen mit dem Giirtel wechselseitig in Beriihrung sind. Der in Form von kleinen weisslichen Massen ergossene Samen nimmt in zwei durch eine Vertiefung der Korperdecken gebildeten Langsrinnen die Gestalt kurzer Cylinder an und fliesst so zum Giirtel, um sich von dort in die Samentaschen zu begeben. Die beiden Wiirmer sind alsdann durch einen Ring von Schleim mit einander verbunden, der vom Giirtel und vielleicht auch von den Nebendriisen abgeson- dert wurde, deren Gegenwart in der Nahe der Geschlechtsorgane wir erwahnt haben. Die durch die Miindungen der Eileiter ausgetretenen Eier gelangen zum Giirtel, wo sie von Schleim eingehiilt werden, in welchem man Samenthierchen wahrnimmt und der fiir sie eine Kapsel von eirunder Form bildet.” 2 In these the length of the spine has been found to vary from 4u to 7u, head 24u to 34u, middle-piece 2u to su, tail 544 to 67u. The longest head found in the fertilized egg measured 34u. 498 FOOT. [Vox. XIV. with nearly completed cocoons, and in all these cases the sacs were found to be empty and flattened. In some cases a large mass of spermatophores was aggregated at one end of the slime-tube, covering ‘segments 9 to 11 of one worm, while the same segments of the other worm showed no spermatophores. In the former case the spermathecal sacs were empty; in the latter, full. An examination of the spermathecae of twenty worms that were not copulating showed only one with empty spermathecae. This indicates that the sacs are not filled and emptied during one copulation. The above facts seem rather to necessitate two copulations, one to fill the spermathecae, and one to form the cocoons. I have several times watched copulating worms from three to five hours consecutively, and seen them separate without being able to identify a cocoon in any stage of formation. Copulating worms are, however, so y if ) SS WN SQ ei Zp NS Fic. 2.— Spermatophore. x 150. easily disturbed that it would be necessary to repeat this observation a great many times, as well as to examine the spermathecae after the worms have separated, before accepting such observation in proof of a second copulation. SPERMATOPHORES (text Fig. 2). The spermatophores are formed after the spermatozoa leave the spermathecae. The contents of the latter when stirred with a needle in water will disintegrate, each spermatozoon No. 3.] ALLOLOBOPHORA FOETIDA. 499 becoming individualized and the mass forming an approximately homogeneous fluid, whereas the spermato- phores do not disintegrate in water, even after twenty-four hours’ immersion. This suggests that the spermatozoa, after leav- ing the spermathecae, come in contact with some adhesive substance which welds them into masses and confines them within the area which is occupied later by the cocoon. Removing the slime-tube from worms with partly formed cocoons, I found on the surface of segments 9g to II, inclusive, tiny opaquely white specks (some- times as many as nine) apparently issuing from integumental orifices. These, like the spermatophores, did not disintegrate in water, and when stained and mounted they showed a very definite granular structure (text Fig. 3). The glands which secrete this substance can be seen on dissecting the worm from the ventral surface and removing everything but the spermathecae, leaving the inner surface of the integument exposed. On segments 9 to 11, inclusive, are tiny opaquely white swellings. If these are pricked with a needle, an opaquely white substance can be pressed from them, which, when stained and mounted, entirely resembles that described above as found on the exterior of the same segments. In some worms these tiny swellings are quite numerous, one being close to the stem of each spermatheca and at least four in each of the three segments, these being distributed in the center of the segment as well as close to the dissepiments. If the center, more dense part, of a spermatophore is crushed on a slide, stained and mounted, we find the same granular secretion as in the above-mentioned integumental glands of seg- ments 9 to 11, and on their exterior surface. Thus it appears to be demonstrated that the spermatophores of this worm are formed by the spermatozoa aggregating around the gran- ular— probably nutritive — substance secreted by these glands. Archoplasm.