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TN: re ' . <- : aie an of gir, ie rtd ri wheats > Ae AE Vee og eT se wi a ' “4 Git a 1) ms ile eres Behe eh a ae Laie iar 19 —+> THE ANATOMICAL RECORD EDITORIAL BOARD IrvING HARDESTY WaRREN H. Lewis Tulane University Johns Hopkins University CLARENCE M. JACKSON Cuar.es F. W. McCuure University of Minnesota Princeton University Tuomas G. LEE WiuraM 8. M1iLuer University of Minnesota University of Wisconsin Freperic T. Lewis FLORENCE R. SABIN Harvard University Johns Hopkins University GEORGE L. STREETER University of Michigan G. Cart Huser, Managing Editor 1330 Hill Street, Ann Arbor, Michigan VOLUME 11 AUGUST, 1916-JANUARY, 1917 . a 4) \\ WS? - % PHILADELPHIA THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY COMPOSED AND PRINTED AT THE WAVERLY PRESS By tHe Witurams & Witktns Company Barrimore, Mp., U.S.A. CONTENTS No. 1. AUGUST ' Exior R. Ciark. A study of the reaction of mesenchyme cells in the tad-pole’s tail toward smiequed oll globules... Five: figures «jcc ec0couno rans eee eae ad ee 1 Louis H. Kornper. An anomalous urinogenital system in a dog. Two figures....... 19 W. Souter Bryant. Sensory elements in the human cerebral hypophysis. One figure. 25 Emity Ray Gregory. A method for micro-injection. One figure.................... 29 No. 2. SEPTEMBER Witiiam B. Kirxuam. The prolonged gestation period in suckling mice.............. 31 HELEN Dean Kina. On the postnatal growth of the body and of the central nervous Buntenr in albino rats that are undersized at birth... ... 2... scepter os ces ehenccun 4] E. L. Jupan. Mounting specimens under Petri dishes and clock glasses.............. 53 No. 3. OCTOBER P. E. Smirx. The effect of hypophysectomy in the early embryo upon the growth and Bomnement-or thenrog. Nenfigures:... 002%. <0. cosas > +. Seek e eek Bee ere oe 57 Carpon GILLASPIE, Lewis I. Minter AND Morris Baskin. Anomalies in lobation of Panett review of literature. Five fimures... 00.25.05 be os des staesapen dss sere 65 CarBON GILLASPIE, Lewis I. Minter anp Morris Baskin. Anomalous renal vessels andsuherm surcicalusioniticanee.s Nine figures. o- .. os... - loosen ies eae othe os OscarR RippLe. Size and length relations of the right and left testes of pigeons in DE LISIG PING) (WE GOSTOE Goat EIREN eS Ne ean arse Pare ge en eee Pee See Er eee nea 87 J. A. Lone. Hygienic cages.for rats and mice. Two figures..........:..0--:eceeeeec 103 No. 4. NOVEMBER ALWIN M. PAPpPENHEIMER. The Golgi apparatus. Personal observations and a review MERRCULeEaiines echwenty-twOmirUles:.¢ . sss, c siateeiiet ..5-: qaseenweeke das eaee + os ss se oe 191 G.S. Horxins. The innervation of the muscle retractor oculi. One figure............ 199 Espen Carey. The anatomy with especial consideration of the embryological signifi- cance of the structures of a full-term fetus amorphus. Nineteen figures........... 207 Ray Henry Kistiter. The thoracic duct in the rabbit. Six figures.................... 233 FRANKLIN P. ReaGan. Some results and possibilities of early embryonic castration. Six figures, (our plates) Pei ice is. ccecie ss oe RE Bee LO tole sedstest 2. ee 251 Heten Dean Kina. The relation of age to fertility in the rat. Three figures........ 269 TEecHNIQUE Norss. I. The application of Benda’s neuroglia stain. II. Some uses of Mallory’s connective tissue stain. By H. M. Kinegsry. III. The use of the Van Wijhe method for the staining of the cartilaginous skeleton. By Gustave J. NoBAcK. IV. A convenient method of orientation in paraffin imbedding when paper trays or boxessare used> -By B.-B. KINGSBURY 204. (46406 eee ee sore 289 ke Oxasia., On’ the elective staming of the erythrocytes. ---- em: -ceeeee a} eee 295 J.B. Jounston. Neutral red as a cell stain for the central nervous system............ 297 No. 6. JANUARY H. H. Donatpson. Biological Problems and the American Association of Anatomists. Address of the President at the Annual Meeting... .....:5.........0.0+8 +08 299 Proceedings of the American Association of Anatomists, Thirty-third session........ 311 Proceedings of the American Association of Anatomists. Abstracts............... 317 Proceedings of the American Association of Anatomists. Demonstrations........... 439 American Association of Anatomists, Constitution... ....<> Jel... aan see 446 American Association of Anatomists, List of officers and members................. 449 Proceedings of the American Society of Zoologists, Fourteenth Annual Meeting...... 467 Proceedings of the American Society of Zoologists, Abstracts ..............-.-04. 473 American Society of Zoologists, ‘Constitwtom 22... eae ee ace se = =e ein iy - e eiete foe 543 Atnerican ‘Society of Zoologists, By-laws: 2 2 apes eee ese ole) s oes ee 545 American Society of Zoologists, Historical Reviews an 2. )- so oles cesta > « - ee 546 American Society of Zoologists, List of officers and members..................-.+. 570 A STUDY OF THE REACTION OF MESENCHYME CELLS IN THE TAD-POLE’S TAIL TOWARD INJECTED OIL GLOBULES! ELIOT R. CLARK From the Anatomical Laboratory of the University of Missouri FIVE FIGURES In their later studies on the mode of development of the lymphatic system, Huntington and McClure have reiterated the view that the lymphatics are formed by the transformation of mesenchyme cells in the following manner. They hold that fluid accumulates in the tissue spaces, forming small lakelets; that the mesenchyme cells are pressed upon by the fluid col- lected; and that, as a result of this mechanical pressure, the mesenchyme cells react by flattening out and by forming mem- branes around the blisters. The main evidence presented in favor of this view consists in the finding, in microscopic sections, of clear spaces in the tissues, unsurrounded by any membrane, of other spaces which have the appearance of being partly surrounded, and of others with a complete covering. Some of these appear to be com- pletely isolated while others are connected with one another. The numerous possibilities of error in the interpretation of the appearances described have been pointed out by Miss Sabin (1), E. L. Clark (2), and myself (3), and will not be reviewed at this time. The importance laid by these investigators on the part played by the mechanical action of fluid on mesenchyme cells may be 1A preliminary report of these studies was published in the Proceedings of the American Ass. of Anat., Anat. Rec., vol. 10, no. 3, 1916, p. 191. 1 THE ANATOMICAL RECORD, VOL. ll, No. 1 AuGust, 1916 2 ELIOT R. CLARK seen from the following quotation. Huntington (4) says (page 289): If two embryonal mesenchymal cells are separated from each other by the accumulation of fluidin the resulting intercellular space, then the opposing aspects of the two cells involved will be subjected to the mechanical and hydrostatic influences of accumulated intercellular fluid, which will react upon the surfaces of the cell still held in syncytial relation to the surrounding mesenchyme. The cells whose opposing surfaces have become freed by the development of an intercellular space, and are subjected to fluid pressure, will react as a whole, be- come flattened, and be transformed into endothelial cells, forming the parietal limit of an originally intercellular mesenchymal space, which is the font and origin of all vertebrate vascular development. This should be supplemented by a quotation from McClure (5), for no explanation is given here for the accumulation of fluid in the tissues. As soon, however, as the haemal vessels begin to function, lymph begins to collect in the intercellular spaces of the embryo and, as we know, is subsequently collected by a set of newly formed vessels, the lymphatics, which convey it to the venous circulation. Those who maintain that the lymphatics sprout centrifugally and continuously from the veins, would necessarily hold that the lymph in the intercellular spaces patiently awaits the arrival of close and hol- low outgrowths from the veins, the lymphatics, before it can be re- ceived into any portion of the lymphatic system. The combined picture, then, is as follows: as soon as the blood-vessels function, ‘‘Iymph begins to collect in the inter- cellular spaces.”’ It collects apparently because it cannot get back into the blood-vessels. The mesenchyme cells are sub- jected to “mechanical and hydrostatic pressure” exerted by the accumulated lymph, and respond by flattening out, and becoming endothelial cells. This comprises the “font and origin of all vertebrate vascular development.” Some of the theoretical objections to this series of assump- tions may be pointed out. First, McClure states that ‘‘as soon as the haemal vessels begin to function, lymph begins to collect in the intercellular spaces of the embryo;” to the pressure exerted by this accu- mulated lymph is assigned the réle of acting as the formative MESENCHYME CELLS IN TAD-POLE’S TAIL 3 stimulus for the transformation of mesenchyme cells into lym- phaties. In another place, McClure argues that blood-vessels and lymphaties differentiate in the same manner. Now if the col- lection of intercellular lymph begins only after the blood-vessels begin to function, how are we to explain the collections of fluid which are supposed to have served as a formative stimulus for the differentiation of blood-vessels? Again, if both blood- vessels and lymphatics are determined solely by the action of mechanical pressure, we are left with the same puzzling problem which confronted Goette (6) in 1873, namely, why the two sets of vessels do not everywhere form communications with one another. The puzzle differs only in that Goette conceived of the circulation from blood to lymph capillary as being in- tracellular, and tried to explain why the same mesenchyme cell was not sometimes demanded by both the blood-vessel and the lymphatic, while Huntington and McClure conceive of the fluid as being extra-cellular and must explain why it does not happen that the same lakelet does not connect with a blood capillary on the one side, and with a lymphatic capillary on the other. Goette’s puzzle has been eliminated by the proof that in the tail of amphibian larvae, mesenchyme cells take no part in the growth of blood or lymphatic vessels. Again, it is hardly necessary that ‘‘those who maintain that the lymphatic sprout centrifugally and continuously from the veins, would necessarily hold that the lymph in the intercellular spaces patiently awaits the arrival of closed and hollow out- growths from the veins, the lymphatics,’ since it was demon- strated clearly by Magendie, over a hundred years ago, and has been proven so many times since that it cannot be questioned, that absorption of substances may take place through the blood- vessels as well as through the lymphatics. Another assumption which is quite unwarranted on the basis of facts is that with an increase in intercellular fluid, this fluid will collect in definite lakelets, in the intercellular spaces. All our knowledge of intercellular spaces indicates that they form an irregular, intercommunicating net-work of spaces filled with fluid. It would be expected that, if this fluid were 4 ELIOT R. CLARK increased, the increase in the intercellular spaces would be a general one, the distention, or edema, being regulated in its intensity only by the relative amount: of resistance offered by the tissue. This resistance would, of course, be greater in dense tissues, in which the cells are bound firmly to one another, and less in tissues where cells are less firmly attached to one an- other. In any region, however, where the tissue is uniform the pressure and separation of cells must be uniform, and no accumulation of tissue fluid in the form of lakelets would seem to be possible. Microscopic examination of embryos bears out this theoretical consideration; there are regions in which the intercellular fluid is large in amount, proportionately, and others where it is small. Where large, the mesenchyme or other cells are uniformly separated from one another, as for example in the umbilical cord, and the ventral body wall. It is unjustifiable from our knowledge, or better, lack of knowledge, to speak positively about the stimuli which are re- sponsible for the primary differentiation of organs or tissues, since with, perhaps, the exception of the formation of the lens as the result of the contact of the optic cup with the epidermis (7), there is hardly an instance in embryology of the satisfactory demonstration of the stimulus responsible for the primary differentiation of any organ or tissue (8). In this connection, results obtained in recent studies made on tissues, grown by the ‘tissue culture’ method of Harrison, are interesting. Har- rison (9) found that primitive nerve cells send out processes into a medium consisting of coagulated lymph. The Lewises (10) obtained similar results with sympathetic nerve cells, in a medium of Locke’s solution. Shipley (11) has found that undifferentiated heart muscle cells differentiate and start rhyth- mic contractions, in a medium of coagulated plasma. In all of these cases the cells were removed from their normal environ- ment. Their continued development makes one sceptical of hypotheses as to the nature of formative stimuli, when such hypotheses are not supported by fact. The only experimental evidence which has been proposed in support of the hypothesis that collections of lymph furnish MESENCHYME CELLS IN TAD-POLE’S TAIL 5 the stimulus to the formation of lymphatics, consists of the re- sults of studies on ‘experimental mesothelium,’ by W. C. Clark (12). This investigator found that if solid blocks of celloidin are placed in the subcutaneous tissue of dogs, they became sur- rounded by flattened cells, which show, when treated with silver salts, black intercellular lines typical of flattened meso- thelial or epithelial tissues. Solid globules of hard paraffin, injected into the cornea of rabbits, were surrounded by a layer of flattened cells. Flattened cells were also found to line ‘dead spaces’ in the tissue and artificial channels, such as may be in- duced by the ligation of the cystic duct, with formation of a mucous fistula. Interesting as are these studies, in spite of the absence of evidence as to the source of origin of the cells which formed the flattened lining membrane, there appears to be no justifica- tion for the conclusion that we have thereby gained any informa- tion as to the mode of differentiation or growth of blood or lym- phatic vessels. And yet, W. C. Clark concludes (page 316) that “Therefore the second hypothesis, premised in this article, is tenable, namely that the flat cells of serous surfaces and those lining blood vessels may regenerate from deep connective tissue cells, and do not necessarily arise from adjacent intact meso- thelial or endothelial cells.”’ Again McClure (13) refers to the results of W. C. Clark as bearing out ‘“‘in a most decisive manner” the view that “the gradual increase in the amount of lymph received by the subocular sacs (in the trout) during the stage of their independence, results in the application of a con- stant and continuous pressure to the mesenchyme cells forming their walls, which in itself must be a positive factor in causing these cells to flatten out and gradually assume an endothelial form.” It is difficult to conceive how the results obtained by W. C. Clark can have any bearing on the problem which confronts McClure, unless the assumption is made that all mesothelial, endothelial, and epithelial membranes which line spaces or ducts have the same properties—an assumption for which facts furnish no justification. Surely blood-vessel endothelrum has 6 ELIOT R. CLARK properties which differ from synovial membranes, peritoneal membranes, or the lining membrane of the urethra, the bile ducts, or the gall-bladder. There is, perhaps, a suggestion of support for the hypothesis in question in some of the results obtained in tissue cultures. Several observers—Harrison (14), M. R. and W. H. Lewis (15), Lambert (16), W. C. Clark (17) and others—have found that in bits of tissues, explanted to plasma or Locke’s solution, mem- branes may be formed around solid bodies—such as alone the cover-slip and around solid threads, as the threads of spider web, used by Harrison, and around droplets of fluid—such as may be formed, occasionally, in plasma preparations, by the retraction of the fibrous threads. The explanation of the for- mation of membranes around droplets of fluid is associated with the apparent inability of cells to grow into a purely fluid medium, without mechanical support, or as expressed by Harrison, their dependence on ‘stereotropism.’ The formation of such a mem- brane in tissue cultures is not to be interpreted as a reaction by flattening out, on the part of the culture cells, but rather, in all probability, as due to the fact that cells grow around the periph- ery of such a droplet. Moreover, it has not yet been possible to determine the origin of the cells which form membranes in tissue cultures. At present there appears to be no ground for claiming that the formation of membranes in this manner, fur- nishes us any information as to the mode of differentiation of blood-vessel or lymphatic endothelium. In order to put to the test the hypothesis that the differentia- tion of blood or lymph vessel endothelium may be stimulated by the mechanical pressure exerted on mesenchyme cells by accumulations of fluid, and to plan the test in such a way as to make it approach as nearly as possible the actual conditions supposed to exist, the present studies. were started. The aim was to inject an inert fluid, in globules of size sufficient to press against the mesenchyme cells, into a region of embryonic tissue, in which the reaction of the cells could be watched in the living animal; to see whether a membrane were formed, and, if so, by what type of cell, and what would be the properties of such a a | MESENCHYME CELLS IN TAD-POLE’S TAIL membrane, especially its reaction toward blood-vessel and lymphatic endothelium. In order to simulate as nearly as possible the fluid whose presence is thought to excite the transformation of mesenchyme cells into lymphatics, and at the same time to have a fluid which would be inert, which would not be absorbed, which would merely exert a mechanical pressure, paraffin oil was selected for injection. The object chosen was the transparent fin expansion of the tail of young frog and toad tad-poles where it is possible to see the individual mesenchyme cells, as well as blood-vessels, lymphatics and leucocytes, and to watch their reactions in the living larvae, from day to day. The tad-poles were anaesthe- tized with chloretone (1: 4000 to 1:5000) and small globules of oil injected into both fins, through fine glass cannulae, under the binocular microscope. The oil was sterilized by heating. In some cases the tadpole was washed in several changes of sterile water, but the results did not differ materially from those obtained when the only antiseptic precaution consisted in steriliz- ing the oil» The observations were made by a method previously described in detail (18)—the larva, anesthetized, was placed in @ micro-aquarium, in chloretone of the proper strength, and the tube of the microscope tilted to the horizontal, to enable the tadpole to retain its normal upright position. The oil globules were of varying sizes, from 20 to 100 micra in diameter. The larvae used were those of Rana catesbiana (bull-frog) and of Fowler’s toad (Bufo lentiginosus Fowleri). The latter have beautifully clear tails, with very few pigment cells at the stages used, while the mesenchyme cells are far apart and stand out most clearly. Since the oil could not be injected without a certain amount of injury, there were always some temporary effects of the in- jection, not attributable to the presence of the oil. These consisted principally of a more or less intense leucocytosis, probably caused by greater or milder degrees of infection. In some cases large numbers of leucocytes gathered about the glob- ules, many of the leucocytes containing pigment. Several! of the globules, around which the leucocytosis was most intense ELIOT R. CLARK MESENCHYME CELLS IN TAD-POLB’S TAIL 7) were extruded. In case this did not occur, the leucocytosis gradually subsided until in most cases three or four days after the injection, the region surrounding the globule contained no more leucocytes than the other parts of the tail. In some in- stances, a slightly increased number of leucocytes near the globule continued for several days longer. From now on the globules remained apparently inert, so far as could be judged from the behavior of leucocytes in their vicinity. The longest time over which a globule was watched was 12 days. In appearance the oil globules, when present in the tail, form spheres with the central portion clear and transparent and a dark per:phery, with a sharp outline. Structures over the cen- tral portion can be seen most clearly. Thus it is possible, in case the diameter of the globule is sufficient to distend the skin slightly, to see the nuclei of the cells of the epidermis, and the details of other structures most distinctly. Many of the globules were oval in shape, immediately after injection. Later, after a day or two, they usually rounded up to a spherical shape though sometimes remaining slightly oval. The behavior of the mesenchyme cells will now be described. In order to follow them with accuracy camera lucida records were made of all mesenchyme cells in the neighborhood of the globules, and their changes from day to day were noted. When a mesenchyme cell happened to be in the outer dark zone of the globule, it was difficult to make out its outlines. Occasionally only one or two of its processes could be clearly seen. The following of such cells, however, was made possible by the fact Fig. 1 From larva of Fowler’s toad, 9 mm. long. Four globules of paraf- fin oil injected into fin expansion of tail, on Aug. 19. Much leucocytosis about three of them, and all three extruded within forty-eight hours. There was very little leucocytosis about the fourth globule, of which three drawings are shown, two, four, and six days after injection. This globule was in the ventral fin. The mesenchyme cells in the immediate vicinity of the globule are shown. The letters, a, b, ete., indicate the same cells. * leucocytes against the globule; pig.L., pigmented leucocyte against the globule. The mesenchyme cells were in three different planes: those nearest the observer are represented in solid black, those furthest away are dotted, while those in the midst are cross-hatched. Enlarged 267 times. Drawn with camera lucida. 10 ELIOT R. CLARK that the globules from time to time shifted their position slightly, so that a cell, at one time not clearly seen, later could be clearly outlined. This applied to only a very few cells, particularly in the toad larvae, because of the relative rarity of their mesen- chyme cells. In view of the descriptions in the literature of the reaction of connective tissue cells to the pressure exerted by foreign substances, the behavior of the mesenchyme cells was a great surprise. It was expected that the cells near the globule would flatten out on its surface and form a membrane. On the contrary, the mesenchyme cells apparently paid no particular attention to the globules. They maintained their identity as ‘star-shaped’ cells, with thickened central portion and branched processes, and their property of slow progression, described in an earlier paper. That the mesenchyme cells are not influenced by the pressure exerted by the globules was brought out quite strikingly in one instance in which the globule shifted its position in such a way as to come to lie against a mesenchyme cell which had been at a slight distance from the globule. For a day or two it was rather difficult to make out the outlines of the cell. The globule then shifted its position in the opposite direction, and the mesenchyme cell could now be seen clearly, apparently unchanged, at a slight distance from the globule. Occasionally there are to be seen, over the clear part of the globule, if the globule is of sufficient size to distend the skin shghtly, one or two flattened cells which, at first glance, might be interpreted as cells flattened out by the pressure of the globule. Such cells were seen over only a few of the globules, and their explanation was obvious on studying other parts of the tail, at a distance from the globules. Such flattened out mesenchyme cells appear more or ess evenly distributed, lying just below the epidermis, and are not particularly associated with the oil globules. That they are associated with the skin and not with the oil globules, is shown by the fact that none are present over the majority of the globules, and also by the fact that, if the globules shift, these cells maintain the same posi- tion with reference to the skin, while they are left behind by the oil globules (fig. 4). There are no other appearances on MESENCHYME CELLS IN TAD-POLE’S TAIL Lt Fig. 2. From larva of Rana catesbiana, 10.6 mm. long. Two globules of paraffin oil injected into the ventral, and a small globule into the dorsal fin; all three remained. Much leucocytosis around each. The first drawing (Aug. 3) was made immediately after the injection. Small mass of cellular débris is shown, in the path of the injection. Mesenchyme nearest the globule lettered as in fig. 1. In drawings Aug. 4, 5, and 7, some of the leucocytes about the globule are shown. In drawing of Aug. 5 are shown the successive positions taken by a leucocyte as it moved to the globule, moved along the surface of the globule a short distance, and then moved away. The shifting of the globule is clearly seen, by comparing its relation to the blood vessel. The cells d and e, which lie close to the globule Aug. 3 and 4, are left behind, while the cells ) and c are approached by the shifting of the globule. Enlarged 267 times. Drawn with camera lucida. 12 ELIOT R. CLARK the part of the mesenchyme cells which even remotely suggested a flattening out. The ‘act that some of the globules watched shifted their position would also indicate that no surrounding membrane had been formed, for a membrane would prevent such movement. The behavior of wandering cells towards the globules was watched with interest. As already stated there was a leucocy- tosis of greater or less intensity following the introduction of the globules, for the first two or three days. Leucocytes, most of them small and clear, others larger, and containing pigment, collected around the globules. Many of them flattened them- selves out on the surface of the globule, or formed irregular humps on the profile. Occasionally such a flattened leucocyte formed a thin, circular structure, with nucleus visible over the clearest, central portion of the globule. Such a cell, coupled with the irregular humps on the profile, if seen only at one stage, and not followed, might well give the impression of membrane formation by wandering cells. When, however, such cells were followed, it was seen that they gradually moved away (fig. 2). The humps on the profile changed shape, with each drawing, even when the records were made several times daily, while the flattened cells on the clearer part moved away. After four or five days most of the globules were quite free from the presence of leucocytes. In order to be sure of the behavior of the wandering cells, some were watched intensively. They were seen to move, w th the typical amoeboid type of progression, up to the oil globule, to flatten themselves out on its surface and again move away. The impression was gained, that, had sections been made at a time when the leucocytes were flattened on the globule, they might have been interpreted as forming a membrane, an interpretation which the study of the living shows would be quite unjustifiable. Chromatophores which are present in large numbers in the dorsal fins of bull-frog larvae and to a somewhat less extent in the ventral fins of the same larvae, sometimes wrapped around the globules with their Jong branched processes, when the glob- MESENCHYME CELLS IN TAD-POLE’S TAIL 13 ules were injected near them, but did not form a definite membrane. Since the mesenchyme cells failed to form a membrane around the globules, the second part of the inquiry, namely, the reaction of such a membrane, if formed, toward the lymphatics or blood- vessels in their vicinity, could not be followed. It was of in- terest, however, to observe the reaction of blood-vessel and lymphatie endothelium to the oil globules. In one case a par- ticularly favorable opportunity was afforded to study the re- action of the blood-capillary, since the globule pressed against a blood-capillary, forcing it to make a bend in its course (fig. 4). Fig. 3 From same Rana ecatesbiana larva from which figure 2 was taken. To show relation of pigment cells to globule. This small oil globule was in- jected into the dorsal fin, on Aug. 3; the drawing was made Aug. 5. Enlarged 267 times. Camera lucida. The capillary then appeared to wrap around a part of the glob- ule. In this position the capillary showed no tendency to give off cells which might grow around the globule, but instead remained as a distinct vessel. The circulation of blood cells through it, which was at first interrupted, was later resumed. Blood-capillaries and lymphatics near the oil globules showed no tendency to