Riles Roenge gee SBI. nie 1 tat \ if 7 Wahl ta, 4 Vinnie CEN i Dl bi eS La vt: TARR fee JOURNAL OF MORPHOLOGY FounpDEp By C. O. WHITMAN EDITED BY J2 Se KINGS DBY University of Illinois Urbana, Ill. WITH THE COLLABORATION OF Gary N. CALkINS Epwin G. CoNxKLIN C. E. McCuune Columbia University Princeton University University of Pennsylvania W. M. WHEELER WILLIAM PATTEN Bussey Institution Harvard University Dartmouth College VOLUME 33 DECEMBER, 1919-MARCH, 1920 THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA IAUVAUOL 1O YVDOIOHTAOM. vawrTinY! O . ra aganvod Ya daTidga Ya€T@aoula .a .t sionilld to ytisieviaU fil .anadiU 70 UOITAHOGALIION ANT ATIW ougdOolM A.D wvlWaKoO . urvad avindaAD .Wyvaan ninsviveaned to wietsviaU wtiereviaU aoissa2rmrd wiiaioviaU sidmsloOD VaTTAY MALLILV! sguqadHW VM .W 7 svolfoO divomiirG gdievinU brevieH soisjuitienl vaeer4 66 dMJTOV 1") (Al (-@fei AA Hfé AMV AOHd YNOLOIA GUA YMOTAYA TO ATUTITAAL AATAIW AHT ATHAIGT ATLAS CONTENTS No. 1. DECEMBER, 1919 GrorGceE T. Hareirr. Germ cells of coelenterates. VI. General considerations, discussion, conclusions. Thirty figures (three plates).................... Cart L. Husss. A comparative study of the bones forming the opercular Series Of fiSWeS.... ss: PONY PUR ROT PEL ee) VR. AS es cl aN sete ont, LE PT GrorGE Ortnay SHtnsI. Embryology of coccids, with especial reference to the formation of the ovary, origin and differentiation of the germ cells, germ layers, rudiments of the midgut, and the intracellular symbiotic organism. One hundred thirty-three figures (twenty plates)...................0.... CHARLES L. PARMENTER. Chromosome number and pairs in the somatic mitoses of Ambystoma tigrinum. Thirty-seven figures (nine plates)...... No. 2. MARCH, 1920 Hacuiro Yuasa. The anatomy of the head and mouth-parts of Orthoptera and EKuplexoptera. One hundred and sixty-three figures (nine plates)......... Wm. A. KeEPNER AND FRANK HELVESTINE, JR. Pharynx of Microstoma cauda- hime One text iimure and three, plates)... « saeeiee «es + ve La ote ee H. D. Resp. The morphology of the sound-transmitting apparatus in caudate Amphibia and its phylogenetic significance. Eighteen text figures and six DISteS erty meee tea, Some A... 7: es Se Rae, GEORGE W. TANNREUTHER. The development of Asplanchnia ebbesbornii (Rotifer). Twenty-one text figures and seven plates.................. Tracy, Henry C. The elupeoid cranium in its relation to the Seannicndides feeeonlaen and the membranous labyrinth. Three figures and four | SLAPS SS ai co eee erie Re ke 8 a” SRR ty ees O. W. Hyman. The development of Gelasimus after hatching. Twelve plates [STP SUN 8 1 es CTT 2h) A ec kA 8 cl Louise Smita. The hyobranchial apparatus of Spelerpes bislineatus. Fifteen 61 73 169 251 309 325 . 389 439 485 PIAES MEDLEY Se VEN OES). Yam oie: An siert’s |< «de nde Sp 5abS le cite better 527 AUTHOR’S ARSTRACT OF THIS PAPER ISSUED BY THE BIBLLOGRAPHIC SERVICE, OCTOBER 13 GERM CELLS OF COELENTERATES VI. GENERAL CONSIDERATIONS, DISCUSSION, CONCLUSIONS GEORGE T. HARGITT Zoological Laboratory, Syracuse University THIRTY FIGURES (THREE PLATES) CONTENTS LMNELOOUUGETOTIN ES Seren etree te eae elec aeelas siatereeta ie arena Os efor spenaters sumac de 1 filer Orieineor eerm cells 255 520/520 adele ite oc ope SE lS cle do shtte ee aye ote 2 Pep reeam igor mire. tone ke ackvas aie Cgc ates.) 3S hale chek tye eo endosa Ice terns 2 Ady Wheaten ieee a Fea 1) See nC CRS Ca eRe ee cee stn cree eans feces 3 2 (Clone hinerornes Se et Ge io Sic BRE Me SiGe 12 Mik Phe eerm-—plagm theory... 22. ee. c-fos ois + + ol elete misters 2 ee sm ewialo ne see 12 1. General statement and discussion of the theory.................. 12 DE GaGeMGG ATOM ERY GTOZOR. 02-5 oy So.5 ease - tere ee as lee» = else 14 Mea COTITINCE lie ee ee ce tokcntte >: oo. RRR veins © ateveiey 14 [hs Eek TaN aed ee ORS 2 tae Seo AoE eo! aNd Ae Soe 15 t RE PENERAbION. e422 ds ant eAR A Tite 2s «dee bate = shake id (oso ae ho's 21 ak, IDTSRGvermniGG! Calle co oe Cac oan ob umoneeaebo ++ oOnoeeaoe Ob ber 23 3. Evidence from germ cells, outside of Hydrozoa................... 26 A Bvidence from tissue culitmes..+....i....--.--satsaus-s. eo sane Bo. 27 Seebi vid encemmonmi cancernce lsu rerriy slat. « Sekar sts) ie iets rere 29 6: Summary, and canelusions). 446.04... 5. 80 BM te rae. Pah). et tae oes setae geet. one eee mas ce Baer 84 PEG erage ta. si sa lashes fable es acces 9 ot SSA Iepe rea eee Pe 87 5 Establishment of the external form of the body seer. «ee isl 90 6 The formation of the germ layers and embryonic envelopes....-...----- 97 7 The formation of the nervous system.......------+-+srrrrrererettte 101 Br Intracellular symbiosis. {25.0 ¥e-% 222 Wen = + se + oh oeeiease meri e a * Aine c's 105 9 The origin of the germ cells........-..-----s+esseree erste tert 112 10 The formation of the digestive tract........-.---.+seerrseretretsr tees 118 iy Coppedge) Sok a See ees OA ices neo a te at ately 121 Bbliaprapliye, teeta. ake eee heed oe « iS eald yelp ig IS 127 1. INTRODUCTION Historical On account of their economic importance, scale insects have been the object of extensive observations by several inves- tigators. The literature pertaining to these insects is, however, limited in its scope, being mostly concerned with the external morphology, general accounts of the life history, habits, and methods of control. The accounts of the formation of the egg and subsequent history of the development of coccids are mostly 1A thesis submitted to the faculty of the Graduate School of the University of Missouri for the degree of Doctor of Philosophy. 73 Vt: ae GEORGO ORIHAY SHINJI fragmentary. One of the earliest works of this kind was by Leydig (’54), who described and figured the general appearance of the ovaries of Lecanium (Coccus) hesperidum and its eggs with three nurse cells and an egg cell. Although he did not actually describe the process of differentiation of the ovarian elements, he claimed that nurse cells, egg cell, and the epithelial cells must have arisen from undifferentiated germ glands. He also described the formation of the embryo from the egg by multiplication of the single egg cell. The statement that this form is really viviparous was made in this article. He also pointed out the presence of numerous pseudonavicellae in this insect. These organisms, according to him, migrate into the egg at its posterior end and multiply rapidly by budding. Leuckart (’58) has also studied the ovarian structure of Lecanium hesperidum and found that three nurse cells and an egg cell developed from epithelial cells. Therefore he main- tained, as his predecessor did, that the nurse cells and the egg cell are the modifications of the epithelial cells. Lubbock (’59) also came to the same conclusion, namely, the nurse cells, the egg cell, and the epithelial cells are all originally undifferentiated cells of the germ rudiment. The most complete account of the development of coccids was, however, presented by Mecznikow (’66). His work on coccids is not so complete as was that for Aphids, Corixa, and Cecidomia. Nevertheless, it covers the development of the Aspidiotus nerii from a single egg-cell stage to the time of hatch- ing. He described and figured a differentiated egg with its germinal vesicle. The appearance of the ‘Wulst’ prior to the formation of the blastoderm and the invagination of the blasto- derm near the posterior pole of the egg were also mentioned. One layer of the invaginated ‘Keimhiigel’ degenerated to form the amnion while the other developed into the embryo proper. However, he failed to observe the phenomenon of revolution of the embryo. The entire alimentary canal, he thought might be formed by further elongation of both stomodeum and proc- todeum. He described and figured an early appearance of the germ cells and of the pseudovitellus. EMBRYOLOGY OF COCCIDS Tas Brandt (’89), who likewise studied Lecanium hesperidum and Aspidiotus nerii, with special reference to the embryonic cover- ings, stated that the embryo of Aspidiotus nerii was bent, as Mecznikow had already described, with its caudal part over the oral portion. He further observed the process of the revolution of the embryo following the rupture of the amnion. In Aspidi- otus, he observed, as did also Mecznikow, that the ventral plate of the embryo was found lying closely on the amniotic covering, even before the revolution, so that the yolk was rapidly removed from this region. Then followed the work of Putnam (’78) on the cottony maple scale, Pulvinaria innumerabilis. This writer evidently thought the ovarian eggs were In some manner, unknown to him, attached to the body cavity by their free or anterior end, where the dif- ferentiation first takes place. Besides this mistake, his figures are too vague to show anything very definite. However, this much was sure, that the eggs of the cottony maple scale developed within the body of the female. He detected the presence of the pseudonavicellae in the female before mating and also in the older eggs. The method of infection or the possible migration of these bodies into the eggs was not studied. However, he suggested that these bodies, having higher specifie gravity than water, may represent the metamorphosed state of spermatozoa, and that their presence in the egg may be comparable to the phenomenon of fertilization. Witaczil’s (86) work on the anatomy of the coccids contains an account of the differentiation of the nurse cells and of the egg cell from undifferentiated epithelial cells in the ovaries of Leucaspis pini. Although not figured, there is an account of the presence of the so-called nutritive string between the nurse chamber and the egg chamber. He did not find the pseudovitellus in the eggs of several species, but expressed the view that the pseudovitellus of Mecznikow may represent a mass of yolk granules. The investigations above mentioned were entirely made upon fresh material or at least upon material prepared in toto. The eggs were usually studied in water to which acetic acid and sugar were added, or in somewhat similar solutions. The only paper, 76 GEORGO ORIHAY SHINJI the result of the study of the sectioned material, fixed and stained in accordance with modern microscopic technique is the contribution by Emeis (’15). The sole purpose of his article was to present the history of the three ovarian elements, namely, the nurse cells, the egg cell, and the epithelial cells. He did not, however, show whether the epithelial cells, from which the egg and the nurse cells develop, come from the primordial germ cells or from the original mesoderm. He was also not sure whether or not a quan- titative or qualitative cell division takes place among the early oogonial and oocytal cells. His cytological accounts of the ovarian cells do not include the phenomenon of the polar body formation. - Nevertheless, the most interesting feature of the article is the discovery of the symbiotic organisms in the egg as well as in the epithelial cells. As the foregoing brief survey of literature indicates, two phases only of the development have been confirmed. The rest of the accounts still remain to be confirmed or rejected, while the origin and subsequent history of the pseudovitellus and the Pseudo- navicellae demand a new and careful investigation. Again the development of several organs (respiratory, circulatory, sensory, and secretory) remains entirely undescribed. The purpose, then, of this work is to contribute as much as possible toward the embryology of certain scale insects, with, however, especial reference to the history of the pseudovitellus, the germ cells, germ layers, alimentary canal, and nervous system. Before going further, I take this opportunity to acknowledge my indebtedness to Professor Haseman, of the University of Missouri, with whom the work was carried on. My hearty thanks are due to Mr. Hollinger, who not only helped me in the collection and identification of the material, but also gave valu- able information, and, above all, daily encouragement; and to Mr. Severance, of the library, through whose effort many valuable journals in the library of Congress and of other institutions were made available to me. Last, but not least, obligation is due to Professor Woodworth, of the University of California, with whom the study of the cottony cushion scale was originally begun. EMBRYOLOGY OF COCCIDS CE Material and methods Three species of Coccidae belonging to three different genera were chosen for the present investigation. These are: the mealy bug, Pseudococcus medanieli Hollinger (ms.); Hunter’s Lecaniodiaspis, Lecaniodiaspis pruinosa (Hunter); the cottony cushion scale, Icerya purchasi Mask. The material of the mealy bug was obtained from its most favorite host plant, the ragweed (Ambrosia trifida Linn.), on two trips in the latter part of September and another in early October, while that of Lecaniodiaspis was collected from time to time on two elm trees on the campus of the University of Missouri, during the season of 1917 to 1918. The cottony cushion scale was col- lected from Acacia and Pitosporum found in the vicinity of San Francisco Bay, California, during the seasons of January, 1915, to May, 1917. Of the three species each had its own advantages. The eggs of the mealy bug were very easily fixed, sectioned, and stained, but they were so small that it was difficult to dissect away the chorions. The eggs of both the cottony cushion scale and Lecan- iodiaspis are large, and the chorion can be removed easily. The eggs of the Lecaniodiaspis, however, stain with considerable difficulty. In fact, the egg of the cottony cushion scale was the most favorable material, having none of the disadvantages above mentioned. Yet it must be said here that it was with the study of Lecaniodiaspis and to a considerable extent with that of the Pseudococcus that the writer was able to see the true significance of several organs and inclusions. The experimental method of determining the age of the embryo was tried to a considerable extent with the cottony cushion scale. For this purpose, about thirty adult females with the egg sacs were collected, together with the infested twigs. After removing the egg sac with a sharpened bamboo stick, the females were placed in small paper boxes. Every five or ten minutes the specimens were observed, and if an egg was seen protruding from the vaginal orifice, it was transferred into a numbered gelatin capsule. In the capsule, the egg was able to develop even to the 78 GEORGO ORIHAY SHINJI time of hatching. Thus the experiment went along with promise of success until several of the eggs supposed to be of the same age were fixed and mounted in toto. The examination of these prepared specimens, however, showed that no two of them were in the same stage of development. One of them contained an embryo nearly ready