— The appearance of the archoplasm (Foot (6) ) in the living egg indicates that it is at least semi-fluid, and this interpretation is in accord with the history of the archoplasm, 500 FOOT. (VOL. XV). as traced in fixed material. Its relatively rapid change of position in the egg, its accumulation at all the centers of activ- ity, — spindle, cone, and sphere, — its subsequent aggregation at the periphery, and its final massing at the poles indicate that it is not a mere condensation of the cytoplasmic net- work; for the migration of such points of condensation from the periphery to the poles would cause a marked disturbance of the network. The differentiation of a portion of the archo- plasm in both spindle and spheres supports the observations of those investigators who have differentiated a specific sub- stance in either of these two structures. Strasburger (18), Boveri (2), Meves (13-14), George Niessing (16), Carl Niess- ing (15), Henneguy (10), and von Klinckowstrém (12). Whether the archoplasm can be identified with the “hyalinen Grundsubstanz”’ Vejdovsky (19), p. 40, has observed in these eggs, Iam unable to determine. Thus far I have been unsuc- cessful in attempts to differentiate anything else that suggests the substance in question; but this fact can scarcely be regarded as evidence of its identity with archoplasm ; neither have attempts to differentiate the cell-sap of authors proved successful; but I am not at all prepared to say that for this reason the archoplasm must be identified with the cell-sap. The fate of that portion of the archoplasm which forms the polar rings has not yet been ascertained ; neither am I prepared at present to discuss the difference in form between the two rings, nor the lack of constancy in the form of either ring. At the pronuclear stage, — when the two masses are complete, —- their position in relation to each other appears to be constant. The cleavage-planes, however, stand in no constant relation to these structures (the substance is not divided between the cells), and even the first cleavage sometimes assigns both polar rings to one cell, no part of them being consigned to the other. Centrosome. — Since expressing the belief that the cleavage centrosomes are not derived from the middle-piece of the spermatozoon (7), and that the middle-piece (the posterior end of the head of the spermatozoon) produces an effect upon the cytoplasm comparable to the effect produced on it by the No:3:]] ALLOLOBOPHORA FOETIDA. 501 spine (the anterior end of the head of the spermatozoén), I have made the following observations on this point. I have seen very early stages of the development of the sperm attrac- tion-sphere, — stages where the middle-piece of the sperm is still intact, while the rays of the attraction-sphere are focused around it, just as the rays of the cone are at one stage focused around the head of the spermatozodn. I hope to be able to illustrate the morphological resemblance of these two struc- tures in a series of photomicrographs. Cytoplasmic Granules or Microsomes.—In an earlier paper (7) I published a list of structures in these eggs, which select methyl green, among them numerous “ large and small granules or bodies,” also the centrosomes and nucleoli. I have since succeeded in differentiating from these structures the cen- trosomes and the nucleoli, the latter being found in the cyto- plasm of the odcytes, Ist and 2d orders, as well as in the pronuclei. The details of this work will be published later. Spindle.---The phenomena in this egg indicate that at least part of the first cleavage spindle has its origin in the cytoplasm ; as the polar rays are often formed while the mem- brane of both pronuclei is intact. When an isolated male pronucleus has reached its maximum size, its attraction- sphere is seen in the cytoplasm, while the membrane of the pronucleus is still intact. Occasionally a spindle is formed by the meeting of the rays of two male attraction-spheres, or the rays of a male attraction-sphere with those of the egg attraction-sphere at the lower pole of the second maturation spindle. All these rays anastomose, as do the fibers in the first and second maturation spindle.! Reduction. —Twenty-two chromosomes can be counted in the odgonia and only eleven in the first maturation spindle ; thus we have the typical number reduction of chromosomes, — the ‘“pseudo-reduction” of Riickert (17). The position and shape of the chromosomes in the spindle accord with Flem- ming’s (4) heterotype form of division. In many cases four distinct parts can be differentiated (text Fig. 4), the typical tetrad being thus repr