grow toward it, or to send out sprouts to it. The results of this study, then, indicate that, aside from the temporary inflammatory reaction, due probably to the injury and to the bacteria introduced at the time of injection, the pres- ence in the fin of the tad-pole’s tail of injected globules of paraffin oil, of sufficient size to cause a distension of the tissues, fails 14 ELIOT R. CLARK to stimulate the formation of membranes about the globules, on the part of mesenchyme cells, wandering cells, or of blood- vessel or lymphatic endothelium. These results are in disagreement with those of W. C. Clark (19), already mentioned. They are, however, in agreement with the older findings of E. Juckuff (20) who found that soft paraffin, injected subcutaneously, travelled long distances from the injection site, showing that no membrane was formed about Aug. 17 Aug. 20 Fig. 4 Globule of paraffin oil injected into ventral fin of tail of Rana cates- biana larva on Aug. 13. The globule rested against a blood-capillary, fore- ing it to bend slightly. The two sketches shown, made four and seven days respectively after the injection, show the relation of the globule to the blood capillary, to a nearby lymphatic, (lym) and to two mesenchyme cells which happened to be under the epidermis immediately over the globule. Note that the globule has shifted to the right, so that the two mesenchyme cells, which on Aug. 17 are over the right part of the globule, are over the left central portion Aug. 20. Other mesenchyme cells not shown. ad Enlarged 267 times: Drawn with camera lucida. Fig. 5 Same larva as figure 4. Small globule injected in dorsal fin on Aug. 13. Drawing made nine days later—on Aug. 22, d.c., pigment cell. Enlarged 267 times. Drawn with camera lucida. MESENCHYME CELLS IN TAD-POLE’S TAIL 15 it—a finding verified by MacCallum (21). It will be remem- bered that the attempt was made, in selecting the substance to be injected, to find something which would simulate the sup- posed lakelets of tissue fluid whose presence are held by Hunting- ton and McClure to stimulate, merely by the mechanical pres- sure which they are supposed to exert, the mesenchyme cells to form membranes. It is obvious that in point of size and consistency the small globules of oil much more nearly repro- duce the supposed conditions than the relatively enormous pus pockets, or the relatively huge solid blocks of celloidin or the globules of hard paraffin. It is also obvious that in a trans- parent object, like the tad-pole’s tail, where the individual cells may be seen with great clearness and the reaction process watched in the living animal, the conditions for observing what happens are much more favorable than in the case of the other experiments referred to. Since, then, the action of pressure alone fails to stimulate the formation of membranes on the part of mesenchyme cells, an important link in the argument used in favor of the origin of lymphatics from mesenchyme cells, as presented by Hunting- ton and McClure, drops out. A certain feeling of disappointment must be confessed, that the mesenchyme cells failed to respond to the presence of the oil globules by the formation around them of membranes, as it was expected they would, because of the desire to see what would be the reaction of such membranes toward blood-vessels and lymphatics. It is true that cells derived from the middle layer or meso- derm differentiate at various stages into pavement epithelium, or endothelium, other than that which lines the blood and lymph vascular systems. Among such may be mentioned the lining of the large cavities pleural, peritoneal, and pericardial, the lining of bursae and synovial membranes, and the outer layers of tendons and fasciae. It should also be remembered that the same middle embryonic layer differentiates into smooth and striated muscle, into cartilage and bone, into blood cells, and other types of tissues. Each of these tissues has certain 16 ELIOT R. CLARK modes of reaction, a specific life history, the property of respond- ing each in its own individual way, to various stimuli. To trans- fer the modes of reaction of one set of tissues derived from the mesoderm to another set is quite unjustifiable. To be more specific, it is not justifiable to claim that, if connective tissues, in adult animals, are capable of forming membranes about solid foreign bodies, or large accumulations of fluid, or if membranes form around liquid vesicles in the midst of coagulated lymph in tissue culture preparations, then lymphatics arise as the result of the pressure exerted by accumulated lakelets of lymph. It is even unjustifiable to transfer to lymphatics the properties of structures which resemble them morphologically so nearly as blood-vessels, for, while there are many points of similarity between the modes of reaction of the two, there are also strik- ing differences. It is conceivable that future studies may reveal the various stimuli which are responsible for the primary differentiation of tissues and organs. For the lymphatic endothelium it would seem a more hopeful field to investigate the chemical nature of the intercellular fluid, to see whether any evidence can be gained as to the collection there of especial chemical substances which stimulate its differentiation. To propose such an hypoth- esis at the present time, however, would be pure speculation. Much confusion has arisen because quite different structures have been grouped together under the name of endothelium or mesothelium. It would seem that the time is ripe to sepa- rate these different forms of flattened lining cells under differ- ent names. If, for example, we could speak of blood-vessel endothelium as Haem-angiothelium, and of lymphatic endothelium as Lymph-angiothelium, or some equally specific names, and if distinctive names could be selected for the other forms of pave- ment epithelium, much of the confusion would disappear. In conclusion, it is a pleasure to express my gratitude to the Marine Biological Laboratory at Wood’s Hole, where these studies were made, for generously granted laboratory facilities, MESENCHYME CELLS IN TAD-POLE’S TAIL ty LITERATURE CITED (1) Sasryn, F. R. 19138) Johns Hopkins Hosp. Reports. Monographs. New Series, no. 5. (2) CuarK, E. L. 1912 Anat. Rec., vol. 6. (3) Cuark, BE. R. 1911 Anat. Rec., vol. 5. (4) Huntinaton, G. 8. 1914 Am. Jour. Anat., vol. 16. (5) McCuure, C. F. W. 1915 Anat. Rec., vol. 9, no. 4, p. 281. (6) Gorrre 1875 Die Entwickelungsgeschichte der Unke. Leipzig. (7) Lewis, W. H. 1907 Am. Jour. Anat., vol 6, p. 473. 1913 Stockard Am. Jour. Anat., vol. 15, p. 253. 1910 vol. 10, pp. 393-423. (8) Herssr 1901 Formative Reize in der Thierischen Ontogenese, Leipzig. (9) Harrison, A. G. 1910 Jour. Exp. Zool., vol 9. (10) Lewis anpD Lewis 1912 Anat. Rec., vol. 6, p. 7. (11) Surptey, P. G. 1916 Anat. Rec., vol. 10, p. 347. (12) Cuark, W. C. 1914 Anat. Rec., vol. 8. 1916 Anat. Ree. vol. 10. (13) McCuurz, C. F. W. 1915 Mem. of Wistar Inst., no. 4, p. 29. (14) Harrison, R. G. 1910 Jour. Exp. Zool., vol. 9. 1911 Science, vol. 34. 1914 Jour. Exp. Zool., vol. 17. (15) Lewis, M. R. anp W. H. 1911 Anat. Rec., vol. 5. (16) Lampert, R. A. 1912 Anat. Rec., vol. 6. (i)iCrarE, W. ©: 1916 loc: cit:, p. sis: (18) CuarxK, E. R. 1912 Am. Jour. Anat., vol. 13. (19) CrarK, W. C. 1916 loc. cit. (20) Juckurr, E. 1893 Archiv. f. Pathol. u. Physiol., Bd. 32, p. 124. (21) Mac Cattum, W. G. 1903 Johns Hopkins Hospital Bulletin vol. 14, pp. 5 and 6. THE ANATOMICAL RECORD, VOL. 11, NO. 1 AN ANOMALOUS URINOGENITAL SYSTEM IN A DOG LOUIS H. KORNDER From the Anatomical Laboratory of the Northwestern University Medical School TWO FIGURES Anomalies of the urinogenital system are frequent and have ceased to attract much attention. Few, however, present features of such embryological interest as the following case. Because of this and on account of its value, as illustrating the physiological adaptability of one system to the needs of an- other, this case seems worthy of mention. My acknowledg- ment is due Dr. L. B. Arey for valuable suggestions regarding the embryological considerations. On opening the abdomen of a dog it is commonly observed that the bladder is large and lies almost entirely in the abdomi- nal cavity. In a medium sized mature female, selected at ran- dom for the purpose of obtaining certain tissues used in a re- search problem, the bladder lay deep in the pelvie cavity and was rather small, the size and shape being that of a walnut. On palpation it felt extremely firm, much as though it were a solid mass of tissue. A longitudinal incision through its wall revealed but a very small lumen, less than 1 em. in diameter and 2 em. in length. Two broad ligaments passed from this bladder over the rec- tum and gained attachment to the front of the sacrum. One ligament was considerably longer than the other, due to the bladder lying ventral and to the left of the uterus instead of directly ventral as is normally the case. Except for these two ligaments and a slightly shortened urethra which merged into the left wall of the urinogenital sinus, other connections with the bladder could not be established. 1Contribution No. 40, May 15, 1916. 19 20 LOUIS H. KORNDER These findings led to an examination of the kidneys, which were found to be normal in shape, size and position. Each possessed one short ureter, the right being 6.5 em. and the left 7.2 em. in length. Originating in the pelvis of the kidneys the ureters coursed downward over the psoas muscles and passed one on each side into the horns of the bicornuate uterus. This union occurred about a centimeter below the place where the short Fallopian tubes merge into the uterine horns. The uterine horns and the uterus were not soft and pliable as is usual but were hard and rigid and on making a longitudinal incision through their walls, were found to be filled with débris, composed mainly of desquamated epithelial cells. The structures in this region were surrounded and some deeply imbedded in a mass of fibrous and adipose tissue. This appeared to form a common capsule which covered the union of the ureters with the uterine horns and extended over the Fallopian tubes including the ovaries, becoming at this con- nection part of the ovarian bursa. The ovarian bursa exists normally in the dog asa separate fold of peritoneum covering each ovary. This is usually covered by adipose tissue but opens through a small slit-hike opening into the abdominal cavity. It has been mentioned that the urethra was slightly shorter than normal and passed from the left into the wall ofthe urino- genital sinus. This relation of the urethra to the urinogenital sinus and the original location of the bladder explains why in the accompanying figure (fig. 1) the bladder is shown as lying between the uterus and rectum, instead of ventral to the uterus as is normal. Histological preparations of the bladder show a slightly changed epithelial lining, consisting in two to three layers of low cuboidal! epithelium. The uterine surface epithelium instead of being high columnar in type is pseudo-stratified. The deeper glandular epithelium, however, is the same as in a normal dog’s uterus. The ovaries which were deeply imbedded in their ovarian bursae on sectioning showed nothing atypical, with the exception of ANOMALOUS URINOGENITAL SYSTEM IN A DOG 21 ureter ovarian bursa Ky enlrance- of ureler uterine horn- opened ulerus___ lateral ligament- S of the bladder ___# bladder _/ cervix,.____i¥' | ureth PQ__ ————— Fig. 1 Ventral view. Ureters shown as they enter both horns of uterus. Ovarian bursae and upper end of uterine horns opened. Bladder small and abnormal in location. 22 LOUIS H. KORNDER a shghtly more fibrous stroma than is usual. Several large Graffhan follicles present indicated a normal functional activity of the ovaries. Histologically, then, practically nothing unusual exists, the anomaly being one of gross anatomy, this con- sisting in a union of the urinary with the reproductive tract, the fusion of ureters and uterine horns leaving the bladder as a cul de sac which leads through the urethra into the urinogenital. sinus. EMBRYOLOGICAL CONSIDERATIONS The structures involved here are embryological derivatives of the mesonephric ducts, metanephros, Muellerian ducts and cloaca. That the cloaca developed normally is indicated by the presence of a rectum, bladder, urethra and urinogenital sinus. The presence of the uterus, tubes and vagina likewise indicate the normal development of the Muellerian ducts. The anomaly then must be due to a defective embryological growth of the mesonephric ducts and their derivatives, with a deficiency in the development of the nephrogenic cord as a pos- sible causal stimulus. In pig embryos of approximately 5mm. length the mesoneph- ric ducts give rise to the ureteric anlage of the metanephros where the ducts bend to join the cloaca. But that the ureteric anlages do not always originate at this bend is indicated by the frequency of double or triple ureters. In these instances the first ureteric bend develops usually into the ureter most nor- mal, while the rest show evidence of slowed or mal-development. From these cases it may be assumed that the ureteric anlage need not necessarily arise at a definite location but can occur at any point along the ducts. Figure 2, a very diagrammatic sketch, shows both the Muel- lerian and Wolffian ducts leading into the urimogenital sinus. The approximate position where normally a single metanephric anlage arises from the Wolffian duct is indicated by (A). How- ever, IN Man 2s many as six such anlages have been observed. While in these instances the anlage corresponding to (A) de- velops into the adult ureter the possibility exists that a more ANOMALOUS URINOGENITAL SYSTEM IN A DOG 23 cranial anlage, for instance (6), may become the functional ureter. Reference to figure 2 will show the lower part of the Wolffian duet not cross-hatched. This portion which extends from the normal ureteric anlage on downwards is during further develop- ment drawn into the urinogenital sinus. Through this fusion the ureters receive their normal connection with the definitive bladder which develops partly out of this portion of the sinus. UGS Fig. 2 Diagrammatic sketch. M, Muellerian duct; W, Wolffian duct; A, location of normal metanephric anlage; 2, possible upwardly displaced meta- nephric anlage; D, extended ureter merging into the Muellerian duct at U; N, portion of Wolffian duct taken into wall of urogenital sinus; UgS., cross-hatched portion of Wolffian duct degenerates. If in this particular case, however, the ureteric anlage did not develop low enough to be included in that lower portion of the mesonephric duct then just as soon as the