to hatch, another had an embryo with its appendages well recognizable, while the remainder were mostly in much earlier stages. Sectioned material of several adult females of both the mealy bug and cottony cushion scale brought to light the fact that the eggs of these two species of coccids undergo a partial devel- opment in the uterus of the female. The early deposition of the egg was usually noticed in specimens in which the growth of the ovarian eggs was in rapid progress. The eggs were not deposited until the completion of the blastoderm. The eggs of the Lecaniodiaspis, on the contrary, were deposited at the first cleavage stage, and should serve as the most desirable material for this purpose. Unfortunately, I have failed to work with this species during the past year. Thus the determination of the age of the eggs by the experi- mental method alone is not reliable. Therefore the relative ages of the embryos in this study were mostly determined by the number of cells, the position of the polar granules, the length of the embryo and of the appendages, and other morphological features. Most of the material for embryological study was obtained from egg-sacs of the fully matured female scales in the following manner: Several females with their egg-sacs were collected at various times of the day, and the egg-sacs were separated from the body with a sharpened bamboo stick or needle. In some cases the eggs were lightly shaken out of the egg-sac into a watch- . glass containing the fixing fluid, but in many cases the entire egg- sacs were dropped directly into the watch-glass containing the fixing fluid, and the cottony substance was removed afterward with a pair of sharpened bamboo sticks. The use of sharpened bamboo sticks proved to be advantageous, for they can be made of any desired sharpness and they are not acted upon by such corrosive mixtures as Gilson’s. EMBRYOLOGY OF COCCIDS 79 One of the fixing reagents most extensively used was Carnoy’s aceto-aleohol-chloroform mixture, prepared by mixing thoroughly equal parts of absolute alcohol, glacial acetic, and chloroform saturated with corrosive sublimate. Eggs fixed in this mixture for from one to two hours lost their red pigment and became transparent. They were then washed in 30 per cent alcohol for two hours, and passed through 50 per cent to 70 per cent alcohol with an intermission of one hour. They were then either left in 70 per cent alcohol until needed or dehydrated by passing them up through 90 per cent, 100 per cent alcohol to xylol and imbedded in 52° to 58° paraffin. Sections were cut from 5y to 7u in thickness and stained mostly with iron alum haematoxylin followed by eosin, orange G, acid fuchsin, or a mixture of these. The triple stain, saffranin-gentian- violet, and orange G, was also frequently used with very good results. For whole mounts, the eggs were passed from 70 per cent to 30 per cent alcohol, in which the chorion was dissected away under , a binocular microscope. Embryos thus freed of their chorions were stained with a diluted solution of gentian violet for from one to six hours and then decolorized with 70 per cent alcohol. Dela- field’s haematoxylin, borax carmine, and alum cochineal have also been used with fairly good results. The most beautiful specimens, however, were those that were treated with gentian violet. For the study of the history of the germ cells not only the genital organs of the embryo, but also those of several stages of larvae, pupae, and adult scales were necessary. Ovaries were mostly dissected out and fixed in either Fleming’s or Zenker’s solution. In many cases, however, whole larvae, pupae, and adult were put directly into Gilson’s or Carnoy’s aceto-alcohol- chloroform solution, sectioned and stained in the same manner as in the case of embryos. Perenyi’s solution was also tried, but all except the aceto- alcohol-chloroform mixtures were useless unless heated to 70°C., because the eggs as well as larvae are covered with a waxy sub- stance which prevents penetration of fluids. When heated, these JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1 SO GEORGO ORIHAY SHINJI fixing fluids as well as ordinary water will kill and fix the specimen, but in such a case the finer details of nuclear structure are often destroyed or distorted. 2. THE ORIGIN AND DIFFERENTIATION OF THE OVARIAN ELEMENTS The germ cells in the ovaries of the larvae at the time of hatch- ing are similar in size and appearance (fig. 1). By mitosis, these germ cells multiply during the first, second, and third larval stages and form a mass of so-called oogonia (fig. 6.) At an early period in the fourth larval stage, however, all oogonia cease to multiply. Consequently, all appear alike on account of their being in the so-called resting stage. This condition is soon followed by a peculiar phenomenon. A few oogonia situated along the periphery of the ovary suddenly undergo another, the last, oogonial division and begin to grow, not only in size, but also in nuclear complexity. The number of these oocytes of the first order forming a group varies with the species. In Pseudococcus there are four oocytes in a group, but in Icerya the number is five. At first the nuclei of the oocytes in each group appear exactly the same, all being in the so-called synizesis stage (fig. 4). From the contracted nuclear contents fine thread-like chromo- somes emerge (fig. 6). At first these chromosome threads are distinctly doubled, but later appear as single. Meantime a sort of protoplasmic substance begins to be secreted around each of the oocytes except the one situated toward the proximal end. As their later history shows, these secretory oocytes nourish the single oocytes located below or toward the distal end; and thus become the so-called nurse cells. The cytoplasm areas of the fast-growing nurse cells soon come into contact with one another. Being colloidal in nature, the nutritive substances secreted by the nurse cells elongate in the direction of the least resistance, which in this case is toward the egg nucleus, for the expanding force of the nurse cells is much greater than that of a single egg cell situated near the distal end. Consequently, the nutritive substance, which is elaborated by the nurse cells, now literally pours over the egg, causing a rapid increase in its size (fig. 11). EMBRYOLOGY OF COCCIDS 81 Epithelial cells which surround the nurse chamber above never multiply, but those around the egg multiply rapidly and help to accommodate the protoplasmic substance which pours from the nurse chamber above (fig. 15). Soon a constriction becomes evident at the junction of the two chambers (fig. 16), due partly to the ingrowth of the epithelial cells at the base of the nurse chambers and partly to the rapid expansion of the egg and nurse cells. The epithelial cells surrounding the egg chamber are cubical or elongate ovoid in shape and actively divide, while those around the constriction are smaller and spindle shaped. No mitosis was observed among the latter. As the constriction progresses, the space through which the two chambers communicate becomes smaller. Consequently, the protoplasmic substance, which has been flowing homogeneously toward the egg nucleus or the germinal vesicle, now flows out of this small passage in minute streamlets as molasses does when poured through a funnel into a flask. The nuclei of the nurse cells still increase in size, and change from spherical to egg-shape, the narrow end being directed toward the egg chamber. The chromatin threads become broken into numerous chromatin bodies of different sizes. The nucleus of the egg proper moves toward the center of the egg chamber, but the chromatin threads are still in the paired condition as last described. Their affinity for iron-alum haematoxylin is changed. They now stain so faintly with this dye as to seem almost achromatic in nature. From this time on, the germinal vesicle begins to migrate from the center toward the periphery of the egg. During this migra- tion the chromosomes lose their paired appearance and form several small, spherical bodies which become scattered along the nuclear membrane (fig. 17). There is, however, no indication of their being passed out through the nuclear membrane into the surrounding protoplasm, as several investigators of other insects have stated. The place where the germinal vesicle reaches the peripheral layer is on the ventral surface, midway between the equator and the posterior pole of the egg. As soon as the clear transparent nucleus reaches the periphery, the surrounding protoplasmic 82 GEORGO ORIHAY SHINJI substance becomes compact, thereby causing an indentation of the surface. In the next stage, the nuclear membrane of the germinal vesicle disappears and the vesicle itself loses its clear appearance. This change is due to the appearance of the spindle fibers about the chromosomes (fig. 26). The oocyte is now in the metaphase of the maturation division. Following this stage the chromo- somes divide into two groups and move to the opposite poles. Soon one of the daughter cells gradually protrudes from the egg proper (fig. 31). In this manner the first polar body is formed. ° The process of the second polar body formation has not been studied, but that the egg undergoes the second maturation division may be established by the presence of three polar bodies. Meanwhile the epithelial cells not only cease to multiply, but they also become reduced to a membranous structure. The nurse cells also cease to grow. Their size becomes very much reduced. The contents of the nurse chamber is withdrawn into the egg and its epithelial layer shrinks to a small mass. This, together with the remains of the epithelial cells of the egg, closes over the opening left by the egg entering the oviduct. The earliest egg found in the oviduct (or the uterus, as it is often called) shows a large nucleus at the center of the egg. Since this large nucleus divides, it must be the first cleavage nucleus. It follows, therefore, that the union of the male and the female pronuclei must have occurred during the passage of the egg pro- nucleus to the center of the egg after the formation of the last polar body. Thus my observations on the three species of coccids are in accord with those of Leuckart (’53) and Emeis (716). It may also be added that the ovary of Icerya purchasi is the most favor- able material for the study of this problem, since the cells are large and numerous. The origin of the three ovarian elements in aphids has been differently described by different writers. Lubbock claims that the egg and nurse cells are modified epithelial follicular cells of the end chamber. Recent observations of Tannreuther (’07) are to the same effect, for he declares that the egg cells do not arise EMBRYOLOGY OF COCCIDS 83 from the inner mass in common with the nurse cells or ovarian glands, but grow out of the follicular epithelial cells at the base of the end chamber. Balbiani (’82) states that the germ rudiments of partheno- genetic aphids undergo a process of budding previous to their differentiation into the nurse cells and oocytes. Mecznikow (’66) derived the end chamber from a mass of cells, ‘“Die am untersten Pole des endfaches legenden Zell sich bedeutend vergréssert, wobei sie in ein, aus dem Endfachepithel entstandes Follikel eingeschlossen wird und hier ihre weitere Entwicklung vollzieht.”’ Stevens (’05) insists that the contents of the end chamber are of two kinds, possibly corresponding to the summer and winter eggs, and that those situated on the lower portion of the end chamber degenerate in the ovaries which produce the agamic eggs. In insects (other than Hemiptera) the results of several inves- tigators differ with the species with which they have worked. The prevailing idea, up to 1905, was that the three ovarian elements, nurse cells, epithelial cells, and oocytes, are all derived from the germ cells. Paulcke (’00) who studied the development of the honey-bee, however, discovered for the first time that the nurse cells and oocytes are originally the same, but later become differentiated by a certain irregular cell division. Similar dis- coveries of the existence of a quantitative difference between the nurse ceils and oocytes have since been reported in several Coleoptera. In Dytiscus, for example, Gunthert (710) found that the chromatin eliminated from the nucleus passes, in each successive mitosis, into the pole of a single daughter cell, and that the cell having this extra chromatin substance becomes the oocyte and those lacking this the nurse cells. Somewhat similar observations were made by Giardia (’01) and Debaisieux (’09) in Dytiscus, and Govert (’13) in Carabus, Cicindela and Trichiosoma. The oogonial origin of the oocytes, nurse cells and follicular epithelial cells was clearly established in Polistes and Platy- phylax by Marshall (07); in Podura by de Winter (713), and in Leptinotarsa by Hegner (714). In these cases, the nurse cells 84 GEORGO ORIHAY SHINJI and follicular epithelial cells are regarded as abortive cells. No differential mitosis was observed. Hegner (’12) states that the germ cells in Miaster give rise to nothing but the true oocytes, and that the nurse cells and epithelial cells are both derived from somatic cells. He deduces this from the fact that in Miaster americana altogether sixty- four oogonial cells are formed by six successive divisions of a single primordial germ cell and that the number of the young larvae produced is also about sixty-four. The foregoing survey of the more important literature per- taining to this subject, brief as it is, indicates that, even among the same order of insects, there is no definite law governing the differentiation of the three ovarian elements. The accounts of the lineage of the ovarian elements in Leptinotarsa (Wieman, ’14; Hegner, 712), and Hydrophilus (Korschelt, ’89) are good ex- amples. However, it should be mentioned that in all insects the oocytes and also the nurse cells, when present, are all derived from primordial germ cells. As yet no case has been found in which the primordial germ cells of the insects entirely degenerate and the secondary or functional germ cells are formed de novo at a much later period of development. 3. THE EGG The eggs of all species of coccids studied, at the stage last mentioned, consist of the following substances: 1. Chorion—the outermost covering or membrane. 2. Protoplasm—the ground substance. 3. Corticular layer—a thick protoplasmic layer next to the chorion. 4. Fat globules—oily droplets suspended in the protoplasmic network. 5. Yolk granules—protoplasmic suspension. 6. Pigment oil-fluid filling interspace between fat globules. 7. Germinal vesicle with its nuclear membrane. 8. Yolk membrane—membrane next to, and, in fact, almost apposed to the chorion. EMBRYOLOGY OF COCCIDS 85 The chorion is a very thin membranous structure which en- closes the substances above mentioned. It is formed shortly before the passage of the egg from the egg chamber into the uterus, and is secreted by the follicular epithelial cells. The protoplasm or cytoplasm fills, so to speak, most of the space between the other inclusions of the egg, with the exception of the space occupied by the nucleus or the germinal vesicle. As already stated, this ground substance of the egg is elaborated by the nurse cells and is literally poured on the egg. At first the protoplasm is a homogeneous mass uniformly surrounding the central clear region, the nucleus. Later, however, it becomes mesh-like, owing perhaps to the more rapid expansion of the egg than the flow of the nutritive or protoplasmic substance from above, to the intrusion and consequent suspension of other sub- stances, and also to the physiological change due to the metabolic activity of the germinal vesicle. What seems to me a sort of yolk substance is found in the egg of the mealy bug of the giant ragweed. ‘This substance may be spherical, but it is more often irregular in shape. It first appears at about the time when the germinal vesicle reaches the periph- ery. The exact origin of this substance remains to be studied further. The fact that similar granules are abundant in the body cavity surrounding the ovarioles, and also in spaces between the chorion and the epithelial cells, strongly suggests that these particles may actually migrate from the body of the mother through the epithelial layer into the egg. The presence of similar substances in the body cavity of the mother is another evidence in favor of the view just stated. Several investigators of other insect eggs state that they have observed the migration of chromatin matter from the germinal vesicle of the egg. No indication of such migration was observed in the case of the scale insects studied. . In the ovarian eggs of these species of coccids, the nucleus or the germinal vesicle was always found. The pigment-oil or coloring matter appears in the mature, but not in the ovarian egg. I have had occasion to observe this fluid-like matter flowing into the egg at the posterior or pointed 86 _ GEORGO ORIHAY SHINJI end. This substance was found literally filling the oviducts of the adult during her egg-laying period. The presence of the yolk membrane cannot better be illus- trated than by figure 28. On account of the migration of the symbiotic organisms after the formation of the yolk membrane, the latter is pushed in and remains separated from the chorionic membrane (which is the last to envelop the egg). A fully matured egg with all its components is elongate oval. The pointed end corresponds to the cephalic and the blunt end to the caudal end of the insect. The ventral surface near the pointed end is slightly indented. Thus not only the antero- posteriority, but also the dorsoventrality are marked in the eggs of the coccids, but not so clearly as in the eggs of the Orthoptera and Coleoptera reported by Heymons (’89), Wheeler (’93), and others. Besides such a difference in shape, the anteroposteriority is well marked by the presence of a dark-staining substance, the position of which varies with the species. In cottony cushion scale, it is, at first, visible near the posterior pole, but later becomes pushed gradually toward the anterior pole by the invaginating germ-band; while in the case of Pseudococcus and Lecanodiaspis, it is found always near the anterior end of the egg. The presence of these polar granules or symbiotic organisms is a great service in the determination of the position of the sectioned material. Later on, I shall treat of the history and significance of this substance under a separate heading. No micropyle was found. The longest and shortest diameters of the eggs of the three species of coccids are respectively as follows: LONG SHORT 2S) (O29) (SEL NaS DIAMETER DIAMETER mm mm. ER UEE AEE OR GSB, Wioc.u.0 5 5s oben nee ae Rete ae eT 1 8.5-9.0 4.5-5.0 PSCC OCOCCUSRINACUUNTEL cc hele intr ei ae eee 4.0 220 Lecemodtas pus PrUInOst... .. . sere seus cs wan egos vee 6.0-4.0 235 EMBRYOLOGY OF COCCIDS 87 4. CLEAVAGE The type of cleavage in the coccids studied is the pure super- ficial type which is so common among the Arthropoda. The first cleavage spindle lies at right angles to the shorter axis of the egg, so that one of the two daughter cells arising from the first division wanders toward the posterior pole while the other cell remains near the position formerly occupied by the mother nucleus (fig. 43). This behavior of the first two cleavage cells in coccids is exactly like that of the termite studied by Knower (00). At first, all the cleaving cells were in the same mitotic state, but gradually some lag behind others in division so that in a later stage of cleavage, e.g., at the thirty-two cell stage, more than one cleavage figure is noticeable among them (fig. 78). Up to about the eight-cell stage in the eggs of Lecaniodiaspis, and to a still later stage in Pseudococcus and Icerya, these cleavage cells are all at some distance from the cortical layers. Although in each cell the nuclear membrane is distinct, the cytoplasm presents numerous pseudopodial processes which con- nect with those of neighboring cells. On account of their somewhat isolated appearance, they are usually known as pro- toplasmic islands. In eggs containing a large amount of yolk, as, for example, those of Chrysomelid beetles studied by Hegner (14), the winter eggs of plant-lice investigated by Tannreuther (07) and Webster and Phillips (12), these protoplasmic islands literally cut up the yolk into blocks. I have noticed this block- like appearance of the egg contents in the living eggs of Lecanio- diaspis, but upon sectioning them, I become convinced they were not comparable to the yolk-blocks found, for example, in the ova of aphids, because the eggs of Lecaniodiaspis contain no yolk granules. The eggs of the mealy bug contain a darkly staining substance resembling the yolk of the ova of aphids, but they are never cut up into blocks by the cleaving cells (fig. 42). Weismann (’82) stated that in Rhodites and Biorhiza aptera (eynipids), the first two cleavage nuclei move apart in the direction of the longitudinal axis of the egg. One of them, upon reaching the posterior pole of the egg, remain inactive and _ probably degenerates, while the other, upon arriving at the anterior pole, 88 GEORGO ORIHAY SHINJI produces, by rapid multiplication, all of the embryonic cells. As stated above, the first cleavage products of our scale insects do not behave in this way, but both cleavage cells continue multiplying, and some of their products later form the blasto- derm, while the others remain in the interior of the egg and constitute the so-called yolk-cells. In this respect, the develop- ment of the eggs of coccids resembles that of the silkworm and of Neophylex and Gryllotalpa, studied, respectively, by Toyama (02), Patten (’84), and Korotneff (’84). Silvestri (11) recently discovered that in a parasitic Hymenopteran, Copidosoma, one of the two nucleoli escapes from the nucleus at the end of the growth period of the oocyte. Later, this escaped nucleolus passes into one of the two cleavage cells. During a series of cleavage processes, only one cell remains in possession of this nucleolar substance and becomes the germ cell. In another parasitic Hymenopteran the escaped nucleolar bodies become localized at the posterior end of the egg until one of the first cleavage cells reaches out and takes them up into its protoplasm. In both cases the cell which becomes possessed of this escaped nucleolar substance, differentiates into the germ cells. In the scale insects I have studied no escape of the nucleolar substance into the egg was observed and the germ cells do not appear during the cleavage period. — All cleavage cells divide mitotically. No case of amitotis, as described for Blatta by Wheeler (’93), has been observed. The fact that very many cells are in the process of division during the early stages indicates the rapidity with which cells divide. Nelson (’15) states that no case of a single spireme stage was found in the cleavage cells of the honey-bee. On this point my specimens agree strictly with his observation. An abundance of spireme figures are, however, found among the blastoderm cells. As the number of cleavage cells increase, they migrate, one by one, toward the periphery and become imbedded in a thick cortical layer of the protoplasm. In figure 44 the condition of a loose blastoderm is shown. Although the cells are arranged in a peripheral layer, they are very far apart from one another. The spaces between these blastoderm cells are gradually filled by the EMBRYOLOGY OF COCCIDS 89 division of the blastoderm cells as well as by a further migration of cleavage cells from within. The point at which cleavage nuclei, or cells as they are often called, reach the surface of the egg varies in different groups of insects. In Muscidae Graber (’79) found the first arrival of cells at the posterior end of the egg, while in Pieris Bobretzky (78) observed the appearance of the first blastoderm at the anterior end. Wheeler (’93) described the first blastoderm cells on the ventral side, while Heider (’88) stated that the blastoderm in Hydrophilus was first formed around the middle of the egg as a transverse girdle, somewhat nearer the posterior pole and that the development occurred last at the poles. Again, accord- ing to Nelson, the cleavage cell first reaches the cortical layer on the ventral side near the cephalic pole in the egg of the honey- bee. In the winter egg of the aphid, Melanoxanthus (Ptero- comma, salices), according to Tannreuther (’07), all of the blastodermic cells spread uniformly over the entire surface except at the posterior pole of the egg. Therefore, I agree with Nelson ('15) that the point at which the first cleavage cells reach the surface has little significance so far as the formation of the blastoderm is concerned. The condition of the egg at the time the geet process has ceased among the cells within the egg and the blastoderm forma- tion is completed, is shown in figure 80. At the poles and sides the blastoderm is similar in appearance. A short distance within the blastoderm is another loose layer of cells. This is irregular in shape, and the nuclei are much clearer and coarser than those of the blastodermic cells. These are the so-called yolk cells of Will (’84), and are no other than the cleavage cells that failed to migrate to help form the blastoderm. At about the time invagination occurs at the posterior end of the egg, these cells move toward the periphery and become closely apposed to the blastoderm cells. 90 GEORGO ORIHAY SHINJI 5. ESTABLISHMENT OF THE EXTERNAL FORM OF THE EMBRYO The development of the embryo was traced up to the com- pletion of the blastoderm as shown in figure 80. I first describe the development of the embryo as seen mostly from surface views. The first change externally visible after the completion of the blastoderm is a depression or an invagination near the posterior end of the egg. It is, at first, very shallow, but gradually deepens forming a V- or U-shaped structure (fig. 45). This condition is much more pronounced in the case of the cottony cushion scale than in the other two species studied. The portion of the blastodermic layer constituting the bottom of the blasto- pore and its near-by area increases greatly in thickness, while toward the anterior pole and in the area surrounding the blasto- pore it becomes thin. In the cottony cushion scale, the colony of parasitic organisms, originally found at the posterior pole of the egg, is later pushed, so to speak, toward the anterior pole by the elongation of the invaginating germ band.? During the same period of development, the mass of the parasitic organisms in the eggs of Pseudococcus and of Lecaniodiaspis has also migrated a short distance from its point of entrance, the anterior pole, towards the posterior pole. goe 20,00 0500s Sia 2; < eg 959.8059 95 —_-mup Rp, ww a 3 38 ra 89 90 91 92 PLATE 13 EXPLANATION OF FIGURES Longitudinal section of an egg of Pseudococcus. A magnified view of figure 99 between vp. and my. Posterior half of a longitudinal section of an early Lecaniodiaspis embryo. A portion of figure 96, much magnified. 152 EMBRYOLOGY OF COCCIDS PLATE 13 GEORGO ORIHAY SHINJI ” | " midint 153 PLATE 14 EXPLANATION OF FIGURES 93 t096 Four consecutive longitudinal sections of anembryo. Pseudococcus. 97 Transverse section of an embryo, Lecaniodiaspis, approximately in the stage represented in figure 89. 98 Oblique longitudinal section of an egg of Pseudococcus. 99 Longitudinal section of a Pseudococcus embryo. 190 A portion of transverse section through the blastopore of Pseudococcus. 154 PLATE 14 EMBRYOLOGY OF COCCIDS GEORGO ORIHAY SHINJI - prot 155 PLATE 15 EXPLANATION OF FIGURES 101 Oblique transverse section through the posterior end of an egg of Icerya. 102 A portion of longitudinal section of an egg like the one represented in figure 66. 103 Transverse section through the second maxillae of an Pseudococcus embryo like the one represented in figure 50. 104 Transverse section through the second maxilla region of an embryo like that represented in figure 50. 156 EMBRYOLOGY OF COCCIDS PLATE 15 GEORGO ORIHAY SHINJI meso ect ' : i \ ! 4, PFO A ) PLATE 16 EXPLANATION OF FIGURES 106 Oblique frontal section of the embryo like the one represented in figure 55. 107 Transverse section of a Pseudococcus embryo somewhat older than the one represented in figure 55. 108 Transverse section through thoracic region of the embryo represented in figure 105. 109 Transverse section through the last thoracic region of an embryo some- what older than the one represented in figure 55. 158 PLATE 16 EMBRYOLOGY OF COCCIDS GEORGO ORIHAY SHINJI 106 -gangb 108 159 JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1 PLATE 17 EXPLANATION OF FIGURES 110 Longitudinal section through the brain of an embryo like the one repre- sented in figure 51. 111 Transverse section through the thoracic region of a Pseudococcus embryo like the one represented in figure 72. 112 Transverse section through the salivary glands of Pseudococcus. 113 Transverse section through the third abdominal segment of the embryo almost ready to hatch. 114 Transverse section of a Pseudococcus embryo like the one represented in figure 51. 115 Another section of the same embryo as figure 114. 116 A portion of figure 117 magnified. 117 Oblique longitudinal section of an embryo. 160 EMBRYOLOGY OF COCCIDS PLATE 17 GEORGO ORIHAY SHINJI “~-meso gangbh 161 PLATE 18 EXPLANATION OF FIGURES 118 Median longitudinal section of a Pseudococcus embryo a short time before the completion of the alimentary canal. 