normal degeneration of the upper part of the Wolffian duct occurred, the upwardly displaced anlage (B) which developed into the ureter was without connection with the urinogenital sinus. Being thus isolated it seems probable that the ureter (D) extended to the nearby Muellerian duct and merged into it at (U). This established an outlet into the urinogenital sinus. The above is offered as one possibility to which the present anomaly may be due. It is entirely hypothetical as any con- 24 LOUIS H. KORNDER sideration of this case must be. Because of this a further ex- planation may possibly be found in the following. In addi- tion to those known embryological facts mentioned above it should be recalled that in embryos of & to 11 mm. length the Muellerian ducts develop caudalward beneath the epithelium of the mesonephric fold. Reference to dissections of the pig embryo show the Muellerian ducts lying very close to the Wolf- fian ducts so that the occurrence of a more or less complete longitudinal fusion of these two ducts seems not impossible. The establishment of this anomalous union of ureters and uterine horns presumably occurred early in the development of the animal. Since in the female, during the normal develop- ment the mesonephric ducts degenerate and disappear almost entirely, it may be assumed that in this case these ducts gradu- ally fused with the Muellerian ducts. It may be that in this way an early connection occurred on either side between the Muellerian duct and an upwardly displaced ureter. The question may be raised why a metanephros thus dis- placed should have abandoned the mesonephric duct and appro- priated a new outlet by way of the Muellerian ducts? It may be that the functional need of maintaining the patency of the meso- nephric duct was ineffectual compared with the tendency to- ward atrophy and consequent occlusion which the cranial portion normally shows. This query becomes all the more pertinent in view of the recent report by Bremer (Jour. Anat., vol. 19, 716) that in the cat the mesonephros maintains its activity until the permanent kidney assumes the excretory function. This being true of the cat it is more than probable that it also exists in the dog since both belong to the Carnivora. SENSORY ELEMENTS IN THE HUMAN CEREBRAL EPYPOPHYSISs W. SOHIER BRYANT ONE FIGURE In the past few years, the greater part of the work relating to the cerebral hypophysis has been of a therapeutic, a clinical or a surgically experimental nature, and the interest aroused in these aspects of the pituitary has tended to obscure the fact that certain histological elements in the structure of the organ still remain a mystery. The following report re-introduces the subject of the sensory elements of the hypophysial cavity, of which I have made a careful examination in human speci- mens: These sensory elements occur in maculae, which, in sagit- tal sections of the pituitary are seen situated on the posterior wall of the cavity, and sometimes, apparently, on the anterior wall. The maculae are composed of tall columnar ciliated sen- sory cells interspersed with bipolar cells, which have their nuclei towards the periphery; whereas in the ciliated cells, the nuclei are near the base which terminates in a caudal prolongation. Between these caudal processes of the ciliated cells, there is a layer of round cells, resting on a thin basement membrane. An area of ciliated cuboidal cells occurs at the margins of the maculae. I have found these sensory cells in all the freshly hardened human hypophyses that I have examined, except those in which the parenchyma had been almost completely replaced by connective-tissue; the sensory cells are moreover encountered even In pituitaries which have undergone very extensive pathologi- cal change. In their gross arrangement, the sensory elements of the hypophyseal cavity are suggestive of the sensory ele- ments of the maculae acousticae. Gentés (3), in his examination of the hypophysis of cats and dogs, found in the juxta-nervous layer, a stratified cylinder ~ 25 26 W. SOHIER BRYANT Fig. 1 Sensory epithelium of the hypophysial cavity of a human adult. Stained with hematoxylin-eosin. This material was procured through the kindness of Dr. William Mabon and the assistance of Dr. Clarence O. Cheney, Pathologist of the Manhattan State Hospital; the work was done in the New York Psychiatric Institute. Special thanks are due Dr. Charles Bates Dunlap for his technical assistance and super- vision of the work. SENSORY ELEMENTS IN CEREBRAL HYPOPHYSIS 21 epithelium resembling certain sensory epithelia. Cajal (1) has noted numerous special bi-polar cells in the epithelium adjacent to the nervous lobe in the hypophysis of the rat. Pi- rone (4), on the basis of his examinations in the cat and the dog, confirms the findings of Gentés: the cylindrical epithelium, in its superficial layers, presents the structure of the support- ing cells of sensory epithelia. Gemelli (2) finds that the pos- terior wall of the hypophysial cavity, in the guinea-pig, is com- posed of a layer of cylindrical cells, and other cells which he ealls supporting cells. The only reference in the literature to sensory elements in the human hypophysis is given by Tello (5), who mentioned that he found sensory epithelium in his human specimens, but omits to give any adequate description of it. LITERATURE CITED (1) Casau 1911 Hypophyse ou Glande Pituitaire. Histologie du Systeme Ner- veux de l’ Homme et des Vertébrés, II, p. 437. (2) Gemetitr 1908 I Processi della Secrezione dell ’ipofise nei Mammiferi. Arch. Scienz. Med., vol. 30, p. 521, no. 27. (3) Gentés 1903 Structure du Feuillet juxta-nerveux de la portion glandu- laire de l’hypophyse. Bull. et Mém. de la Soc. de Biol., vol. 55, p. 100. (4) Prrone, R. 1905 Sulla fina struttura e sui fenomeni di secrezione dell ’ipo- fise. Archiv. di Fisiologia, II, p. 60. (5) Tetto 1912 Algunal observaciones sobre la histologia de la hipofisis humana. Trabajos del Laboratorio de Investigaciones Biologicas, De La Uni- versidad de Madrid, Tomo X, p. 145. (Illustrated.) Dire Me | ; A METHOD FOR MICRO-INJECTION EMILY RAY GREGORY Zoological Laboratory, University of Pennsylvania ONE FIGURE After spending several months in an effort to secure satisfactory results and economy of material with methods used by other workers, the writer devised the following apparatus which is very easily arranged and does the work without waste except for accidents due to inexperi- ence or carelessness in handling. Direct pressure is secured from a good (50c.) De Vilbiss atomizer bulb. The bulb lies on the floor and is kept from rolling by a crocheted net cover. The pressure is transmitted by a red rubber tube 3; of an inch in diameter. After the transmitter reaches the table a short glass tube (h) is inserted in it and a cord (g) run from this to the upright of a small stand with a heavy base to keep these parts in position. Eleven inches further on a glass T (r) is inserted, to the stem of which a thistle tube is joined by a short piece of rubber tubing (p). Eighteen inches more of the transmitter end with a short glass injection tube (c) which has the outer end drawn into a capillary tip at right angles to itself. A sliding rod (i) passed through the pivoted top (b) of the upright of the stand makes a convenient support for the end of the transmitter. A piece of cork (a) on the pointed bent tip of the rod gives enough friction to keep the tube from slipping and still allows it to be readily moved in either direction or removed for use, with one hand. Il 26e'F 61 ree T 18° €68'T sAup sup 0S-0F ian 08 IHDIGM AGO NI GASVANONI AODVINGOUUd ATIVAG | 89°€ 1810S '9 68 L|S8 OT\S8'S |0Z"ET/S9 ST|ZZ 0Z|80' ST\OS ATIF9 LT/Se Tle ITF | ow OL'F |IS'F\16'% |L1'6|60'8 |S9°9 [SF OT/ES OT\TS 6TIOL GL\S9 ZTIES 1320 CT\es ILe | Ee. 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In the total of 69 records for the three series, 42, or nearly two-thirds, show that the daily percentage weight increase is greater for the individuals with a low birth weight than for those with a heavy birth weight. In other words, regardless of sex, rats that are very small at birth tend to grow more rapidly than do rats that have a heavy birth weight, although their actual body weights are always less at any given period. On computing the percentage increase in body weight at 150 days over the birth weight for the various individuals it was found that the rats that were small at birth had gained a greater amount than had the rats that were heavy at birth. For each series, as shown in the last column in table 2, these percentage values stand in inverse order to that of the birth weights, with the one exception in the second series (rat no. 4). The results of this investigation are in accord with those obtained by Dunn (’08) in her study of the weight increase in a ‘group’ of seven albino rats (three males and four females) which had very unlike body weights when they were fourteen days old. Dunn found that, with one exception, the order relation of the weight at fourteen days of age was maintained until the end of the experiment; the rats having the heaviest initial weights were also the heaviest at sixty-six days of age when the weighings were discontinued. The lighter rats, on the other hand, while putting on less absolute weight, had gained at the end a greater percentage of their original weight than had the individuals with the heavier initial weight. In addition to the undersized young which, as shown above, are capable of developing into adults which are only slightly below normal, a litter sometimes contains individuals of a much lower grade to which the term ‘runt’ properly applies. In these individuals, which apparently are indistinguishable from the other young at birth, the normal action of the growth factors is inhibited from the very beginning of postnatal life by unknown constitutional causes, not by environmental conditions. When the young rats are old enough to leave the nest the runts can THE ANATOMICAL RECORD, VOL. 11, No. 2 AS HELEN DEAN KING easily be distinguished from the other members of the litter, not only because of their very small size, but also because of their slower movements and apparent lack of normal vitality. Runts grow slowl for a certain time, but no matter how favorable the external conditions, they never exhibit normal vigor and they are always dwarfed and stunted in their body growth. In such animals growth is not merely retarded, as it is in the case of rats experimentally stunted (Hatai, ’07; Osborne and Mendel, ’14), but it is permanently checked at an early age. Several attempts have been made in our colony to increase the size of runts by special feeding, and to breed them for the production of a dwarfed race of rats. Only a few litters could be obtained from such stock, and these contained a very small number of young which were puny from birth and which died at an early age. The reproductive powers of these animals are apparently never developed in a normal way, as the males rarely mate and most of the females are sterile. All of the rats used in this study were killed at the end of 150 days, the body weights and body lengths determined, and. she brains and spinal cords removed and weighed. This was done in order to ascertain whether the central nervous system in adult rats that were small at birth bears the same relation to body weight and to body length as that found in adult individ uals that were of average size, or above, at birth. Table 3 gives the body lengths of the individuals, the observed weights of the brains and of the spinal cords, and the brain and cord weights corrected according to table 68 in “The rat: data and reference tables’ (Donaldson, ’15) which gives the com- putations for the ‘standard’ weights of the central nervous system in albino rats of various body lengths. In addition to the above data, table 3 shows the percentage deviations of the observed weights of the brains and of the spinal cords from the correspond- ing standard weights. It is evident, from the percentage values given in the fifth and in the eighth columns of table 3, that ‘the observed weights of the brain and of the spinal cord in all of the rats are consider- ably below the standard weights for these organs in animals GROWTH OF BODY AND NERVOUS SYSTEM IN RATS 49 TABLE 3 Showing the body lengths, with the observed and the ‘standard’ weights for the brain and for the spinal cord, of the albino rats whose weight data are given in table 1 3 - l zm x =) Ab Ze a 2 9 o8 a BS 2g | e [a ja, |ebal © [os | gee rm aj A, al a4 AK ar) RAT NO. an 2 e ele a oe el G 2 | Ee | dee | edt] be | 28a | g8 »S | oe | cea] eel on | 282| fe Rete | eee ee 1 183 | 1.461) 1.773|—17.5) 0.455} 0.524|—13.1 Series 1 (Females) 2 185 | 1.611) 1.782}|— 9.5} 0.473} 0.532)/—11.0 3 182 | 1.679} 1.768)/— 5.0) 0.499) 0.520|— 4.0 1 175 | 1.511) 1.735)—12.9} 0.418) 0.492)—15.0 Serea 2 (Females) 2 184 | 1.616} 1.778!