119 Longitudinal section of the embryo of Pseudococcus at the time of the completion of the alimentary canal. 120 Longitudinal section of an Iceryan embryo. 121 Transverse section of an Iceryan embryo like the one represented in figure 119. PLATE 18 EMBRYOLOGY OF COCCIDS SHINJI GEORGO ORIHAY N, 3 > maces ae foes me ra Ors & i SUE aa el dint ate, 163 PLATE 19 EXPLANATION OF FIGURES 122 Transverse section of Iceryan embryo like the one represented in figure 119. 123 and 124 Hair glands of a newly hatched Pseudococcus larva. 125 Transverse section through the eyes of an embryo like the one represented in figure 119. 126 Caudal portion of an embryo Pseudococcus like the one represented in figure 70. 127 and 128 Stages of the growth of the rudiments of the midgut of Pseudococcus. 164 PLATE 19 EMBRYOLOGY OF COCCIDS GEORGO ORIHAY SHINJI 124 123 at 2 Do0G GL: < Denke Be ‘So 089 +) eo! e ° — meso +3) =———— meso midint t 165 PLATE 20 EXPLANATION OF FIGURES 129 and 130 Stages of the growth of the rudiments of the midgut of Pseudococcus. 131 Longitudinal section of an Iceryan embryo a short time after the com- pletion of revolutions. 132 Longitudinal section of an Iceryan embryo like the one represented in figure 119. 133. The colony of symbiotic parasites at the time of the hatching of larva. 166 EMBRYOLOGY OF COCCIDS PLATE 20 GEORGO ORIHAY SHINJI 22 te Soe 26 @o e®, icy set e midint® Gna. Resumen por el autor, Charles L. Parmenter, Universidad de Pennsylvania. Numero de cromosomas y parejas de cromosomas en las mitosis somiaticas de Amblystoma tigrinum. El autor dd a conocer en el presente trabajo un estudio del numero de cromosomas y sus relaciones de longitud en las células de varios tejidos somdticos de Amblystoma tigrinum, cuyos resultados suministran pruebas en favor de la teorfa de la indi- vidualidad de los cromosomas En sesenta células pertenecientes a veinte y tres individuos diferentes el ntimero de cromosomas es constantemente veinte y ocho. Las medidas lineales de los cromosomas de un ntimero limitado de complejos seleccionados cuidadosamente indican que los cromosomas de una célula forman una serie duplicada en tamafio y forma, lo que presta apoyo a la suposicion de que estan formados por pares de cromosomas homdlogos maternos y paternos. También existe una constancia aproximada en la relacién de tamafo entre los pares de cromo- somas de los complejos de diferentes individuos. Los datos mencionados favorecen la teorfa de la individualidad de los cromosomas y no confirman el aserto de Della Valle, que supone que la variacién del nimero de cromosomas es la regla y que las longitudes de dichos cromosomas en una célula se deben meramente a una casualidad. Translation by José F. Nonidez Carnegie Institution of Washington AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 13 CHROMOSOME NUMBER AND PAIRS IN THE SOMATIC MITOSES OF AMBYSTOMA! TIGRINUM CHARLES L. PARMENTER Zoological Laboratory, University of Pennsylvania THIRTY-SEVEN FIGURES (NINE PLATES) CONTENTS TURTON 9. dts 2s Ho SUS Ae Boba toe otto oid Cloke toe neo ero mace ORCC aca Reena so 7A 56 ZS 5 AlzAG} meal vi7 so Mer Sis b. Possible variation in number in uncounted romp lene: foe Bae . 182 Technique.. (nee amons.. of Pees A. The eves a Pinemocauese : Saint a. Method of determining ine. “aerial ere 1. Procedure. , Anis 2. Clearness and apecii cota oF ae spl cee. c. Abnormal complexes......... Bees OMA tie, CHLOMOSOME PAIS! serps bo. 2:44 eles ss oe oo Ae A Dae ee ee CmlMtrOdUGtonyastatementinwins cece sein ao cela ae ee eee ee . 185 b. Mensuration.. aS: 1. Types of cells Be ea ey nee RR AA tM un ate D. ie ek DE a Ae Oi | an NO a aah Debvidence fon theyexissencerol PAlnsee.... sce acca seer ae Ore OUMTT Any ee ear ee T eeers yeee hr tty ttk foe Gone oe oe een Discussion. . week A. imeeeductany Premiere weer B. Constancy of Tinta en univ uae wo 8 RY OOM ROMs OMY Gry arigbions mother Wrodeless..-..tsse ss cnc. 0% «2 so) Davie he nie ek choevaaiees 1D), WENO SOI IM ClaienioMnis > ae te poem ene Ga Deebod be odocoo uaHe ona ees E. Fragmentation. . F. Existence of pairs.. a. Pairs in the germ ncelise. b. Pairs in the somatic SU Le oe Ee ake eee ae 1. Meves’ measurements............ PewWella Vallessamedsurements. 2 os cc cacao « tek acct Si Jeesiuillush tm VNamongsiorane) inlsenMOwON, | aa ooono ne ooo bn gosuooKe ene emConstanienelapiversizeure avionss s-sneeetenere meee seine oe eee nee GuSuniun a Gy Olemiensuneme nda)..." d4 ao «Facto ckeoharslerg reise Ge eiec SuanN Ty gO tGOMCIUSLOMS sf, Y8t) a: Gio sells wun o% oelcgre oeeoreteatait aletns: daueie se-se ee 169 181 184 184 186 . 186 SE SOURCESEOMELROL ya rere t Tie Aon ee aes EEL ihan coe Toe Guhesulliszot measurements cacent meat cok tc ose ciees, cuclouysl > dimers Jee Cribkenia ford eherminimoe paisa eee). scl a te satan eee 186 191 191 194 199 . 200 200 200 206 206 . 207 208 . 208 210 211 215 216 220 220 221 1 Also known as Amblystoma. 170 CHARLES L. PARMENTER INTRODUCTION It is believed (McClung, 717, pp. 536-38) that the chromatin of an organism is, for the most part at least, the idioplasm, and consists of a definite linearly arranged series of differentiated materials which is perpetuated from generation to generation. The chromosomes which are essentially constant in number in an individual are thought to constitute the visible mechanism for this perpetuation. This conception is known as the theory of the individuality of the chromosomes, which is quite generally accepted by all who have an intimate acquaintance with chro- mosome behavior. However, there are a few not so acquainted who strenuously oppose the theory. Among these is Della Valle (’09, 711, ’12), who presents some data and a large amount of discussion in an effort to disprove this theory upon the claim that the chromosome number in an individual is not constant, but is simply the quotient of the quantity of chromatin divided by the average size of the chro- mosomes. ‘This removes from them any constancy of organiza- tion and contradicts the above theory. These observations have been cited by other opponents of the theory as cytological evidence in favor of their contentions. Della Valle’s conclusions are based upon observations made upon dividing cells of the peritoneum and blood-cells of Salamandra maculosa, together with a large amount of data taken from the observations of . others. Meves (11) and Della Valle (12) further oppose the theory upon the basis of linear measurements made upon the spermat- ogonial and somatic chromosomes of Salamandra maculosa in denying Montgomery’s (’01) and Sutton’s (’02) claim that the chromosomes occur in pairs whose homologues are of equal length, and that approximately constant size relations among chromosomes are maintained from one cell generation to another. In the spring of 1916 I was fortunate in obtaining peritoneal and other somatic tissues of Ambystoma tigrinum. This made it possible to repeat Della Valle’s observations upon the somatic CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA HEL cells of the same and other tissues of this closely related species and thus to determine whether such a variation as he claims is present in the somatic tissues of other Amphibians. Also the chromosomes of some cells in my material are sufficiently favorable for measurements to permit a reconsideration of their length relationhips. Since this paper is regrettably controversial, it is necessary to give careful attention to all the methods and conditions under which the preparations and the observations were made. Della Valle also lays much emphasis upon this point, and therefore considerable space is devoted to this problem. For facilities in collecting and preparing this material I am indebted to the courtesy of the Department of Zoology of the University of Minnesota, and to Prof. C. P. Sigerfoos I owe the loan of several very excellent preparations. The work was done under the direction of Prof. C. E. McClung, of the University of Pennsylvania, toward whom I feel especially grateful for constant encouragement and valuable criticism, and for his characteristically generous and kindly interest at all times. I am also greatly indebted to other members of the department, especially to Dr. Eleanor Carothers and Dr. D. H. Wenrich, for helpful suggestions and very painstaking criticisms. TECHNIQUE The material used was obtained during the spring of 1916 from larvae of Ambystoma tigrinum, which were abundant in the ponds and lagoons near the College of Agriculture of the University of Minnesota. Mitotic figures in epithelial cells of the tail, gill plates, and lung, and of the endothelium from peritoneum and mesentery were studied. Tail epithelium Very excellent preparations of this tissue were kindly loaned me by Prof. Charles P. Sigerfoos, of the University of Minnesota. These were made from the tails of larvae 2 to 14 inches in length obtained during the last of May and the first of June during several years. | 172 CHARLES L. PARMENTER The living larvae were thrown into Flemming’s stronger solu- tion. After about four hours of fixation, the tails were split dorsoventrally? into two thin plates of cells. These two plates of cells were then fixed twenty hours longer. After washing in running tap-water for twelve hours or more, the pieces were stained in toto in Heidenhain’s haematoxylin, carefully dehy- drated, and cleared in xylol and mounted in damar. Gill-plate epithelium The most successful gill-plate preparations were also obtained from larvae ? to 13 inches long. In each larva there are eight gill plates, one subtended from each gill arch and another behind each posterior gill cleft. These gill plates contain very numer- ous mitotic figures. They are composed of two epithelial lamellae with connective-tissue cells and capillaries lying between them. The two layers, unseparated, are so thin that they give very excellent preparations. The material was fixed in situ by dropping the living larvae into the Flemming’s stronger solution as soon as they were taken from the net. They were fixed in situ twenty-four hours and 2 Haecker (’99) describesa very successful method of separating these two plates of cells. The posterior end of the larva is cut off after fixation just in front of the cloaca. With a sharp scalpel the thick cephalic end of the tail is split dorso- ventrally through the middle of the vertebra to a depth of an eighth of an inch or more. By grasping with the forceps the ends thus made free, the two layers of epithelium can be pulled apart in a manner similar to separating two sheets of fly-paper with adhesive surfaces sticking together. Professor Sigerfoos advises separating the two layers after about four hours of fixation and then allowing to fix about twenty hours longer. The numerous large mitotic figures in various stages with clear cell walls which can be studied without an immersion lens makes this material excellent for the class-room. The gill-plate preparations are equal or superior to those of the tail epithelium. Those of larger larvae are too thick when mounted in toto, but give very satisfactory preparations when separated. Peritoneal prepa- rations of larger larvae contain fewer mitoses and the cell walls are indistinct. However, preparations can be made from the more rapidly growing shorter larvae from which the gill-plates were taken and would probably contain more divisions. Preparations of Ambystoma punctatum are less favorable than those of A. tigrinum because there are fewer figures, more pigment cells in the tail epithelium and the gill-plates are small and thicker. CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA Ls washed in running tap-water. The larvae from which the gill- plates were taken were fixed for other purposes, and no special effort was made to insure good fixation of the plates. They were preserved_in position in 5 per cent formalin, which was later gradually replaced with 80 per cent alcohol. The fixed gill-plates were carefully removed from the larvae in 80 per cent alcohol and were left attached to the gill arches, which, in subsequent handling, were grasped by forceps to prevent injury of the plates. Hydrogen peroxide was added to the 80 per cent alcohol drop by drop through a fine glass capillary siphon until the solution amounted to equal parts of each. In this the plates were bleached for four to twelve hours and then transferred to the mordant by the above-mentioned drop-process and stained in iron haematoxylin. They were dehydrated by this drop method, cleared in cedar-wood oil followed by xylol, cut from gill arches, after being transferred to the slide, and then covered with damar and a thin cover-glass. While the damar was hardening they were kept for twenty-four hours or more under slight pressure to insure flat preparations. The peritoneum, mesentery, and lungs These preparations were made from larvae 3 to 4 inches long. All the tissues of a given individual were not only fixed together in the same fixative for the same length of time, but also received the same treatment in all subsequent processes. They were put into the fixatives within an estimated maximum of two minutes after the first incision. Two methods of procedure were used in preparing these tissues for fixation: 1. In order to avoid any possible unfavorable effect of cap- tivity, the tissues were fixed in situ in the field as soon as the larvae were taken from the net. The animals were prepared for fixation as follows: With sharp scissors the body wall was cut open along the midventral line and also lateral incisions were made on each side at right angles to the first incision behind the pectoral girdle and in front of the pelvic girdle, so that the two halves of the body wall fell away from the viscera and opened 174 CHARLES L. PARMENTER wide the body cavity. The folds of the viscera were pulled apart and the whole larva was plunged into the fixative. This secured immediate and uniform fixation. The operation requires less than a minute and the incisions are apparently painless, for the larva does not often struggle. 2. The body walls, lungs, and viscera were removed from the body of the larvae before fixing, either in the field or at the laboratory. The peritoneum was fixed in situ on the body walls. Only normally inflated lungs were used, and these were ligated anteriorly before removal from the body to prevent them from collapsing. After fixing one or two hours, they were cut into two or more flat longitudinal strips and returned to the fixative. The mesentery, attached to the intestine, was spread out flat on a piece of glass and the whole immersed in the fixative with the tissue beneath. Fixatives The fixatives used were: 1) Flemming’s stronger solution, thirty hours; 2) Rouin’s solution, forty-three hours; 3) Bouin’s solution, to which was added 14 grams of chromic acid crystals per 100 ec., twenty to twenty-four hours; 4) Hermann’s solution with two parts of osmic acid (Lee, ’13, p. 38) twelve to eighteen hours; 5) a solution of saturated picrie acid 75 ec., formalin 15 ee., glacial acetic acid 10 cc., urea crystals 2 grams, thirty to forty-three hours. The urea should be added gradually to the solution warmed to about 40°C., otherwise a precipitate is formed. It is a difficult matter to decide which solution gave the best fixation. The prettiest cells were fixed in Hermann’s and the chromic acid modification of Bouin’s fluid. However, the peri- toneum preparations of the osmic fixatives were a little thicker and less transparent than the others. If any fixative should be exclusively chosen, I believe it should be the chromic acid modi- fication of Bouin’s solution, because of its excellent fixation, convenience, and economy. The peritoneum was removed as follows: The two sides of the body wall were detached by an incision along the back CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 175 close to the spine. Under the binocular lens, in water, the peritoneum was carefully loosened from the underlying tissue by scraping it with a sharp scalpel, first along the edge cut from the back. Sections of this loosened edge were then grasped by the forceps and relatively large sheets were easily pulled off from the underlying tissue. _The peritoneum covering the dorsal and lateral portion of the body wall is deeply pigmented and to it adhere considerable muscle and connective tissue when the peritoneum is removed. This portion was grasped with the forceps in removing the peritoneum from the body wall, as well as in all subsequent handling. Consequently the cells in the ventral transparent region available for study have been undis- turbed by instruments. However, there still remains a possi- bility that the strain of pulling the peritoneum loose might disturb some cells. Peritoneum fixed in Flemming’s and Hermann’s solutions was stripped from the body wall after four hours of fixation and then fixed twenty hours longer. That treated with the various picric acid mixtures was stripped immediately after fixation. However, the peritoneum fixed in the chromic acid modification of Bouin’s solution may be preserved in alcohol for as much as a year before stripping. That of Ambystoma punctatum, fixed in Flemming’s stronger solution and preserved in 5 per cent formalin, can be stripped at least six months after fixation. Material fixed in osmic acid fluids was washed five to fourteen hours in frequent changes of tap-water. Picric acid preparations were gradually transferred to 70 per cent alcohol, beginning with 10 per cent and progressing through successively stronger grades differmg by 10 per cent. They remained in each grade five to ten minutes. The tissues remained in 70 per cent alcohol con- taining a few drops of saturated aqueous lithium carbonate solution until the picric stain was removed, and before staining they were returned to water by reversing the above process. All of the material was stained in Heidenhain’s haematoxylin after mordanting in a 23 per cent solution of iron alum for four to six hours. No counterstains were used. JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1 176 CHARLES L. PARMENTER Dehydration was accomplished by passing the material through the above grades. The fluids were removed from, and added to, the containers without handling the material. Alcohols were _ followed by half xylol and half absolute alcohol, and finally by xylol. The pieces of peritoneum were transferred from xylol to a slide where the above-mentioned pigmented area, with the at- tached muscle fibers, was removed quickly with a sharp scalpel just before mounting. After mounting in damar under a cover- glass, they were put under a light pressure for twenty-four hours or more while drying to insure as flat a preparation as possible. OBSERVATIONS It should be emphasized that the preparations upon which these observations were made are unsectioned surface mem- branes. This makes it possible to study the mitotic figures with the confidence that all of the chromosomes are present and that none have been cut and are being counted more than once. This is an important consideration in determining whether the — number of chromosomes is constant. A. The number of chromosomes There are twenty-eight chromosomes in the somatic complexes of Ambystoma tigrinum. In forty-five unquestionable enumera- tions and in eighteen which contained either one or two chro- mosomes that might possibly be considered subject to interpre- tation, there are none which vary from twenty-eight. In three complexes, because of the alternative interpretations possible at one or more points, the number cannot be definitely determined and is interpreted to be either twenty-seven or twenty-eight. The fact that these numbers are so close to twenty-eight is strong evidence that these cells contain the usual number of chromosomes. The counts as indicated in the accompanying table have been obtained from twenty-three different individuals varying in age CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 177 approximately from six to ten weeks. The preparations of tail epithelium loaned by Professor Sigerfoos were taken from a collection which he has been accumulating for a number of years. It is probable, therefore, that the counts in these preparations represent the chromosome number present during a series of years and that the number is constant from year to year. Table showing for each tissue studied, the number of different individuals repre- sented and the number of complexes with their distribution into classes as de- scribed on page 178. The total number of different individuals represented is twenty-three NUMBER CLASSES TISSUE —9RD EE — EE ————————EE————— es TOTAL VIDUALS I II III IReniioneumey ons aah eee wore oen 7 14 6 1 PAY INTESemibe type tcr te cievaiticstises sists te, ahese 1 1 0 0 1 UIT Meare etnrcte chars aia tccucra Bisgeter aa 7 2 il 10 “LU ey OTH CC) Ws a ee A 5 9 4 0 13 CUB DIRTESHREL State ood oh vtec age ast 8 14 6 1 21 USTED ie SA a aa eee 45 18 3 66 a. Method of determining number. Since one of the chief pur- poses of this study is to determine accurately whether there is any variation in the number of chromosomes, considerable care has been taken to eliminate from the evidence every possible source of error. An important part of the presentation of this evidence is, then, a concise description of the exact procedure employed in obtaining it. 1. Procedure. In order to avoid overlooking any mitotic figures, the entire surface of every piece of tissue was completely surveyed systematically before beginning to count any of the chromosomes in any of the complexes. The survey was accom- plished with a 4-mm. objective and an 8x ocular supplemented by a mechanical stage. | In determining the number of chromosomes in each complex, a camera lucida sketch of it was first made at a magnification of 2633 diameters. This sketch was carefully compared with the cell in order to make certain that no errors had been made in 178 CHARLES L. PARMENTER sketching it. The chromosomes were then numbered consecu- tively, the number being placed on both ends of each chromo- some. This method avoided any possibility of overlooking any chromosome or of counting any chromosome twice. 2. Clearness and classification of the complexes. All the com- plexes counted were polar views of late prophases and of meta- phases and have been divided into three classes on the basis of their clearness. The first class consists of forty-five complexes in which every chromosome was so Clearly separated from adja- cent chromosomes that it could be optically traced continuously over its entire length, without losing sight of it at any point. Only the counts from complexes of this group are submitted as data which are unquestionably free from objection and uncertainty. In the second class of cells there are eighteen complexes in which the chromosomes are all exactly as clear as those of the first class, with the exception that either one or two chromosomes cannot be clearly traced over their entire length as they could be in class I and therefore might possibly be hypercritically considered to necessitate interpretation. The three cells of the third class differ from those of the second class in that they each contain places in which the number of chromosomes cannot be determined with confidence and consequently are actually subjects for interpretation. Complexes of the first class. Complexes of this class are represented by figures 1 to 8 which have been made in carbon and are attempts to represent the actual appearance of the chromosomes and their relative positions inthecomplexes. Rep- resentative cells from each of the tissues studied, except the mesentery and lung, have been so drawn. Other complexes of this group have been outlined in ink, figures 9 to 20, to give a further assurance of the nature of the complexes constituting this class of conditions. Since it is impossible to represent chromosomes in a drawing as clearly as they are seen in a cell, it is necessary to consider briefly this situation in order to prevent misunderstanding, and incorrect impressions concerning the clearness of the cells and CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 179 the faithfulness of the description of the conditions under which the number of chromosomes was determined. The difficulty lies in the necessity of representing on a plane surface chromo- somes which in the cell occupy several levels. The effect can be produced by shading, but at the same time at points where chromosomes cross or overlap each other for various distances they might create the impression in the drawing that they cannot be ‘‘optically traced continuously over their entire length.’”’ There are such cases in every drawing. This is especially true of the late metaphases of the tail epithelial com- plexes (e.g., figs. 7, 8) where every chromosome in the cell can be clearly and faithfully traced as described above. There is also the condition in which parts of the same chromo- some are so related to one another that their appearance in the drawings might create a doubt as to their clearness in the cell. Examples of this are represented in figures 6 and 8, chromosome ‘a,’ in which the two arms of the same chromosome turn abruptly upon one another and the appearance might be subject to the criticism that there are two different chromosomes involved—a portion of one lying exactly upon another with their ends termi- nating at the same point. Such cases were carefully examined end the two arms can clearly be seen to follow into each other. In four of this first class of cells there is another condition that needs mention. These cells contain one or two chromosomes which appear to be broken into two parts (e.g., figs. 19 and 20, f). The parts in each case are separated by very short spaces and are exactly in line with each other. Della Valle (’09, fig. 11) shows two cases of this sort as one chromosome, but discusses them (p. 116) as uncertain. That there is a single chromosome concerned in each of these cases is further evidenced by the fact that there are twenty-one similar cases in other cells of this class (e.g., figs. 5, 7, 14 and 15, f) and thirty-five cases in cells of class II in which the parts are connected by various amounts of chromatin. In some instances the connection is seen as faintly staining chromatin, in others as a single or double darkly stained thread. 180 CHARLES L. PARMENTER Complexes of the second class. In fourteen cells of this class there is one point in one chromosome and in four cells there is one point in each of two chromosomes which, to persons hyper- critically inclined, might possibly appear uncertain. To one acquainted with the material, each of these points is entirely clear, and even when accepted as subject to interpretation it is very plain how the interpretation should be made—so plain that I am certain that the count of twenty-eight chromosomes is accurate and dependable. But for the sake of unquestionable fairness I have placed these cells in a separate group. As to the exact nature of the interpretations in these eighteen com- plexes, four of them have some small portion of only one chro- mosome so covered by others that it cannot be traced over its entire length without losing sight of it as stated above (p. 178). Two other cells had two chromosomes of this nature. Five complexes have a single chromosome lying in such a relation to another chromosome that it might possibly be interpreted as a part of the other chromosome (e.g., fig. 23, chromosome 7), and in three more cells there were two such chromosomes. In the remaining five complexes a single chromosome was so situated or otherwise involved, that it might be interpreted that there were two chromosomes present (e.g., fig. 21, 2). In considering all the interpretation possible in each of these eighteen cells the minimum number in any one of them would be twenty-seven and the maximum number thirty. Even grant- ing this much variation, it is far removed from that expected in a series of chance variants as Della Valle claims them to be. The points in question were sketched as described above before the chromosomes were counted, so that the determination of the number of chromosomes was not influenced, either con- sciously or unconsciously, by a knowledge of how many chromo- somes were present or by how they should be sketched in order to produce the expected number. This procedure and the fact that the number counted always agreed with the number present in the forty-five cells of class I make it practically certain that the enumeration is correct. It should be emphasized again that these cases are only subject to question when hypercritically CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 181 considered and would otherwise constitute a part of class I. In fact, an experienced cytologist of this laboratory, in examining these, without a knowledge of the number of chromosomes present, could see no reason for considering them as subjects for interpretation, and it seems almost absurd to place them in a separate class. : ft Complexes of the third class. There was a very large number of cells which were beautifully clear everywhere except in regard to one or two chromosomes. However, only three of these were sketched, because the number of clear counts was so large that an increased number of these uncertain counts is of little value. Each of the three cells drawn contains two points of uncer- tainty as to whether there are one or two chromosomes present. The number is interpreted as either twenty-seven or twenty-eight. The minimum number