— 9.1] 0.467) 0.528/—11.5 3 192 | 1.653} 1.814)/— 8.8] 0.485) 0.560/—138.4 L 4 188 | 1.643] 1.796|— 8.5) 0.475) 0.544|—12.6 1 198 | 1.697} 1.849|— 8.1] 0.529) 0.556)— 4.8 Greenlee. (Miales) 2 210 | 1.765) 1.903)/— 7.2) 0.579} 0.603)— 3.9 3 217 | 1.955) 1.933/+ 1.1] 0.631) 0.630/4+ 0.2 { 4 212 | 1.999) 1.911/+ 4.6) 0.669] 0.611/+ 9.4 of like body length, except in the case of the two largest males where the observed weights slightly exceed the standard weights. That the weights of the central nervous system in all individuals of a given litter, having the same sex and about the same body weight, should deviate from the standard weights in the same direction was not an unexpected result. In the rat variation within the litter unit is usually in the same direction and much less than that in the general population as regards body weight (Jackson, 713; King, ’15 b), and doubtless this rule holds for the central nervous system and other organs as well. In every series, as shown in table 3, the rats with the smallest birth weights are the ones whose brain and cord weights show the most marked deviations from the standard weights, regardless of their body length and body weight with which the weight of the central nervous system is, as a rule, closely correlated. Thus, in the first series, female no. 1 had a body length of 183 mm. and a body weight of 145 grams while female no. 3 was shorter and heavier, 50 HELEN DEAN KING yet in the former individual the brain was 17.5 per cent and the cord 13.1 per cent below the corresponding standard weights; in the latter individual the brain and cord weights were only about 5 per cent less than the standard weights. All of the rats in the second series had cord weights that showed relatively greater deviations from the standards than did the brain weights; the lowest weight for both brain and cord being found in the rat that had the shortest body length and the smallest body weight (no. 1). The most interesting result is found on comparing the records for the rats belonging to the third series. The brains of the two rats that had birth weights much lower than the aver- age birth weight (nos. 1 and 2) were each about 8 per cent less ‘than the standard and the cord weights showed a minus deviation of some 4 per cent from the standard, yet in body measurements rat no. 1 was below and rat no. 2. was above the average for stock males of like age. The brain and cord weights of their brothers, each of which was unusually heavy at birth, were above the computed standard weights for the central nervous system in animals of like body length. That the postnatal growth of the central nervous system is influenced to some extent by the factors that determine the size of arat at birth seems to be a conclusion warranted by the analy- sis of data given above. SUMMARY AND CONCLUSIONS The results obtained in this investigation seem to indicate that the undersized individuals which are sometimes found in a new- born litter of rats are not necessarily ‘runts’ in the generally accepted use of that term. Some of these small individuals, as shown above, attain an adult size that not only is within the normal limits of variation:in the body weights of standard stock rats of like age, but may even exceed the average body ' weight of a large number of stock animals (rat no. 2, series 3). All rats in a litter are not born with a like capacity for growth, as the data in table 1 indicate, and even when environmental conditions are as favorable and as uniform as it is possible to make them, individuals having unlike birth weights show marked differences in their rates of growth from birth to the adult state. GROWTH OF BODY AND NERVOUS SYSTEM IN RATS 51 In the cases studied the individuals having a small birth weight seemed to possess a’very great capacity for growth from the very beginning of postnatal life. Female no. 1 of the first series increased 2 per cent more in body weight during the first twenty- four hours after birth than did either of her sisters; for the same period the gain in body weight of the two smallest males in the third series was over 6 per cent more than that of their brothers. In the second series, however, the weight increase for the first day was greater for the rats that were heavy than for those that were small at birth. With an early acceleration in the rate of growth there is seem- ingly correlated an early cessation in the extent of growth, as in the adult state individuals that were undersized at birth are always smaller than the other members of the same litter. On the other hand, rats with a heavy birth weight tend to grow more slowly at first than do the smaller individuals, but they continue to grow for a longer time and eventually reach a greater size. This rule seems to apply to females as well as to males. Not only does body weight at birth indicate the probable capacity of the individual for subsequent growth, but it also indicates the probable size of the central nervous system, since rats that are undersized at birth tend to have a much smaller central nervous system when they become mature than do other rats. The factors, whatever their nature, that determine the body size of arat at birth seem to have a marked effect on the subsequent postnatal development of the individual, influencing the ultimate body weight as well as the size of the central nervous system. A very small weight at birth indicates that a rat has a handi- cap in its organization that environment, however favorable, cannot overcome. Such animals, although they appear vigorous and healthy during their growth period and after reaching the adult state, are unquestionably sub-normal in regard to the size of the body and of the central nervous system. If allowed to breed these rats would probably produce young having a weaker constitution than their own, and from such stock one would ultimately get ‘runts’ and an increasing tendency towards sterility that would soon bring disaster to the colony. 2 HELEN DEAN KING Judging from the results obtained in this study a newborn litter of rats may contain individuals of three kinds as regards their inherent capacity for body growth. As arule, only young rats having a normal birth weight and a normal capacity for growth are found in a small or medium sized litter produced by a female rat in good physical condition. Occasionally rats are born which have a very small birth weight, and in these individuals, if they are able to survive, the growth capacity is lessened to some extent but not sufficiently to prevent them from being classed as ‘normal’ after they have reached maturity. If a litter is very large, or if the mother is not in good physical con- dition during the gestation period, some of her young may be born with their growth capacity so impaired that it is impossible for them to grow beyond a certain stage. These individuals are true ‘runts’ and, fortunately, they are lacking in repro- ductive vigor as well as in growth capacity so that they are usually unable to reproduce their kind and so prove a menace to the colony in which they live. LITERATURE. CITED Donaupson, H. H. 1906 A comparison of the white rate with man in respect to the growth of the entire body. Boas anniversary volume, New York. 1915 The rat. Data and reference tables. Memoirs of The Wistar Institute of Anatomy and Biology, no. 6. Philadelphia. Dunn, EvizasetH 1908 A study of the gain in weight for the light and heavy individuals of a single group of albino rats. Anat. Rec., vol. 2. Haral, 8. 1907 Effects of partial starvation followed by a return to normal diet, on the growth of the body and central nervous system of albino rats. Amer. Jour. Phys., vol. 18. Jackson, C. M. 1913 Postnatal growth and variability of the body and of the various organs in the albino rat. Am. Jour. Anat., vol. 15. Kine, Heten Dean 1915 a .On the weight of the albino rat at birth and the factors that influence it. Anat. Rec., vol. 9. 1915 b The growth and variability in the body weight of the albino rat. Anat. Rec., vol. 9. Osporne, T. B., AND MENDEL, L. B. 1914 The suppression of growth and the capacity to grow. Jour. Biol. Chem., vol. 18. PoPENOE, PAuL 1916 Experimental inbreeding. Jour. Heredity, vol. 7. Watson, J. B. 1905 The effects of bearing young upon the body-weight and the weight of the central nervous system of the female white rat. Jour. Comp. Neur. and Psychol., vol. 15. MOUNTING SPECIMENS UNDER PETRI DISHES AND CLOCK GLASSES EK. L. JUDAH McGill University, Montreal Since obtaining a suitable cement for the sealing of square museum jars, the mounting of thin sections of pathological and anatomical specimens under Petri dishes and clock glasses has been made both easy and cheap. Several years ago these mounts attracted a great deal of attention, being put on the market as paper weights, etc., but were gradually adopted for the exhibition and display of museum specimens. The method, however, being patented was expensive and beyond the reach of the average museum; besides it was necessary to send your material for mounting to the manufacturer. In 1906 Dr. Hutchinson of the Royal Victoria Hospital, Montreal, read a paper before the British Medical Association at Toronto on this method; but unfortunately he did not have a suitable cement and the process was slow and laborious. METHOD The fluid used for mounting should be brought to a boil in the same dish that is to be used for mounting and allowed to stand over night, or until cool, to get rid of as much air as possible. The dish should be deep enough to come well up over the mount to allow of easy manipulation of both Petri dish and specimen. Get a Petri dish of suitable size to hold the specimen so that when the sheet of glass which is to form the cover of the mount is in position it will not quite touch it. Great care must be taken that the Petri dish or clock glass fits perfectly on the base and does not rock. In placing the Petri dish in the mounting fluid, do so without causing any air bubbles. When the dish and fluid are ready, wash the speci- men in several changes of the same fluid that you are mounting in, to get rid of any loose particles or dirt. In the last change of fluid, work out all air and remove quickly to the Petri dish, face downwards, again eliminating air bubbles. A small piece of looking-glass in the bottom of the mounting dish is very convenient, as by tipping the Petri dish over and on its side slightly, any bubbles under the specimen may be seen. The specimen now being in position, put on the base or cover and remove from the mounting-dish, holding it firmly so that it cannot slip 53 54 E. L. JUDAH and admit air while turning the mount right side up. Over the junc- tion of Petri dish and cover pour hot cement! to the thickness of about one quarter of an inch, and allow the mount to stand on a flat surface for a few days, or until the cement is perfectly adherent to both Petri dish and cover. If after several days it is desired to finish the mount, remove any bubbles of fluid between the cover and the cement with a very hot knife, working the knife to the outer edge of the base. This must be carefully done, and the knife kept hot enough so that the cement will be kept liquid and not be drawn away from the Petri dish. All the fluid must be removed from under the edge of the Petri dish in this way. The best results are obtained, however, ie allowing the mount to stand for several weeks when most of the excess fluid will work out by itself. I usually mount several dozen specimens at a time and let them stand until they are ready to finish. If any air bubbles should happen to get under the edge of the Petri dish they will have to be worked into the mount in the same way that fluid is removed, only work your knife inwards instead of outwards. Even with the greatest care air is often retained in the specimen itself, and only detected after the mount has stood for several days. Should the bubbles be very small they will quite frequently be absorbed by themselves; if not, shake the mount until they are all in one large bubble, and resubmerge in mounting fluid which must be heated as hot as you can comfortably stand your hand in. When the cement has become pliable enough so that it is possible to move the Petri dish with the fingers, insert the point of a sharp knife between the dish and base, gently “forcing them apart and allowing a little fluid to enter. The mount must then be tilted on its side to bring the air bubble to the opening, where it will escape when you remove the point of the knife; then press the Petri dish up against the base, expelling all superfluous fluid. Close the hole by pressing the soft cement together with the fingers. Air bubbles may have to be removed several times depending on the specimen. Sections of lung give the most trouble. When sure that there are no more bubbles in the mount and that all fluid has been removed from between the cement base and the Petri dish, coat the cement over to the thickness of about one-eighth of an inch with re- fined asphalt, being careful that it does not burn; I usually melt it in a tea-spoon over a Bunsen burner. When the asphalt has been all applied, reheat the whole with a very hot knife and apply a bezel ring which must be heated red hot, and pressed down into the cement so that the lower edge will rest upon the base. Clean with gasolin and polish with bon ami soap. The Petri dishes used are manufactured specially from 2 to 3 mm. thick, as the ordinary ones sometimes break with the expansion and 1 See Muir and Judah, Sealing of museum jars. Bulletin 5, Inter. Assoc. Med. Mus., page 87. MOUNTING SPECIMENS UNDER PETRI DISHES D0 contraction of the fluid. The bezel rings which are used to give a finished appearance to the cement can be made out of any metal that will stand being heated red hot. They are not, however, absolutely necessary, as you can use a sheet of cardboard with a hole cut in the centre and passe partout the edges. Dr. Higgins of the Experimental Farm, Ottawa, has a very convenient cardboard case into which he slips the mount. If a bezel ring is to be used, the base or cover should be made out of plate glass with the edges bevelled. Clock glasses are inferior to Petri dishes for this method because they magnify and distort the specimen. The following list of sizes have been found to answer all requirements. While 36 in number they only require ten different sized bezel rings and plate glass bases, a desired advantage when the glass ware is made to order. Sizes (Outside measurements) SIZE NO. WIDTH HEIGHT SIZE NO. WIDTH HEIGHT cm. cm cm. cm. 1 ae 3 19 8 2.5 2 18 2 20 8 2 3 18 7 21 8 1.5 4 16 3 22 8 1 5 16 2 23 6 2.5 6 16 1.5 24 6 2 7 14 3 25 6 1.5 8 14 2.5 26 6 1 9 ppl4 2 Q7 4 2.5 10 14 1.5 28 4 2 11 12 3 29 4 1.5 12 12 2.5 30 4 1 13 12 2 31 3 2 14 12 1 32 3 1.5 15 10 2.5 33 3 1 16 10 2 34 2 2 17 10 1.5 35 2 1.5 1S) 10 1 36 2 1 ANNOUNCEMENT An Authors’ Index has been prepared and printed for each of the following journals: JOURNAL OF MORPHOLOGY 25 volumes—1887-1914 — Price per copy, 50 cents THE AMERICAN JOURNAL OF ANATOMY 18 volumes—1901-1915 Price per copy, 50 cents THE ANATOMICAL RECORD 10 volumes—1906—(July)1916 Price per copy, 50 cents Sent post-paid to any address THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY Philadelphia 56 THE EFFECT OF HYPOPHYSECTOMY IN THE EARLY EMBRYO UPON THE GROWTH AND DEVEL- OPMENT OF THE FROG A PRELIMINARY REPORT P. E. SMITH From the Anatomical Laboratory, University of California TEN FIGURES The extirpation of the hypophysis in the adult frog has not given uniform results. Caselli (00) and Gaglio (’02) who re- ported no changes following hypophysectomies were followed ‘by Boteano (06) who reported a neuromuscular asthenia in the operated animals. Houssay (10) came to the conclusion that the removal of the gland was followed by death. Adler (14) burned out the hypophysis of a 20 mm. Rana temporaria Jarvae with the electric cautery. Out of the 1200 operated ani- mals three were found to have been hypophysectomized, not, however, without great injury to the surrounding soft parts, particularly the brain. In not one of those three animals did hind legs develop beyond a small bud, and transformation did not take place, the specimens remaining as neotonic tadpoles. This work was commenced in the Spring of 1914, repeated in 1915, and again in 1916, Diemyctylus torosus, Rana pipiens, and Rana boylei being successively used. In this paper the results obtained with the California yellow-legged frog, R. boylei are reported. Shortly