of chromosomes possible of interpreta- tion in one cell is twenty-six, the maximum is twenty-eight; in the other two cells the minimum is twenty-seven, the maximum is twenty-nine. These three cases were interpreted while the sketch was being made and before it was known how many chromosomes were present. It is not true, therefore, that the interpretations were prejudiced nor- that any cases which did not agree with the expected numbers were cast aside and consequently ignored. On the contrary, they are here included as part of the evidence in forming the conclusions drawn from this study. Rationally considered, then, of the cells sketched there are sixty-three in which the enumeration of chromosomes is accurate and dependable and three in which there are unavoidable interpretations necessary. These sixty-six complexes constitute very strong evidence that the number of chromosomes in Am- bystoma tigrinum is constant. b. Possible variation in number in uncounted complexes. As to whether or not there was any variation in chromosome number in this species can be judged from the results obtained from the sixty-six cells which were studied. If as few as 2 per cent of the total complexes studied varied from: the usual number, at least one of these should have made its appearance. Furthermore, 182 CHARLES L. PARMENTER Della Valle (’09, p. 117) claims that variation of chromosome number is probably a general law and (p. 120) that his counts strikingly bear out the expectation expressed by Newton’s theo- retical binomial curve. Were this the condition in Ambystoma tigrinum, a good proportion of the sixty-six complexes should have shown variation in number. Since no variation was found, it is safe to conclude that there is none in the cells that could not be counted. c. Abnormal complexes. Seven apparent variations from the usual number were found. These were groups of chromosomes in which the number was clearly other than twenty-eight (figs. 22, 24, 25, A.B., 26, A.B.). But when these groups are thoroughly analyzed it is certain that they are nothing else than cases of a very unusual behavior of four cells and do not constitute a variation from the usual number of chromosomes. Figure 22 shows a peritoneal cell which has lost a part of the chromosomes. Chromosome a is but part of a chromosome, showing very unmistakable evidence that a portion of it has been broken off and there is a conspicuous depression in the tissue from which it is evident that the remainder of the chromosomes of this cell have been lost. The cell lies close to a tear in the peritoneum. It is a bare possibility that the tear and the loss of the chromosomes is due to the same cause. The second case isa very early metaphase from the peritoneum. It consists, as represented in figure 24, of one group of twelve chromosomes and another group of sixteen immediately adja- cent to it.’ These two groups and figures 2 and 20 are very similar to Della Valle’s dicentric cell (’09, fig. 6) and Flemming’s (91) figures 31 to 39, table 40. ) Zs FP AW \\ C PLATE 5 EXPLANATION OF FIGURES 17 to 19 Two prophases and one early metaphase of gill-plate epithelium. 20 A peritoneal metaphase. 236 © JAS WE Ba eecss CO ——) Vin cSSS ‘a ——5 PLATE 6 EXPLANATION OF FIGURES 21 and 23 Gill-plate prophases of class Il. Chromosomes ‘/,’ figure 21, inter- preted as one; ‘i,’ figure 23, as two (p. 12). 22 A peritoneal complex with part of the chromosomes missing. 24 A peritoneal complex separated into two parts. The pair numbers are duplicated in figure 31 and_in the legend of figure 37. 238 WM |) . 14 i 5 if < PLATE 7 EXPLANATION OF FIGURES 25A Another peritoneal complex separated into two parts which are drawn in their relative positions. 866. 25B The chromosomes of figure 25A. X 1755. Chromosomes 14 to 17 are interpreted; note that the chromatids of these four chromosomes are well separated. 26A A lung epithelial cell separated into two parts which are drawn in their relative positions. XX 866. 26B Chromosomes of 26A. X 1755. Pair numbers duplicated in figure 32. 240 PLATE 7 CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA CHARLES L. PARMENTER PLATE 8 EXPLANATION OF FIGURES 27 to 32. Chromosomes of figures 1, 3, 9, 10, 24, and 26, respectively, arranged in pairs with pair numbers and figures indicating their lengths in millimeters at X 2633. The amount included in any figure for foreshortening is indicated above that figure. The pair numbers are duplicated in the above figures and in the legend of figures 33 to 37, respectively. CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA CHARLES L, PARMENTER Seu C LS Ub 12.5 13 15.1 16.2 22.5 233 265 26.8 27.2 27.2 9 ON o6 ML 28 14145 178179 25.5 26.9 278 28.6 316 32.1 acl 29 ig a5 139 195 eos 30 145 175 18 20 iv av vb vu LY Sl 85105 14 165 20 20 22) 22712315 23.5 S2) ia: a I 14 15 7 175 IS 19 | 2 a 4 5 $3.5 355 wy CL ai t¢ hd 404 562.8 44.4 45.3 45.7 45.8 Lo ve SY 33.6 33.6 iv a” JV 36.5 39.5 VA Vv yy 36.5 42 44.8 44.8 4g 49.5 25 26 29 33.5 39 4\ pe) 26 26 26 o7 244 $2.3 55.4 568 55.5 55.5 565 56.5 \ +12 573 58.8 61.3 ¥ ey PLATE 8 48.9 49.3 51 52 3 58.7 58.9 62.2 64 +1 2 585 585 60 62 % 47 47.5 48 49.5 30 30 30 33 13 14 245, PLATE 9 EXPLANATION OF FIGURES 33 to 37 Representing the lengths of the chromosomes in the cells indicated in the table below at a magnification of 1316. The differences in the lengths of the lines and also the spaces between the lines represent relative differences in chromosome lengths. For convenience the width of the spaces between the lines are made eight times the differences in lengths. The lengths of the chromo- somes and their percentage of the average length in the cell is shown in the table below. The amount of foreshortening in any chromosome is indicated in plate 8. FIGURES PAIRS 33 (1, 27) 34 (3, 28) 35 (9, 29) 36 (10, 30) 37 (24, 31) Milli- Per Milli- Per Milli- Per Milli- Per Milli- Per metres} cent | metres| cent | metres|} cent | metres; cent | metres| cent 12.5| 37 | 14.0| 35 | 18.0| 44 | 14.5] 34 8.5 | 27 13.0 | 38 | 14.5 | 36 | 18:35} 45 | 17.5) 41 | 10.5! 338 15.1} 44 | 17.8) 45 | 19.0} 46 | 18.0] 42 | 14.0] 44 Z 16:2) 47 | 47.9) 45 | 1975.) 47 "| 2070 | "47 | AGsoiee2 3 22.5 | 66 | 25.5 | 64 | 26.7] 65 | 24.7) 58 | 20.0] 68 23.3 | 68 | 26.9], 68 | 27.5 | 68 | 25.0] 59 | 20.0] 638 ie ZO | EC | 2H 10" 29090.) el 2Oe| © Gs! eee 26:8 | 78 | 23/6.) 72.) 29-5 | “72—) 29:0) | 68" 4), 22.00 70 5 2h.2 | 80, \-Sl-6 3) 7-|| '2uQ uol$a77-] Ly IVOIWOLSAW I FVOINIUIS -AVGIHONWH@OL a LyO | : Ley ey, AVOIMNLOIN cGy ‘ IVUSTOWOTHd AL igus 3 ca iP B | IVOILNOGOHL 1d 9 Sy AVQIHIVNIOWS 30 VLVaNVO TO SALVYVddV ONILLINSNVUL-GNNOS SIG BOA ata oN Oa alae 9 ULV Td Resumen por el autor, G. W. Tannreuther. Universidad de Missouri. El desarrollo de Asplanchnia ebbesborni (Rotiferos). La formacién y segmentacién de los évulos puede seguirse paso a paso dentro del animal vivo a causa de su transparencia. La segmentacién, en muchos puntos, tales como la direccién, orden y marcha de la misma, presenta el mismo caracter que la de los anélidos, formandose tres generaciones de ect6meros. La regidn ventral de los ect6meros corresponde al futuro extremo anterior, mientras que la que ocupan los macrémeros, A, B y C corresponde mas al extremo posterior. Durante la gastrulacién el macrémero grande 3D pasa al centro del embrién. A, B y C permanecen en la superficie del extremo posterior del embrién y sus derivados estén relacionados mas directamente con la pro- duccion del voluminoso pié embrionario. 38D (E) origina el sis- tema reproductor con unas cudntas fibras musculares y todo el sistema digestivo, con la excepcién del stomodaeum. Los deri- vados de los ect6meros producen las demas estructuras. Todos los 6rganos, (con excepcidn del sistema digestivo del macho) son funcionales y estan bien desarrollados en ambos sexos. El ani- mal produce 6vulos machos, hembras y otros en estado de reposo. Todos los 6vulos, excepto los en reposo, son transparentes; los ultimos poseen abundante vitelo. Los machos y las hembras no son originados nunca por el mismo animal, mientras que los em- briones machos y 6vulos en reposo son producidos por la misma hembra. Los évulos machos y los ultimamente mencionados difieren en estructura y solamente los en reposo son susceptibles de fecundacién. La naturaleza y marcha del desarrollo indican que los rotiferos no son larvas troc6foros de los anélidos, que han persistido, sino mas bien que los rotfferos y anélidos se han origi- nado a expensas de una misma forma ancestral, habiendo alcan- zado los anélidos un estado mas elevado en la historia de su evo- lucidn. Translation by José F. Nonidez Carnegie Institution of Washington AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 2 THE DEVELOPMENT OF ASPLANCHNA EBBESBORNII (ROTIFER) GEORGE W. TANNREUTHER Zoological Laboratory, University of Missouri TWENTY-ONE TEXT FIGURES AND SEVEN PLATES CONTENTS eT ROU Gt OTM my ias y- rh aot a Ere tuber Gove een ces ticked Seyler ek estes Sas Sak 389 INEM! LONGO sid gig cS OMe aie d oO ants BED OOS c SEG Glos cana ian Gana ae 391 MiahenialeanGemevnOUseme mayan Gece cat cancion foe cs oe scie Le ean a ubine ss 394 ir eMC VOTEEAR ON, Ol OVA... Ure saan esos eens. ELS ea ERs 394 Hee rR ETOP OT EIN hy acr reese fain e.be 15 3 awh tc osios ans ior ae vot SS MRI etee ss be 394 2, SSRIs Foo's Sod GaSe oom a ie DORIS BO ROS EOE eee eR Gata Comoe ye ee 395 CHS ETDS 03 is Bates GR chee RCN eRe gE ee I 2 Co a 395 eM estonatroniom cleayagercellss ss ssaece 2 Jc 0s so. came Rema ees 395 Pay DELO AALULetOhiGleayareh se os ERG sel E sleicts anc SRR ea 396 Sa chet ONT AI Ld) RUE PRES aE es ery ci ray Pop PGS oy) 0 oy =soi din. s-sbainkes aoa EYE ek 400 Sesnep ain OMMOm Perm ly ens mre seit pire ae irc ices ccihs.s « o,clbie.+ che emetic ele ees 401 PH CUOCELI Aster te ON An hen teen are ee eh LS ir ee ea an ee 401 BPRIVICHOGCEEG Le Ase SHOt ae Ann SR RIL E SMe YON. 2. Red Pee 402 Sir ibiipere bee socks ws ORNS am eben eM QP Ces on a ae eae (ee 404 Borne OmuOl bhOeM AM TEPLOM Ns er ee co threes Oh ss oe ened dig Ae esos aroma 406 ROPER ME My SOCIO ote on ee eee ee a IG. ac bas hae wim alors Sere 407 REPLOGUCUIVE SYSTEM ss 0M eeA ts ne yoebaate dn a wtde yc cd oe dation Hecke LEE ATS HOt Ue 410 BI eTULOEYS SV CLOTS Sp yn 5 ce erate eres FAY cc's «god Sl Hane wae Motes Sere 411 INIGEAVOISUGH ASSENT sees bot aie Sechis 36.00 Bee ee ee eee ee 414 POON On TOLLeTS 10, Ghe aMiMAl KINgOOM.......... 1.00.2 .20000 2020s sn eos 415 RS EMERESIBIEY ie ish 5 an RNS So yo ial oes wk OY DRA RD SeTA SEO hs 419 INTRODUCTION The size of the adult rotifer, in many instances, has made the study of development a difficult problem. A great descriptive mass of literature has grown up around this group of animals, but with the exception of a very few detailed accounts of their structure, the writers have contented themselves with the description of the external form. 389 JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2 390 GEORGE W. TANNREUTHER The morphology of the Rotatorian family Flosculariidae was worked out by T. H. Montgomery (’03) in considerable detail. Zelinka (’92) published an account of the development of Calli- dina ruseola. Jennings (’96) traced the cell lineage of Asplanchna herricki, for a few generations, but did not correlate the cells of the early embryo with the adult structures. Some rotifers are very transparent; this makes possible, not only a detailed study of the position and relation of the various structures, but an investigation of the origin, growth, and cleavage of the eggs. Where complete development occurs within the parent, a sequence of the different stages from the first appearance of the egg to the time of birth can be followed with a considerable degree of accuracy. The extreme trans- parency of the adult Asplanchna ebbesbornii, makes possible a detailed study of the successive stages in development. The more important points in development may be summarized as follows: 1. The cleavage is unequal and regular. A small cleavage cavity is present. The gastrula is formed by a modified epiboly. The blastopore occurs at the posterior end of the developing embryo. 2. The first cleavage plane is at right angles to the future median longitudinal axis of the adult. It divides the egg into two very unequal cells. The smaller cell is ectodermal and mesodermal, while the larger cell contributes to the three germ layers. 3. The gastrula consists of an outer layer of epithelial cells enclosing an inner cell mass. The outer layer includes all of the derivatives of A, B, and C and the three cells d!, d?, and d? derived from D. The inner cell mass includes all of the remain- ing cells. 4. The outer layer or ectoderm gives rise to the cuticle, hypo- dermis, brain, excretory system, trochal disc, cilia, buccal pouch, and musculature. The inner cell mass derived from 3D produces the remaining parts of the digestive system and glands, the reproductive system, and a few of the muscles which control it. ASPLANCHNA EBBESBORNII (ROTIFER) 391 5. The origin of the various organs cannot be traced back to any definite cell or cells of the early cleavage stages, but can be directly associated with definite regions of the early gastrula. It is true, however, that the products of any one cell A, B, C, or D, in the production of the ectoderm, can be definitely followed and localized in the gastrular ectoderm, but there are no struc- tural differences to mark off these regions. The ectodermal cells of any region possess the same potentiality in producing muscle fibers, regardless of their origin. It is impossible to tell definitely the differentiation of the digestive and reproductive systems until late cleavage. 6. The derivative of the blastomere B form the ventral, those of D the dorsal, those of A the left lateral, and those of C the right lateral ectoderm of the adult animal. 7. The brain on the median dorsal side is derived from the ectoderm at the anterior end. The urogenital sinus is formed by a solid ingrowth of the ectoderm on the median dorsal side at the posterior end above the base of the embryonic foot. Its position is considered as being ventral in the adult, due largely to the disappearance of the foot in later embryonic life. The embryo in its early development is curved ventrally, making the dorsal side appear quite long in comparison to that of the ventral. The mouth is ventro-anterior. NATURAL HISTORY The rotifer Asplanchna ebbesbornii is not very abundant in this immediate locality. The material was collected from small rain pools and placed in aquaria, filled with tap-water. This particular rotifer was first observed in one of the freshly prepared cultures in January, 1916. They persisted about two weeks and disappeared. They reappeared in the following March and continued for two weeks. The rotifers appeared again in the following May. In each instance about two months elapsed between the times of their appearance. This periodicity con- tinued until December, 1918, when this paper was completed. In each cycle of appearance males, females, and resting eggs were formed in about the same proportions. The cultures were kept indoors at laboratory temperature. 392 GEORGE W. TANNREUTHER The January and March cycles were not studied very exten- sively. The studies proper were begun with the May cyele, 1916, in tracing out the cell lineage. The adults are extremely transparent, and by holding them in any desired position under a supported cover-slip the cleavage of the individual cells can be traced step by step. Two distinct kinds of adult females exist, which are structurally similar as far as can be determined under a magnification of 100 to 150 diameters. The one repro- duces females parthenogenetically, and the other males parthen- ogenetically or resting eggs, which carried the cycle of one period to their next reappearance. The resting eggs pass through their early stages of cleavage before deposition. ‘There are, however, two kinds of resting eggs: a very thin-shelled one, with a single shell membrane, and a thick-shelled egg, with a double shell membrane. The thin-shelled egg develops with the same rapidity as the parthenogenetically produced individuals and hatches out immediately after deposition. Two polar bodies are formed in each kind of resting egg, which do not develop unless fertilized. Females and resting eggs or females and males are never produced by the same individual. The parthenogenetically produced males and females are sexually mature at birth. The uterus of the young females often contains embryos in the late cleavage stages at the time of their birth. The two kinds are practically the same size at birth. Copulation occurs almost immediately after the birth of the males. The male may copu- late with either kind of female. The uterus and the oviduct of the parthenogenetic producing female often contain sperm, but neither males nor resting eggs are produced by this particular individual. On the other hand, the uterus and oviduct of the male-producing parent may contain an abundance of sperm, and yet produce males only, or again, they may produce both males and resting eggs. There is no definite sequence in the production of males and resting eggs by the same parent. A single male may be produced and all the remaining become resting eggs, or vice versa. Or, on the other hand, it is not unusual to find the two alternating. The following is a good example: the sequence ASPLANCHNA EBBESBORNII (ROTIFER) 393 was as follows: two resting eggs, one male, one resting egg, one male, one resting egg, one male. In case of the female which produces the resting eggs the vitellartum undergoes a marked change after impregnation. The yolk spherules become larger and more abundant and give the vitellarium a very dark color. The yolk is first produced at the point where the oviduct takes its origin. This process continues until the vitellarium is completely filled with yolk. Where males and resting eggs are produced by the same parent, the yolk is produced at intervals just before the resting egg begins its growth in the ovary. The male eggs are free from the dark yolk and remain transparent. Impregnation has no effect on the vitellarium of the female-producing individual in the production of yolk. Figures 7 to 11 show the single thin-shelled and the double thick-shelled resting eggs. In figure 12 the spermatozoa are shown in different stages of development. In many instances the sperm of the sexually mature male (before birth) would escape from the testis and become deposited in the uterus of the parent and bring about the production of resting eggs. In other cases the male was little more than a large sperm sac. The male embryos developed normally until the early differentiation of the reproductive organs. At this stage of development all of the cleavage cells, except those directly concerned in the production of the sperm, ceased dividing, took on a vesicular appearance, gradually deteriorated, and functioned as food for the developing sperm. The sperm, when. mature, escaped through the egg membrane into the lumen of the uterus. This condition accounts, to some extent, for the few free-swim- ming males in the cultures. The males are structurally degenerate at birth. ‘The digestive tract is very rudimentary (figs. 5 and 6) and never opens to the exterior. ‘The males are very short-lived, and few free-swimming individuals are present at any one time. The males do not increase in size after birth (figs. 5 and 6). On the other hand, the females increase to at least four to six times their size at the time of birth (fig. 1). The embryology of the male and the female developing individual is practically the same throughout 394 GEORGE W. TANNREUTHER the different stages of development. One polar body is formed in the female egg, while in all others two polar bodies are formed. The individuals hatching from the resting eggs are always parthenogenetic females. But the next generation is of two kinds, one producing females parthenogenetically and the other producing males parthenogenetically or resting eggs. MATERIAL AND METHODS The rotifers were removed from the different cultures with a pipette and placed in watch crystals. The excess water was then removed and the fixing fluid added. SBouin’s fluid gave the best results. The animals were preserved in 70 per cent alcohol until used. If allowed to stand indefinitely in alcohol the individuals turn brown and are not very satisfactory for study. For whole amounts Delafield’s haematoxylin and eryth- rosin were used. Either stain gave good results. The changes from alcohol to xylol and from xylol to the mounting medium must be made very gradually. A few drops of carbo-xylol and clove oil will help considerably in the process. The cuticle is very resistant and will cause considerable shrinkage and dis- tortion if the change be made too quickly. The same caution must be taken in clearing specimens that are to be imbedded and sectioned. Iron-alum haematoxylin and erythrosin gave the best results for sections. In the case of whole mounts, the cover- slips should be supported. ORIGIN AND FORMATION OF OVUM The reproductive organs are composed of the vitellarium, ovary proper, oviduct, and uterus. The uterus opens into the urogenital sinus. The vitellarium is somewhat U-shaped (fig. 4), very transparent when free from yolk, and contains many large nuclei. -The ovary (figs. 1 to 4) occupies a very small area at the base of the vitellarium. Parthenogenetic ova: The ova are very small, and asingle ovum at regular intervals begins its growth. The contents of the growing egg is derived directly from the vitellarium. The ASPLANCHNA EBBESBORNII (ROTIFER) O90 passage of cytoplasmic and yolk granules into the egg is visible under a low power. When the egg reaches the end of its growth period, it is separated from the ovary (fig. 4), and enters the upper end of the oviduct, which, in reality, encloses the greater part of the ovary. A single polar body is formed. In most cases immediately after this maturation, the following egg begins its growth. Two eggs may begin their growth at the same time, but this is very unusual. In the case of the male-producing eggs, two polar bodies are formed, the first of which often divides. The origin, formation, size, and development of the female and the male-producing eggs are identical (figs. 2 to 4). Sexual or resting ova: The origin and growth of the resting eggs are similar to that of the parthenogenetic eggs. The vitel- larium, however, is very dark from the presence of a rich supply of yolk. The yolk passes directly from the vitellarium into the growing egg. Immediately after maturation and fertilization, a very thick inner shell is formed from the cytoplasm. The contents of the double-shelled egg cannot be studied except in sections In many of these resting eggs no inner shell membrane is formed. They are about the same size as the thick-shelled eggs, but are more transparent and contain less yolk. Their cleavage stages can be followed without the aid of sections. The number of resting eggs in the uterus at any one time varies from one to eight. In case of the female or male-producing parent, there may be as many as sixteen embryos in the oviduct and uterus at the same time, ranging from the early cleavage stages to the mature young (fig. 2). CLEAVAGE 1. Designation of the cleavage cells The nomenclature adopted in the designation of the cleavage cells is a modification of the system used by previous investigators on cell lineage. The first four cells (macromeres) are designated by the capital letters A, B, C, and D. The generations of micro- meres (ectomeres) by the small letters a, b, c, and d. The first index number indicates the generation to which the ectomere 396 GEORGE W. TANNREUTHER belongs. Thus a', b!, c!42, or d'1+, all belong to the first genera- tion; c?, b?+, or d?’, belong to the second generation, and a}, b322, ¢32, or d33, ete., belong to the third generation. On account of the peculiar shifting of the macromeres A, B, C, and D in the formation of the first quartette, A, B, and C take a position more anterior and D posterior, instead of at the vegetal pole, as in annelids. When a cell divides, the product receives the designation of the parent cell with the addition of a . 2.1 further index number; thus, 4 mae The cell D, after the for- mation of d? and d?, is designated by the capital letter E; it gives rise to the reproductive system and all of the endoderm (digestive system) except the stomodaeum and the pharynx. 2. Nature of cleavage First cleavage: Immediately after maturation the nucleus passes from the surface toward the center of the egg, but nearer the anterior end. The first cleavage spindle is formed about thirty: minutes after maturation (figs. 13 to 16), the time varying somewhat with external conditions. Low temperature retards the rate of cleavage. The first cleavage spindle occurs in the plane of the long axis of the egg (fig. 7). It passes through the region of the polar body or bodies and divides the egg into two very unequal parts, AB and CD (figs. 15 to 17). The smaller cell, AB, is anterior and the larger cell, CD, is posterior. The cleavage furrow at first is deep and the cells are rounded, but before the second cleavage occurs the cells flatten at their point of contact and the egg becomes more elliptical with the first cleavage plane scarcely visible. The granular content of the cells is uniform, with very few yolk bodies visible. The region immediately surrounding the nucleus is almost free from cytoplasmic granules and makes it possible to follow the nuclear activities in the process of division in the living egg. The first cleavage in the femaleé- and the male- producing egg is the same. It occurs at right angles to the future longitudinal axis of the adult. The second cleavage plane occurs ASPLANCHNA EBBESBORNII (ROTIFER) 397 at an angle of about 45° with that of the first. The two cells divide at different times. These two cleavages combined corre- spond to the second cleavage as it occurs in many of the annelids and molluses. The cell CD divides first into two very unequal parts (figs. 18 and 19). The division of AB is nearly equal (figs. 18 to 20). Shortly after the second cleavage, a slight shifting occurs as the cells flatten (figs. 19 to 24). The largest cell, D, is posterior, B median anterior, C right, and B left (fig. 21), with reference to the median axis of the future animal. The largest cell, D, always divides first in the formation of the quartettes. The orientation of the four-celled embryo is very simple and agrees with that of the annelids at the same stage of development. From this point forward, however, the position taken by the resultant cleavage cells is no longer comparable with that of the annelids, but the sequence of cleavages in the following stages is very similar. Third cleavage: In the formation of the first generation of ectomeres (d!, b!, c', at), the cell D divides first, the new cell is budded off in a dorsal anterior direction on the median dorsal side of A, B, and C (text fig. b and figs. 23 and 24), making a five-cell stage. In this process the polar body is carried forward to the anterior end with the cell d'. While the cleavage spindle is forming for the production of the micromere d!, the cells A, B, and C elongate in an anteroposterior direction (text fig. ec and figs. 24 and 25), so that the cleavage plane of A, B, or C is not hori- zontal, but in a dorsoventral direction at right angles to the long axis of the embryo (figs. 26 to 31). Thus, instead of having the micromeres above the macromeres as in many forms, the cells a1, b!, c1, and d! are on the same level with A, B, and C (figs. 31 and 32). The division of the macromeres A, B, and C is nearly equal in the formation of the first generation of ectomeres. They do not divide simultaneously, but in the invariable order C, B, A. Thus there occurs successively a six-, seven-, and eight-cell stage (figs. 830 to 32). In the formation of the eight- cell stage B and b! are pressed ventrally by d! (figs. 24 and 31). In the eight-cell stage d' is median dorsal, B and b! median ventral, C and c! right, A and a! left, and D posterior extending 398 GEORGE W. TANNREUTHER dorsoventral (figs. 31 to 33). The micromeres form the anterior end. In a few instances, C, B, and A divided in a nearly hori- zontal plane, thus placing the micromeres above the parent cells instead of on the same level with them (text figs. d and e and fig. 33). This mode of division, however, is very unusual, but is comparable with that of the annelids and polyclades. At the completion of the eight-cell stage, the embryo often assumes the shape of the one-cell condition and the cleavage furrows are scarcely distinguishable. The embryo at the dif- ferent stages of development is very plastic and may asssume almost any shape under abnormal pressure. If the egg be removed from the reproductive organs, with the egg membrane intact, after cleavage has begun, normal development will continue. By slight pressure the individual cells of the eight-cell stage can be separated. The isolated cells seldom continue to divide, but begin to deteriorate almost immediately. Fourth cleavage (sixteen-cell stage): A nine-cell stage is reached by the formation of d? from the large cell D on the median dorsal side, in an anterior direction (figs. 34 and 35). As d? is formed, d! is carried around the dorso-anterior end. Next, d! divides in an anteroposterior direction (fig. 36). Following this, the macromeres A, B, and C and their micromeres divide in an anteroposterior direction. The division occurs in the order