after the closure of the medullary plate, Kopsch’s stages d-e, was found to be the size in which the hypophysial invagination could be most successfully removed. About 200 larvae of this stage were operated upon. In speci- mens of this size the hypophysis was successfully removed in over 60 per cent of the operated animals. Approximately 30 per cent of those animals in which the gland was extirpated did ol THE ANATOMICAL RECORD, VOL. 11, No. 3 OCTOBER, 1916 5 o/) P. E. SMITH not give reliable results in the rate of growth as the mouth was wholly or partially removed thus interfering with feeding. Un- operated animals and those in which. the ablation of the gland was unsuccessfully attempted were available for checks. The operation is a simple procedure. The hypophysial in- vagination can be accurately determined from the pit that it early forms or from its location between the protuberance of the forebrain and the stomadeum, which is just forming. This epi- thelial ingrowth was removed with some neighboring epithelium. The wound healed within three hours in most cases, less than 1 per cent of the larvae disintegrating after the operation. The operated animals and checks were kept in boiled water for five days and then transferred toa frog tank where they were in an essentially normal environment. The rate of growth in the hypophysis-free animals has been slower than in the checks. The larger hypophysectomized ani- mals averaged smaller in size than the larger checks, the averages of the two showing a noticeable difference. On June 6 the operated but not hypophysectomized animals had an average length of 40 to 43 mm., the hypophysis-free animals averaging 33 to 35 mm., a ratio constant throughout their growth. The ratio of body to tail length is the same in the two classes, the difference in size being uniform for all parts of the animal. The tail fin did not show an increased width or pleating in the hypophysectomized animals as reported by Adler (14). In activity the two classes of animals showed no marked differences. The hypophysectomized specimens were perhaps slightly more alert, darted more quickly, and consequently were more difficult to capture with the pipette than were the checks. The resistance of the hypophysectomized animals was greater than that of the checks. Towards the close of the experiment the animals were attacked by disease, none reaching the adult stage. The normal specimens succumbed more rapidly to this infection than did the hypophysectomized ones. Some of the intrinsic factors which induce growth of legs and transformation were lacking in the abnormal specimens as will be shown later. The absence of these factors may well be conducive to a greater EFFECT OF HYPOPHYSECTOMY UPON THE FROG 59 hardiness in an animal when compared to the normal tadpole in which the usual rapid changes are taking place. Differences in color began to be noticeable before a length of 15 mm. was reached, and from then on the contrast in pigmen- tation between the hypophysectomized animals and the checks was striking. ‘Those animals without hypophyses were charac- terized by a light grayish appearance; however, the dorsal side was more pigmented than the ventral (figs. 7, 10). These are referred to as albinos. The checks were a brown-black color often showing a mottling (figs. 8, 9). This color difference was more noticeable over the body than on the tail, but was evident in both regions and was the most striking feature up to the time of the appearance of the hind legs in the checks. Sections show that these pigment differences are referable chiefly, if not solely, to the condition of the epidermis. Counts of the melanophores of corresponding areas in the albinos and in the checks show that the number of these cells, in the epidermis, are reduced in the former. Further the melanophores of the albino specimens contain fewer pigment granules than do those of the checks and thus have a distinctly lighter appearance. The melanophores are equally expanded in the two types, consequently, the lighter color of the albinos cannot be due to the contracted condition of the chromatophores but must be referred, in part, to the re- duced number of melanin granules in the pigment cells of the epidermis. In addition to this the free pigment granules which form a distinct zone in the superficial layer of the epidermis in the normal checks are much reduced in number in the albino specimens (figs. 5, 6). It is surprising that in the albinos the deeper or subcutaneous pigment is present Jp as great a quan- tity as in the normal animals, if not greater. The amount and distribution of the retinal pigment seem to be identical in the two. Another important feature was the inhibition in growth of the hind legs of the operated animals. There was only a slight retardation in the time of appearance of the hind leg buds, normally, appearing when the tadpole has reached a length of 25 to 27 mm. In the albino, averages show that the hind limb buds appear when the larvae are from 26 to 28 mm. in length. 60 P. E. SMITH From this state on, however, the hind limbs in an hypophysec- tomized animal grew but little if at all, although the animal’s length increased at a rate but slightly under the normal. The accompanying table shows the increase in length of the hind legs in relation to total length for the albinos and for the checks. (See also figs. 7, 8). Average rate of growth in millimeters in terms of total length, of the hind legs of the checks and the albinos HYPOPHYSECTOMIZED ANIMALS CHECKS Total length Hind leg length Total length Hind leg length’ 26 barely visible 25 barely visible 28 Orn 28 1.0 30 Oat 30 a) 35 Onl 35 3.0 37 (I 38 4.0 40 5.0 45 9.0 Only one exception to the rule that no hind legs grew ou albinos was found. A 386 mm. albino had hind legs 4.2 mm. long when killed. The above is in accord with Adler (14) who found that removal of the hypophysis in a 20 mm. stage in- hibited the growth of the hind legs. Examination of sections of albino and normal animals shows striking differences in the endocrine glands. The sectioned hy- pophysectomized animals show no trace of the anterior lobe of the hypophysis. That part of the floor of the diencephalon which normally abuts against the hypophysis, rests upon the floor of the cranium (fig. 2). This apparently demonstrates con- clusively that the entoderm has not the intrinsic power to form a hypophysis. If it enters into the formation of the gland at all it must be considered as a tissue inclusion which became changed through its adaptability into. glandular parenchyma, a conclusion previously drawn by the writer, Smith (14). The infundibulum shows some structural modifications when com- pared to the checks, although the saccus vasculosus, as far as determined, appears to be normal. In the checks that region of the diencephalon which rests against the pars glandularis is. EFFECT OF HYPOPHYSECTOMY UPON THE FROG 61 Fig. 1 A section through the hypophysial region of a 38 mm. normal tadpole. Fig. 2. A section through the hypophysial region of a 37 mm. albino. Note the much reduced pars nervosa. X 100. Fig. 4 A sagittal section through a lobe of the thyroid of a 37 mm. albino. F 5 6 Fig. 5 A section through the epidermis, in the mid-brain region, of a normal 39 mm. check. The pigment granules are indicated by dots. X 200. Fig. 6 A section through the epidermis, in the mid-brain region, of a 38 mm. albino. A faint melanophore in the left part of the figure. > 200. 62 P. E. SMITH of considerable thickness, that is, in addition to the ependyma there is a rudimentary pars nervosa. Caudad to this the wall is formed almost entirely of ependyma. The pars nervosa is reduced throughout most of its extent to an ependymal layer in the hypophysectomized animals. There may be a small local- ized thickening but nothing to correspond to the normal animal (figs; 1,2). The thyroid shows marked modifications in the albinos. In the accompanying table the size of one lobe of the thyroid of a normal 38 mm. tadpole with 4.0 hind legs and of a 37.0 mm. albino with 0.1 mm. hind legs is given. Size in millimeters of one lobe of the thyroid 388 mm. check 387 mm. albino When other ase rorya von maacr oO Ween eGhi.c:.). ci. aes eee 0.21 Witclthie Actos Se alee coarse 0.3 Witt hi ss. ey a ees 0.15 sRHICIMESS? te pace ee ets OG Fig. 7 Photograph of an albino. X 2. Note the very small hind limb bud. Fig. 8 Photograph of anormal tadpole. Figures 7 and 8 were photographed on the same plate’ ™ 2. Fig. 9 Photograph of a normal tadpole. X 2. Fig. 10 Photograph of an albino. X 2. EFFECT OF HYPOPHYSECTOMY UPON THE FROG 635 The above table shows that the thyroid of the albino is ap- proximately one-third normal size. ‘The contrast is even more striking when the compactness and character of the parenchyma is noted. A sagittal section through the thyroid of a 38 mm. check shows on an average 12 to 15 vesicles, many of which are largely distended with colloid, the parenchyma of the whole gland being compacted together. A sagittal section through the thyroid of a hypophysectomized 37 mm. specimen shows 6 to 8 atrophied vesicles containing but a slight amount, or no colloid, and with large spaces between the vesicles. The cells making up the vesicles of the former are cuboidal and protoplasmic- rich, in the latter little but the nuclei remain (figs. 3, 4). The results from experimental feeding of thyroid by Gudernatsch and other workers suggests that the non-development of the hind legs in the albinos is due not to the hypophysis but rather to the failure of the thyroid. In this connection the 36 mm. albino with 4.2 mm. hind legs, mentioned above, is of interest. Sections of this specimen show that the hypophysis was com- pletely ablated but that the thyroid is normal. This specimen thus gives additional evidence that the retarded development of the hind legs must be referred to the thyroid and not to the hypophysis. Also the reduction in pigment is not due to the atrophy of the thyroids. The modifications of the thyroid obtained by Adler (14) were similar but less striking. An examination of a large number of male and female albinos and checks has, as yet, failed to show any constant variation from the normal in the sex glands of the hypophysectomized animals. The sex glands of the albinos although varying con- siderably apparently do not exceed the limit of variation met with constantly in the normal animals. This conclusion stands in contradiction to the results previously adduced by the author and to the results of Adler (’14) in the hypophysectomized tad- pole and to the conclusions of Hahn (712) in the tadpole with hypertrophied hypophysis as well to the results obtained in mammals by pituitary feeding, notably that of Goetsch (16). The writer wishes to express his appreciation to Dr. H. M. Evans for his generous aid. 64 P. E. SMITH LITERATURE CITED Apuer, L. 1914 Metamorphosestudien an Betrachierlarven. I. Extirpation endokriner Driisen. A. Extirpation der Hypophyse. Arch. f. Entw. mech. d. Organis., Bd. 39. Breput, A. 1913 Innere Sekretion. Zweite Aufl. Berlin. Botreano, E. R. 1906 Contr. la physiol. glandei pituitare la brosca. These, Buearest, zeit. n. Paulesco, cit. f. Biedl. Case.tu, A. 1900 Influence de la fonction de l’hypophyse suz le development de l’organisme. Riv. sper. di fren., vol. 37, cit. f. Biedl. Gacuio, G. 1902 Recherches zur la fonction de l’hypophyse de cerveau chez les grenouilles. Arch. ital. d. Biol., vol. 38, cit. f. Biedl. Goetscu, E. 1916 The influence of pituitary feeding upon growth and sexual development. Bull. Johns Hopk. Hosp., vol. 27. Haun, A. 1912 Einige Beobachtungen an Riesenlarven von Rana esculenta. Arch. f. mikr. Anat., vol.-80. Hovussay 1910 La hypofisio de la rana. Trabajos de Labor de Univ. Nacional de Buenos Aires, cit. f. Biedl. Smiru, P. E. 1914 The development of the hypophysis of Amia calva. Anat. Rec., vol. 8. ANOMALIES IN LOBATION OF LUNGS WITH REVIEW OF LITERATURE CARBON GILLASPIE, LEWIS I. MILLER, AND MORRIS BASKIN Anatomical Department, University of Colorado FIVE FIGURES Judging from the literature, abnormal lobation of the lungs is relatively rare. Lindsay (10) divided the abnormalities in the lobation of the lungs into two classes: those in which the normal number is decreased and those in which the number of lobes is increased. The former is due either to a deficiency of the lobes themselves, or to a deficiency of the fissures, which normally separate the lobes. The latter is due to an increase of lobes, or to an increase in fissures. Complete absence of lobation in definitely formed lungs is rarely if ever found, though its homologue is to be found in the Orang, with two lungs each existing as single lobes. Rokitansky (’61) showed that arrests of development may occur and that this may lead to complete absence or great defi- ciency of one or both lobes. This arrest may be so early that the lungs can scarcely be observed as small round bodies situated at the ends of the bronchi. This condition is generally due to contraction of the volume of the thorax. Pontif (60) in ‘Virchows Archives’ recorded a case in which the right bronchus was connected with an ovoid body, which was imbedded in gelatinous tissue and filled the right half of the thorax. According to Lindsay, cases of class two, that is those which have an excessive number of fissures, are occasionally encoun- tered, and are probably the most common form of abnormality. They do not appear to present any regularity, and lack the interest which is attached to accessory lobes. There are two 65 66 CARBON GILLASPIE, L. I. MILLER, AND M. BASKIN groups of accessory lobes. One which is of considerable develop- mental interest, is composed of completely isolated masses of pulmonary tissue formed between the diaphragm and the base of the left lung; occasionally on the right side such masses are found even in the abdominal cavity. Other masses containing arteries, veins, nerves, and bronchial tissue, but devoid of bronchi, are attached to the oesophagus, aorta or other mediastinal struc- tures by a pedicle. These masses are apparently quite func- tionless. Two such cases are recorded by Vogel (99), in each of which he found a deficiency in the bronchial tree. Simpson (99) described a case of a deficient bronchial tree found in a foetus. In the cases with additional fissures there is a normally placed lung presenting an excessive number of lobes. These abnor- malities are usually very definite in their position, and occur more commonly on the right side. Wrisberg (77), who was the first to notice an accessory lobe in the human lung, recorded a most interesting and unique case of an accessory lobe on the left side produced by the left azygous vein; i.e., the superior intercostal vein which preserved its foetal condition and opened into the left innominate vein. Chiene (’76), described a pear-shaped supernumerary lobe, lying between the upper lobe of the right lung and the bodies of the dorsal vertebrae, having its origin from the angle formed by the junction of the upper lobe with the root of the lung. The supernumerary lobe was separated from the upper lobe of the lung by a double fold of the pleural membrane, which de- scended vertically for seven centimeters from the apex of the thoracic cavity where it was continuous with the pleura costalis. It enclosed in its free border the vena azygous, and formed the outer wall of the cul-de-sac, in which the supernumerary lobe was contained. The left side of the chest was normal; both sides were healthy. Kk. W. Collins (88), recorded a case of an accessory lobe immediately above the posterior part of the root in the angle between it and the upper portion of the right lung. This acces- sory lobe was somewhat pyriform in shape, with a broad pedun- ANOMALIES IN LOBATION OF LUNGS 67 cular attachment. In all, Collins was able to colleet seven cases of accessory lobes in human lungs. A. E. Maryland (’90) described abnormalities in lobes of three lungs. ‘The first was a right lung with no indications of a middle lobe, but a development of a third, or accessory one, on the inner side of the lung. The second was a left lung with a subdivision of the upper lobe. The third was a right lung with an incom- plete separation of a norma] middle lobe. Maryland states: “Cases of more than four right lobes and three left lobes are exceedingly rare.’ Patterson (09-10) described a condition of two additional lobes. One of these was above the root, and separated from the upper lobe by a fissure. This accessory lobe was enclosed within a pleural pouch, which contained the vena azygous. The second additional lobe was below the root, and between the upper and middle lobes. Case I of the present specimens presents features entirely different from those hitherto described. It was obtained in the dissecting room during the term of 1915-1916 from a male sub- ject, aged sixty-one. The cause of death as given on the death certificate was dementia. No clinical history was obtainable. Besides the anomalous condition of the lungs, there was a per- sistent thymus, and a number of anomalous arteries. On gross inspection the lungs presented no pathologic lesions. The left lung apex-base measured 22 em., dorso-ventrally 20 em. The normal fissure (f3), which separated the superior from the inferior lobe, was in its normal position, starting at the junction of the antero-inferior border 25 em. from the apex, and running obliquely upward and backward, dividing the superior and inferior portions completely. The superior portion, however, presented two other fissures, as shown in the diagram, thus dividing this portion into three more or less distinct lobes. Fissure number one (/7/) started at the anterior border 7 cm. below the apex, and ran horizontally backward on the antero- lateral surface of the lung for 6 em. The depth of the fissure was on an average 1.5 cm. 68 CARBON GILLASPIE, L. I. MILLER, AND M. BASKIN Fissure number two (#2) started at the antero-median border 17 cm. below the apex and ran upward and backward for 9 em. ‘This fissure extended through the lung tissue, completely separating the middle from the inferior division of the superior lobe. Neither of the two mentioned fissures extended as far as the main fissure, which separated the superior from the inferior lobe. Three distinct divisions of the superior lobe were evident from an antero-lateral view. The upper division (L7) was pyramidal in Fig. 1 Anterior aspect shape, forming the apex. It measured 8 em. antero-posteriorly, and 7 em. from apex to base. The middle portion (12) was wedge-shaped, wider on the anterior border than on the posterior. It measured 9 em. on the anterior border between the first and second fissures, and 17 cm. antero-posteriorly. | The lower division (13) is lingual in shape. This division measured 6 cm. on the anterior border, and 21 cm. antero- posteriorly. The inferior lobe presented two more supernumerary fissures as did the superior lobe. ANOMALIES IN LOBATION OF LUNGS 69 Fissure number four (/’°4) started 5 em. antero-inferiorly from the supero-inferior fissure and ran parallel with the above fissure for a distance of 10 em. This fissure also extended through the lung tissue separating the upper from the postero-inferior divisions. Fissure number five (5) was on the lateral surface, extend- ing obliquely, superiorly and inferiorly, and incompletely divid- ing the postero-lateral from the postero-inferior divisions. Fig. 2. Posterior aspect The inferior lobe, which was triangular, also presented three fairly distinct divisions. The upper division (14), which is oblong in shape, having a small tongue-like projection on the anterior border, ran obliquely upward, and backward, following the direction of the supero- inferior, or great fissure. This division measured 5 em. antero- inferiorly, and 19 em. antero-posteriorly. The postero-inferior division (14) was more or less quadrangu- lar in shape. It measured 8 cm. on the superior border and 12 em. on the inferior border, i.e., at the base. The postero-lateral division (L6) was irregular in outline. The upper border was made by the supero-inferior fissure, and 70 CARBON GILLASPIE, L. I. MILLER, AND M. BASKIN the lower by a separate fissure between this division and the postero-inferior division. The right lung apex-base measured 19 em., and dorso-ventrally 18 cm. In this lung the fissures were deeper, and went through the lung tissue, completely dividing the lung into distinct divi- sions. The normal fissure (F’3), which divided the apex lobe and middle lobe from the inferior, or base lobe, was a Jittle more irregular than the corresponding fissure on the left lung, ? Fig. 3 Right lung: lateral view and ran obliquely upward and backward, dividing the lung into an upper and lower division. The upper division presented an ir egular outline from a lateral view. ‘There were two large fissures; one which normally divided the apex lobe from the middle lobe, and the other a supernu- merary fissure dividing the middle lobe into two. There was also a small fissure in the apex lobe. Fissure number one (/’7), which divided the apex lobe from the middle on-, started 10 em. from the apex laterally, midway between the apex and the base. ANOMALIES IN LOBATION OF. LUNGS 71 Fissure number two (/'2), which divided the middle lobe into two divisions, ran vertically for a distance of 8 em., starting from the base and running toward the apex. The apex lobe (1’1) was quadrilateral in shape, having the upper border narrower than the base and measuring 9 em. apex- base, and 8 em. antero-posteriorly. The inferior division (L’2) of the upper lobe was lingual in shape, extending obliquely upward and backward 13 em. in its long direction, and measuring 4 em. apex-base. Fig. 4 Left lung: lateral view The inferior division (13) of the upper half of the lung was elongated in outline measuring 4 cm. in antero-posterior direc- tion, and 13 em. apex-base. The lower half of the right lung was triangular in shape, having two clearly visible fissures which ran through the Jung tissue and divided this portion of the lung into three distinct Jobes. There are also two smaller fissures in the inferior border of the lung, merely forming small tongue-like lobules. The fourth fissure (F’4) started 10 em. from the apex midway between apex-base, running downward and backward for a dis- 2 CARBON GILLASPIE, L. I. MILLER, AND M. BASKIN ba | tance of 14 em., and completely separating the upper from the lower division of this half of the right lung. The fifth fissure (/'’5) started from the base, or inferior border, and ran vertically upward for a distance of 10 cm. It extended through the lung tissue, dividing this inferior half into two divisions, a posterior and an antero-median. The superior division (L’4) of this inferior half of the right lung was triangular in shape, measuring 13 cm. on its Inferior Fig. 5 Base border, 8 em. on its superior border, and 11 cm. on its posterior border. The postero-inferior division (L’5) was quadrilateral in out- line, measuring 8 em. on its shortest border and 14 cm. on its longest, or posterior border. The antero-median division (L’6) had the form of an elongated triangle, measuring 11 cm. on the anterior border, 6 cm. on the median posterior border. At the base of the right lung was a very irregularly shaped division distinctly separate from the rest of the ling as shown ANOMALIES IN LOBATION OF LUNGS 43 in the diagram. This basal division (1 7) measured 10 em. in the long direction and 6 em. in the short direction. This divi- sion was separated from the rest of the lung by two distinet fissures (’’6—7) which started at the postero-inferior border, and ran for 9 em. anteriorly and backwards. Case II was obtained in the dissecting room during the term 1915-1916, from a female subject, aged thirty-eight. The cause of death as given on the death certificate was ‘septic meningitis.’ No clinical history was obtainable. The left lung apex-base measured 20 cm., dorso-ventrally 18 em. The right lung apex-base measured 18 em., dorso-ventrally 17 cm. The left lung presented upon examination one accessory lobe, one accessory lobule, and three accessory fissures in the inferior portion of the left lung. The right lung presented upon examination two accessory lobes and two accessory fissures. The first accessory lobe was in the upper portion of the inferior division of the right lung. The other accessory lobe was a basal lobe and was identical with the basal lobe seen in the right lung in Case I. X-Ray! pictures of Case II show separate bronchi going to each of the main lobes including the accessory lobes, as may be seen by referring to the plates. X-Ray views of Case I were unsuccessful on account of a poor bismuth injection. From the foregoing description it is evident that these speci- mens show no distinct azygous lobe, which is the common acces- sory lobe described. These lungs retain their normal shape; but they present, besides the normal fissures, a number of acces- sory fissures which divide the lungs into distinct accessory lobes. Complete absence or deficiency of one or both lobes may be due as Rokitansky points out to arrests of development as con- traction of the volume of the thorax. Supernumerary lobes have been variously explained. Lindsay ascribes a slight adhe- sion of the lungs to the thoracic wall as the cause of the super- 1 Stereoscopic X-ray plates show the separate bronchi as described. Reduced prints fail to show the necessary details and, therefore, are not reproduced here. THE ANATOMICAL RECORD, VOL. 11, No. 3 74 CARBON GILLASPIE, L. I. MILLER, AND M. BASKIN numerary lobe, or possibly an undue curvature of the embryo, so that the Venae Cavae, as it bent down to a position at right angles to its original position, instead of slipping behind the pleura and lung, dragged down a fold of the former and deeply notched the latter. Fischer remarks that it is difficult to conceive of the azygous lobe without an already preéxisting anomalous course of the azygous, and makes no attempt to explain the phenomenon. Collins explains the azygous lobe as a persistent foetal con- dition of the left azygous vein. All previous attempts to explain the origin of the supernu- merary lobes apply only to cases in which there is a definite azygous lobe. The anomalous course of the azygous vein has constricted off a portion of the lung tissue, thereby forming a new lobe. This explanation does not apply to cases in which there are no azygous lobes and in which, however, there are other supernumerary lobes, as the azygous lobes are not in reality true lobes. =. { a ae org 71* | May 30...... { = 4 a 7163 fomaune 208... : 5 ee ee 73* | July 16....... 4 a sone _o07 neh ne nate — 20.0 75* | August 17.... : a oe Et ys * Tuberculosis found. 91 NO, DATE WEIGHT fate, oti ” Sacks hybrids Se oes Pes sox! R ed 0.030 / (oka PNY yan Wei 3 Ae { L = 0.031 | + 3.3 = a R = 0.262 re" July 23... 5 Vn = 9.019 |) Seas 78*| July 30......: { . i a aad wy 79*| August 22.... : = aes 43 . = 2 80 | August 29.... { He e eer 33 3 8i* September 19 { - 5 ee 93.7 = 0.02 82*| September 28 ( = = ae =a0 | 83+| October 7... { Beane ee 84*! October 19 R = 0.007 (Githy-))senaoe L = 0.006 | —16.6 85*| October 24... { - x ata i, 86*| November 21 R = 0.015 (Juv.)...-. || L = 0.020 | +33.3 87*| November 22. { = : Fale 4143 88 | November 25. { % ie Oa — 90.0 Common pigeons ; Ri = 1.260 89 | April 28...... { L = 1.035 See, 90*| August 25.... { 5 = Fae 12.6 ie Gli eae U5 Or | Aprilb....... i| L = 0.990 11.6 | 1 The left suprarenal wholly involved ina tubercle nodule weighing nearly 1.0 gr. 2 Healthy. 3 A hybrid from a family cross. 92 OSCAR RIDDLE TABLE 4 Weight, length and width of testes of young Ring dove (specific) hybrids— killed NO. DATE . ‘ December 4, December 4, December 4, December 4, December 4, December 4, December 4, December 8, December 8, December 8, December 8, December 8, December 8, December 8, December 8, December 8, December 8, December 8, 1915.. 1915.. 1915.. 1915.. 1915. 1915 | 1915 . 1915.. 1915. 1915 1915. 1915... 1915 | 1915. 1915.. 1915.. 1915. 1915.. * Tuberculosis found. PER CENT WEIGHT Bees 2) LENGTH AND WIDTH Be Sel ENCE ENCE = 0.035 = 0.030 = 167 8.0x3.8 = 0.007 (?) 5. sed6 = 0.005(?) —40.0 .6x1.4 —10.9 = 0.004(?) 4.1x0.9 = 0.005(?) +25.0 4.9x0.9 +19.5 = less than 0.005 (?) 5.1x0.8 = 0.005(?) +? yall sts 1h.) =0.0 = 0.005(?) 4.8x1.5 = 0.005(?) = 0.0 5.3x1.4 +10.4 = 0.007 (?) 4.8x1.6 = 0.005(?) —40.0 5.3x0.9 +10.4 = 0.015 6.6x1.9 = 0.010 —50.0 6.6x1.5 =0.0 = 0.025 Ske) kaze = 0.025 = 0.0 9.0x1.9 +8.4 = 0.037 1 OLA Kae. = 0.025 —48.0 le DieXoroiee —16.7 = 0.010 SO Oy Kale = 0.007 —42.9 50 xl =0.0 = 0.320 = 0.280 —14.3 = 0.007 (?) ; AA = 0.005 (?) —40.0 5.0x1.0 +13.6 = 0.025 6.8x 2.2 = 0.020 —25.0 6.7 x 221 —1.5 = 0.010 6.5x1.9 = 0:007 —42.9 O)213) 21S) =0.0 = 0.012 = 0.012 = 0.0 | Summary: = 0.035 Left larger in..... 2 = 0.025 —40.0 Left smaller in.... 13 = 0.100 Two equal in...... 3 = 0.070 —42.9 Left longerin.... 5 = 0.017 Left shorter in.... 3 = 0.015 —13.3 Left equal in...... 4 PER CENT TESTES OF PIGEONS IN HEALTH AND DISEASE 93 is probably responsible for the failure of our weighings to differ- entiate between the masses of several pairs of the testes. In the 30 pairs 18 right testes were larger. 