C and c!, B and b!, A and a', thus producing a twelve-, fourteen-, and sixteen-cell stage, respectively (figs. 34 to 38). The embryo is now composed of four rows of cells with four cells in each row (figs. 38 to 44). The fifth cleavage: The derivatives of D divide in the follow- ing order: d?, di, d'1, thus producing a seventeen-, an eighteen-, and a nineteen-cell stage (figs. 45 to 48). The cleavage spindles in the C, B, and A rows indicate the direction of the cleavages in passing from the nineteen- to the thirty-one-cell stage (figs. 45 to 48). In a few of the embryos, as in annelids, D budded off a third cell, d* (figs. 46, 47, 49, and 50). This extra cell, when produced, has no special significance in the future development of the individuals which bear it; d* and its derivatives will not be considered in the further description of the cleavage stages. ASPLANCHNA EBBESBORNII (ROTIFER) 399 At the close of the fifth cleavage, the quadrants A, B, and C are composed of two rows with four cells each, and the quadrant D of two rows with three cells each, including the large entoderm cell D, making thirty-one cells in all. The comparative sizes of the cells are shown in the different figures. During the fifth cleavage the cells withdraw toward the exterior (fig. 55) and Fig. a Four-cell stage, left side. Fig. b Four-cell stage, left side, showing the cleavage spindle in the forma- tion of the ectomere d!. Fig. ¢ Six-cell stage, ventral side; shows the anteroposterior extension of cells. Figs. dande_ LEight-cell stage, ventral and dorsal sides; shows the position of the first quartette of ectomeres similar to that of annelids. This condition is unusual. Fig. f Seventeen-cell stage, left side. 400 GEORGE W. TANNREUTHER form a cavity, which later is occupied by the large cell D. Before the fifth cleavage is complete, the anterior end of the cell D is partially covered by the cleavage cells immediately in front of it (figs. 45 to 50). Sixth cleavage: After the formation of the small cell d+, the cell d!11 (fig. 51) divides. Next a small cell d*° is formed from D. The first cell of the sixth cleavage to divide is d2!, ete. The sequence of cell formation in the sixth cleavage is similar to that of the fifth, the derivatives of the D quadrant dividing first, then those of C, B, and A. The cell lineage of any one quadrant can be followed indefinitely. Figure 51 represents the beginning of the sixth cleavage. From this stage the surface cells will not be labeled, as it does not contribute to the understanding of the further development. The sixth cleavage doubles the number of cleavage cells on the surface. It does not increase the number of rows in each quadrant, but the number of cells in each row is doubled. The embryo at the end of the sixth cleavage is com- posed of the following cells: the D quadrant contains two rows of six cells each, and d‘ and d*. The C, B, and A quadrants each contain two rows of eight cells each. With the large entoderm cell D, there are thus sixty-three cells in all. 3. Gastrulation Gastrulation, which begins during the close of the fifth cleavage, adheres more strictly to the epibolic type. Immediately after the formation of d2, the surrounding cells at the anterior end of D begin to extend over its surface (fig. 48). The embryo when viewed from the posterior end (figs. 52 and 53) shows the position of the surface cells with reference to D. The spindle indicates the direction in which d‘ is formed. Figures 52 and 54, later stages during the sixth cleavage, show the method of overgrowth on the surface of D (E). Gastrulation is a double process; while the surface cells are extending posteriorly over the surface of E, the large cell itself is migrating into the interior, a result of the pressure of the surrounding cells, and the cavity within the embryo, which began its formation before the sixth cleavage started. ASPLANCHNA EBBESBORNII (ROTIFER) 401 During gastrulation the embryo shortens and increases in width, as shown in figures 55 to 57. The formation of the central cavity (figs. 57 and 58) and gastrulation occurs very rapidly. The entire process requires about fifty minutes and can be demonstrated in the living egg. Figures 58 and 59, a sixty-four- cell stage, viewed from the dorsal and ventral sides, respectively, show the embryo at the end of gastrulation. The blastopore, situated at the macromere end of the embryo, is rather large at first and is surrounded by eight cells, two belonging to each of the four quadrants (figs. 52 to 54). Gastrulation brings about a fundamental change in the re- lation of E to the remaining cells of the embryo. At first it formed the posterior end of the cleavage cells, but at the end of gastrulation it occupies the central region of the growing em- bryo and is completely enclosed by the surrounding cells (figs. 58 and 59). The large cell E is now designated as the mesentoblast, and, on account of its new position is destined to assume a new rdle in the development of the embryo. The embryo at the end of gastrulation contains about 200 cells, which are divided into two distinct regions; the mesentoblast including d‘ and d*, and the epithelial ectoderm, which, at the anterior end, shows the beginning of a double layer (figs. 58 and 59). SEGREGATION OF THE GERM LAYERS In Asplanchna ebbesbornii a distinct segregation of the germ- layers begins with the formation of the cell d? or d*, the nine- or seventeen-cell stage. The cell d? is not usually formed, hence the variation in the number of cells at the time of segregation. The large posterior cell D is destined to become entomesodermal, and all of the remaining cells are ectomesodermal. 1. Ectoderm The dorsal part of the ectoderm is derived from the quadrant D, the right, the left, and the ventral parts from C, A, and B, respectively. The three cells of the quadrant D, d!, d?, and dt, are the first to divide in the formation of the ectoderm during 402 GEORGE W. TANNREUTHER the fifth cleavage. Each cell divides unequally. The anterior cell divides vertically, while the other two divide transversely and parallel to the long axis of the body (fig. 48). For further account of the cleavage process in the formation of the early ectoderm, see description given for the later cleavage stages. Figures 55 to 57 represent the earlier stages in which the cleavage cells become arranged into a definite epithelial layer. The cavity shown in these figures is in reality the early body cavity, since the epithelium later becomes the definitive body wall. A later condition of the ectoderm is shown in figures 67 and 68. From this point onward the division of the epithelial cells is very irregular and their products become differentiated into the definitive ectoderm and its various derivatives. At first the definitive ectoderm is represented by an epithelial layer com- posed of large nucleated cells, and is in immediate contact with the organs forming within (figs. 77 to 90). But as the different organs reach their complete development, the body cavity becomes more and more marked and the cells of the ectoderm are drawn out into a very thin epithelial layer, with the boundary of the cells no longer visible (figs. 96 to 104). The cuticle is a very thin layer formed by a secretion from the ectoderm shortly before birth. The cuticle is usually free from markings and has a smooth surface. Figure 104 represents the condition of these various structures at the time of birth. 2. Mesoderm In the late gastrula stage (figs. 63 to 70), the outer epithelial layer of the embryo at different regions begins the proliferation of cells on the inner surface. These cells contribute directly to the formation of the mesoderm, which later becomes differen- tiated into the muscular system. It is impossible to distinguish in the embryonic ectoderm the cells which directly give rise to the mesoderm. Apparently all parts of the ectoderm possess the same potentiality in the process. These cells, in their for- mation, remain connected with their point of origin. Later these proliferated cells show an apparent connection or fusion with the ASPLANCHNA EBBESBORNII (ROTIFER) 403 inner cell content (the invaginated stomodaeum and the deriv- atives of the mesentoblast) (text figs. g, h, and q). Other rows of proliferated cells at either end are connected with the ectoderm (text fig. k). Many of these cell processes have several attach- ments (text fig. h), but in their further development some of these points of fusion are lost and the developing muscle remains attached to the later formations, which they are destined to control. The muscle fibers, in their early formation, are com- Figs. g and h Early muscle cells connecting ectoderm with inner structures. Figs.iandj Early and definitive stages of muscle-fiber development. Figs. k andl Shows method of attachment in the early and late development of muscle fibers. Fig. m A completely formed muscle, showing attachment to brain and ectoderm. Fig. n A network of muscle fibers extending across body cavity. ABBREVIATIONS b.c., body cavity m.c., muscle cell br., brain m.f., mauscle fiber cp., corpuscle tr., trochus ect., ectoderm w.ph., wall of pharynx 404 GEORGE W. TANNREUTHER posed of cellular processes (text fig. k), which, later, as develop- ment progresses, lose all traces of cell boundaries, with a few nuclei persisting (text figs. 1 tom). The method of attachment in one of the completely formed muscles is represented in text figurel. Itis attached anteriorly to the corona and at its opposite end it is anchored to the ectoderm. Some of these muscle fibers show a distinet cross or longitudinal striation with few nuclei. In the mature embryo (text fig. n), many of the muscle fibers are drawn out and form a delicate network, which extends through the different regions of the body cavity. Some of these fibers are mere lines and are hard to distinguish. Many of the apparent migratory cells within the fluid of the body cavity are directly connected with very delicate processes (text figs. m and n), while others are mere floating corpuscles, which are highly vacuolated. The various structures within the body, as well as the trochal disc, are kept in constant motion by the activities of the various muscle fibers. No attempt was made to represent the position of the different muscles in the various figures drawn. 3. Entoderm During the early part of the seventh cleavage, the blastopore becomes completely closed (figs. 59 and 60). The cells desig- nated as the entoderm include the large cell E and the two small cells d* and d*, which are formed from E during the sixth and seventh cleavages of the embryo. Figures 58 to 60 show the first stages in the cleavage of E, which corresponds to the eighth cleavage. This cleavage is unequal and separates a smaller cell, E!, from a larger cell, E?, posteriorly. Figure 60 is a dorsal view of the embryo and shows the condition of the ectoderm and entoderm at the beginning of the eighth cleavage. The ectodermal cells during the ninth cleavage divide very rapidly and are difficult to follow. Immediately after the first cleavage of E, the cell E? divides equally and transversely, or at right angles to its first cleavage. Figures 61 to 64 show the different stages in the process of division. The embryo, as a whole, is very plastic and becomes more spherical during the cleavage of ASPLANCHNA EBBESBORNII (ROTIFER) 405 E? (fig. 62). Figure 63 represents a 250-cell stage, and by turn- ing the embryo in different positions all of the cells can be recog- nized, but it is impossible to tell the exact boundary of the cells derived from any one quadrant. The derivatives of D always take the initiative in division. Figure 63 represents the eighth cleavage complete and the beginning of the ninth. In figure 64 the entodermal cells do not completely fill the central cavity of the embryo. Next, the anterior entodermal cell E! divides very unequally and forms E!! and e!? (fig. 65). The smaller cell, after a few divisions, is difficult to follow. Immediately after the division of the anterior cell E', the cells E?! and E?? divide equally. The spindles of these divisions are shown in figure 65. The division is equal and takes place in an anteroposterior direction. The entoderm is now composed of five large cells and three smaller ones. Figure 66 represents an embryo with the five large entodermal cells viewed from the left side. Spindles are present in each of the cells for the following cleavage. Figure 67 represents an optical section a little earlier than the preceding, viewed from the right side. The ectodermal cells are somewhat contracted and do not fill the central cavity. The cells of the embryo at the dorsal posterior end multiply. very rapidly, extend backward over the blastopore, and, in a later stage, contribute to the for- mation of the temporary foot. At the next division each of the five entodermal cells divides dorsoventrally and forms two layers of five cells each, as shown in an optical section in figure 68, with the ectoderm removed. Figure 69 shows the same stage with the ectoderm intact. At the next division each of the five upper and the lower entodermal cells divide equally. The cleavage occurs in an anteroposterior direction and produces twenty large entodermal cells, as shown in figure 70, an upper view. From this point forward no attempt was made to follow the individual cleavage cells. Figure 71, a ten-hour embryo, shows about the same stage as the preceding from the right side. The position of the entoderm is indicated by a dotted outline. The beginning of the foot and the first stage in the formation of the stomodaeum is evident from the ventral side. Figure 72, a JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2 406 GEORGE W. TANNREUTHER little later stage than the preceding, from the right side, shows the position of the central entodermal mass and its relation to the embryo as a whole. The ectodermal cells at the anterior end of the embryo now divide very rapidly and later contribute in part to the formation of the stomodaeum and the pharynx (figs. 73 to 75). The central mesentodermal mass of cells becomes differentiated into two distinct regions (figs. 76 and 77), the entoderm proper, which produces the stomach, oesophagus, and digestive glands; and the part which produces the reproductive organs with a few of its controlling muscle fibers. FORMATION OF THE TROCHAL DISC The developing embryo is divided into three distinct regions; the body, the head bearing the trochal disc, and the foot. The foot is an embryonic structure and is absorbed before the birth of the individual. The trochal disc begins as ectodermal prominences or growths, due to a proliferation of cells on the ventro-anterior region. Figure 73, viewed from the ventral side, shows a small lateral fold on either side.