5 were smaller, 7 were not differentiated by the weighings. Size. of testes in hybrids dead of disease Seven of the 10 specifie hybrids of table 2 had larger right testes; 2 had smaller; 1 had the testes of equal size. Of the two generic hybrids (52, 53) represented in this table, one had a larger and one .a smaller right testes. In table 3 are listed 13 specific hybrids; 7 larger right testes, 4 smaller, and 2 equivalents. TABLE 5 Weight, length and width of testes of young Ring dove (specific) hybrids—killed PER CENT PER CENT NO. DATE WEIGHT a Sila LENGTH AND WIDTH saeaae ENCE ENCE (| R = too small to 3.2x1.5 106 November 29, 1915 weigh L = 0.005 + ?.0 4.7x1.8 +46.9 } e R = 0.007 Dox 1.6 107 November 29, 1915 L = 0.007 ao ade 3G i R = 0.009 yoyo AL ats’ ae Seale ere ben, 29) 1915 he .9.010 meiiet || 16:4 18 +10.3 R = 0.020 8.2) & 129 ] 2 5 pemree ey omber 29, 1815 | noi Bee |.) Be ae Teo = 0.0 <, |f]| 1 KOI 1.42.0 110 November 29, 1915 1" 0.000 4411 74x20 GG R = 0.010 9. 9x 252 mt Bigvemier 209,101) yi, 0.016 =00| 7.4x1.6 425.4 if Ra 10190 il7/ a0) eZ meme eee 29, Tae \| eo. 170 11st) Heer 455 a Ee ~ R = 0.007 7 s6 WSS 113 December 3, 1915.. { L = 0.005 40.0 49x09 163 . R = 0.007 See 14 114 December 3, 1915.. { L = 0.005 40.0 Moe 1 6 30.9 115 | December 3, 1915.. { 4 i et gg |) punmary: abs C rt Left larger in...... 3 116 December 3, 1915.. Bs 0005 Left smaller in..... 5 \ ry 0.005 = 080 Two equal in... 4: 4 117 December 3, 1915.. | L & ae 40.0 Left longer in...... 3 \ S Lt Left shorter in..... 4 Two equaling... . 2 THE ANATOMICAL RECORD, VOL. 11, No. 3 TABLE 6 Weights and measurements of testes—birds classified as to kind and disease PER CENT PER No. DATE DISEASE! WEIGHTS wine. || eee lees ENCE ENCE Pure species—Spil. tigrina 118) January 7 ee : SPS AS J _ a es = H.0 eee 438.5 119| January 9 Worms (juy.)....... if " ‘/ Rae 416.6 a : Ae 432.1 120) January 11 Taveat Vere ns . ee 442.5 ee 423.3 121) January 11 | Liver and worms... 4 a et arr, Abe : a = ant 122| January 13. 4|"Worms.eseee. 2... | a eat E98 Pe. 413.9 123) January 15 Worms liver......... { = P ae 412.5 ae ‘ ie | 496.2 124| January 18 Intest. and liver. . a x Feo, “ie ss : a ae | 125), January 25.78) Imtest. | dicen ss i. si Faas 416.6 d let 3 421.1 126) January 29 Worms (old)....... 1 re 5 ae 36 4 bee i an ele 127} March 1 Intest.iGuv.)).52. 20 { + a ere = ra : is a 128} May 17 SOs Jat, (OMCD). 2 = o5- { a cae 34 3 ne _90.8 129} January 28 Liver and spleen. .. { : & eg 90.0 os 3 oh 4 Teg 130| January 28 Wiornmsts. 4422s. 4 ae { ai eae Ai a i as 431.3 Pure species—T. orientalis 131} March 8 itt livelier ae bi se ms Krai eS Ene 132} March 23 Cold (jaye eer if ai BP cag _95 0 ee AG 133) March 23 Cold Guvidiees Ss A We 25.0 ase = —27 6 134] March 23 | Cold (juv.)......... a nae ae Hes i ie Lie 135| March 25 Imtesta(juivs)- seen = < ea 42.9 segs = eae 1 Abbreviations of the names of organs to their first two letters, implies that ad- vanced and very evident tuberculosis was found in those organs (lungs, spleen, liver, joints, mesentery, intestine). Where more than one organ was affected the name of the organ (or organs) apparently most affected is written first. When the word is written out it denotes that this organ was abnormal, but not necessarily tubercular; immature birds are designated—(juv. ). 94 TESTES Or PIGEONS IN HEALTH TABLE 6—Continued AND DISEASE Q5 | PER CENT PER Pure species—St. risoria 136} December 4 | Sp. li. lu............ - s Ah te 90.0 eee | ORCL Lh. «Ascot \ : : a ~70.9 oe : e +14.1 meee 2) ppese lt... aera cL z G10 —50.0 66 : 20 +10.0 139) February 4 IMUCSU...< .\:. ocean - @ Fes _95 0 ies ; an Aaetig 140 March 15 ue Co) livers. a | z Hts 374 ie 5 eae Ga ; E aie —21.1 as = io0 142) May 14 Osea): sp.; liver... ¢ S ec _176 a 42.9 Miscellaneous—pure species 143} November 17 | Canker, li., sp. lu.. E = eae 499 144, November 17 | Fight and hemorr. . : Ez ee 95 3 145) November 18 | Unknown (juv.).... 5 iS Sate 410.5 146 November 20} Cold (?)...........- Beta 99 9 Bgpbecember 3.) Liver)... Fc. ee eae swe Common pigeons l 148) January 3 Unknown (juv.).... - = rae oe : ae _ 38 149) January 17 Unknown (juv.).... { = 3 ante a4 a . i 412.1 (| R = 0.004? about 6.0 150} February 15 | Li. pleura.......... Te ocunae 93 39 Res a eee { mm. long |+138.2 151) April 13 Hemorrhage, liver.. | a sath =40.0 ae Sai 152| April 17 Appar. healthy..... = ui vee aie Bae > ae 418.3 153) May 4 Weakling (juv.).... : x eat 50.0 ee Ono 96 OSCAR RIDDLE TABLE 6—Continued S = aE cENe LENGTH Guet on DATE DISEASE WEIGHTS ero AND WIDTH DIERE- Hybrids—from crosses of species - (} R = 0.000 4.2x0.9 Arv ? 7 2 January 1 ColdeGuv.)iisc..5-+ L = 0.000 5.2x1.2 423.8 January 4 Sp. liver mg Ole he Se, ea ie Picasa Nie Saag L = 0.017 421.4] 8.6x2.5 |+36.5 R = 0.000 Bees ye, 4) lt) January 8 (GiUHa) eee ae eee L = 0.000 es: 450.0 = R = 0.037 Se XS 2a January 15 | DF hee e encrecacs canta eae L = 0.035 =57| S450 ene 5S December oie | lalen (iilive) sores coe ae : Gi =e 00 aw sae ; = 0.027 January 15 So] Omencepees eee me ; So 35.0 January 27 Healthy dwarf; 1, = OKO) Kallledats ses ees L = 0.013(?) +30.0? Hebruaryals: |Spt lus... 22. fess i f ee - 00 = 2 March 6 Space ee. ci ie eee 4 je Hae oe Es R = 0.007 5.0x2.1 January 22 1 iS aes} Oe, Snes Aen ea L = 0.007 = 0.012 5.622.0 ereeD January 24 Lu., liver, spleen... ¢ = z tee ee ae : ce 47.2 = 2 sine}. January 27 Iijivere, Its (Ce ecocs cc : e sets aie fi He. i 126.6 February 13 | Lu., liver, spleen {| R = 0.002? 5.7 QUivVEs aera L = 0.0038+ 5 530)-00) |) te) + 3.5 os 22 March 17 SOsq Wels WAVE, oc co 5 % a ee Bier = 2) 7, 2 Megan SRP. : A aie eae ies Pe = : 4 March 26 Intest. (?) (juv.)... : iti as KG : 9 12.8 March 28 Sp. me. lungs...... < 4 et ae ate ae a af 4 2.0 : : R = 0.031 8.7 x29 Mareh 29 Tis sp, mee see sae \| TL = 0.035 412.9 | 12.2x2.2 |440.2 ' R = 1.015 22.6x 9.0 April 12 Coldmlhnest.. eee L = 0.888 244.3 | 93 5% 8.2 [gag sy R = 0.002(?) 5.2 April 20 (Gita eae b as asda L = 0.002(?) Seto | wae + 3.8 TESTES OF PIGEONS IN TABLE 6—Continued HEALTH AND DISEASE 97 | PER CENT wa Pe > NO DATE DISEASE WEIGHTS OF DIE- LENGIE, |GENTIOB FERENCE AND WIDTH DIETEE= ENCE Hybrids—from crosses of species (con. ) : . ; R = 0.054 10.8 a AK 1, oe oan ee 174) April 23 Liver (intest. ?).... \| L = 0.044 _99 71106 5G ‘e : ; ‘ R = 0.034 8.2 ay De Le Des i nee ee 175) May 6 p. li. pe Ms ene L = 0.032 ee ee ey, Ry— 702020 7.6 76) May 6 A eee : i P L = 0.022 +10.0| 8.0 5.3 Pelee hs R = 0.042 8.3 177| May 10 Li. spleen.......... ‘| L = 0.036 16.71 87 Se aig RF ="0;021 Ue 78} May NS} 0 gh Depa, ont ale E48) May 11 Pe L = 0.018 ~16.7| 6.6 ort = ; R = 0.125 1 Grxe4e5 179, May 16 Healthy (juv.)..... Ht, en os = 0.0| 13.2x43 1413.8 Hybrids—from crosses of genera Re OPO 7202-0 SpA his 3: Cae ae ae ete oer 4) 8p, 13 L = 0.020 F171 G 822.5 |e : 2 R= 02017 9 , 181); December 24 | Liver, pericard..... L = 0.020 417.6 R = 0.005 3.2 x18 ‘ 9 182) January 8 ColdkQ@ ren saee eee L = 0.004 5B 0-3 Oe 1.5 l= 66 R = 0.009 7.3 =1.8 ,12 7 183) January 12 Abdom. wall....... L =0012 433.31 5.7x1.9 |-28.1 R = 0.005? about 4.5 #84) February 8 | Intest...........:.. L = 0.0042 SOR EOH ARO nte Sale Ole R = 0.045 eS 5 cit (2) Nepcees ete Se ety 28. | Intest: (7) L = 0.051 SnOhan Ted S003 is (both teste|s an- 186) March 7 (Cold?) lungs...... - = sae 40.0 gular-glo|bular, like hem|p seed 3 K=O. 014 7.0 ? 187) March 13 IS Soe, Wt (RP) eo scee L =0.019 435.7| 9.0 498.5 R = 0.006 4.6x1.9 / ? 188) March 29 (Cause?) 5 aceeeeo: L = 0.007 +16.7| 5.3x2.0 |415.2 é : R = 0.256 15.7x5.6 189} April 12 Worms, fighting... . L = 0.212 = 99 eis ae eo 2 R = 0.047 8.8x 190} May 15 tas, May cae Be. L = 0.043 = 93 es 10.0 98 OSCAR RIDDLE Six generic hybrids here all show larger right testes. Of 24 birds listed in table 6, larger rights were found in 11, smaller in 5, equivalents in 8 cases. In table 6, 11 generic hybrids are listed; the right testis is larger in 5, smaller in 6. A summary representation of the weight relations of the testes of birds belonging to the several preceding groups is given in table 7. Relative lengths of the two testes The length of 78 pairs of testes was ascertained. These were obtained from the testes of birds belonging to all of the groups discussed in the previous seetion of this paper. The reader is referred to the summary on ‘length relations’ given in table 7 for a first view of the result. Although a high proportion (126 to 39 for all groups) of the right testes are heavier, a reversal of proportions (24 to 42) is found for the absolute lengths of the two testes. In five of the seven groups of table 7 the left testis is absolutely longer. In one of the two exceptional groups— TABLE 7 Summary of the preceding data WEIGHT RELATIONS! LENGTH RELATIONS CLASS STATE L+ L= iy = L+ L= L.— P aoe Healthy... 0 0 5 0 0 3 aia: Gece ag ae oi \| Diseased... 9? ie 31 14 2 2 Chennai Healthy... 5 0 2 1 0 0 ee ge re Diseased...| 2 0 8 2 2 1 SURE eon err Healthy... 6 7 19 8 6 7 Bat ae cura rte Diseased...| 10 | 11 | 24 | 15 1 4 Generic hybridse =. 54-96 Diseased... ia 2, 2 1 6 Total: healltity ae) prtannaee scene cee Til 7 51 9 6 10 otalidiseasedes Ata tarae rs aces | ec 18 74 33 6 14 Grand stotall.rasonee cree. < eee 39 24 126 42 12 24 The cases of larger left testis, are grouped under L+; those of equal size under L=; those with a smaller left testis under L—. 2 Five of the (9), and 3 of the (6), are from 14 Sp. tigrina—all dead at less than 9 months old. TESTES OF PIGEONS IN HEALTH AND DISBASE OY ‘healthy pure species’—it is possible that the smaller length of the left testis is connected with the nearest approximation to uniformity of smaller size in this group. In the second group— ‘diseased generic hybrids’—the testes show the greatest depar- ture in their weight relations from what is elsewhere the rule. An examination of the detailed measurements and percentage weight differences given in tables 4, 5 and 6 is even more con- vineing than the summary of table 7 on the point that the two testes definitely tend to assume two different shapes—the left to be thinner and more elongate, the right to be shorter and thicker. This difference in form is perhaps not without interest since the only persistent gonad in the female—that of the left side—is characteristically ‘thin’ and ‘long.’ The testis that develops on this side is similarly characterized as compared with its mate of the right side. Pure species and hybrids and the relative size of the two testes Table 7 facilitates an expression of a relation which seems to obtain between degree of hydridization on the one hand, and the number of departures from the usual situation—a larger right and a smaller left testis—on the other. There are good reasons for believing that common pigeons—mongrels of many breeds probably not even descended from a single good species—may rightly occupy a place in this classification intermediate to spe- cific hybrids and pure species. There js no question that the generic hybrids are more separated from the pure species than are the specific hybrids. Now, the number of gonad size rela- tions, that depart from the rule, arrange themselves in all of the seven groups of the table, precisely in this order of departure from purity of species. That is, the greatest proportion of larger left testes is found in the group most widely separated from a pure species (generic hybrids); and the two intermediate stages of crossing (specific hybrids and common pigeons) show each its appropriately smaller intermediate number of larger left testes. 100 OSCAR RIDDLE The influence of disease on the actual and relative size of the two testes The detailed data of the several tables and the summary of table 7 demonstrate that diseased birds furnish a higher pro- portion of Jarger left testes—violations of the more general rule— than do healthy birds. Further, the testes of pigeons suffer great reduction in several, or most, forms of disease. Now it probably happens that because of the great reduction in size of the testes in disease that this of itself has rendered the results of a few of the weighings of the smallest glands less certain.! An examination of the detailed data will, however, leave no doubt that disease is a very real and important reason for the observed differences. : It has been found well to designate the cause of death in most of the tables. Advanced tuberculosis is easily, and with much certainty, diagnosed in pigeons. It is the most common cause of death among the birds of our collection. All deaths from this cause are so designated in the tables. A careful com- parison of the size of the testes of birds dead of tuberculosis as compared with those dying of other or unknown causes will show that the gonads of the male suffer greatest reduction under this disease. This is certainly not true for the female gonad; a point upon which data are still being collected. In this connection Hatai’s (’15) observation of the effects of exercise on the size of the ovary and testis of the rat are of interest. Hatai found a like qualitative response—an increase—in both; but quantita- tively the response was quite different; the testes increased only 12.33 per cent while the increase in the ovaries was 84.33 per cent. The several sections of table 6 show that in pigeons the spleen and liver are more often affected by tuberculosis than are other organs. It is the spleen too that suffers greatest hypertrophy and most complete transformation under the disease. ‘This is true of both sexes. 1 Because of imperfect or unclean separation of the gland from body wall, and drying during weight, the greatest care will not always obtain perfect weights of the smallest of these glands. TESTES OF PIGEONS IN HEALTH AND DISEASE 101 Size relations of the testes in the common fowl In table 8 are given the few data we have been able to obtain on the Jungle fowl, common fowl, and the duck. Since these were—with two exceptions—healthy fowls even the few data indicate a situation different from that found in pure species of pigeons. Whether these data are really representative of these forms, cannot now be determined. Whether hydridization (or mongrelization) of these forms is responsible for the apparent predominance of the reverse of the situation found in pigeons is doubtful. They like the sparrows may normally possess a larger left and a smaller right testis. TABLE 8 Weights of testes of Jungle Fowl and common fowl | PER CENT | PER CENT NO. DATE WEIGHT OF DIF- NO. DATE WEIGHT OF DIF- | FERENCE H FERENCE Jungle fowl Common fowl R = 3.203 R= 3.734 eely o. os... . L = 4.217| 431.7 Aa rly bores... 5. { L = 4.219 +12.9 R = 4.700 R= 5.880 2 9 Via) 2) 1 lh a eee L =5.700 | +21.3 2 edly, 16s > .-.., { L = 7.000 +19.0 R = 4.635 R= 6.540 Bipealy S..: 24... i 4 Snot ea Sil ec bia, | eee { L = 5.610| —16.6 {| R = 6.305 R = 11.660 ey ehaby 1Os. 3. 5... paella AA SUV 22 oma! L = 12.815) + 9.9 R = 4.500 p R= 8.820 Sapealy 12s. ...... L=4.900| +89 5 | August 26.... { L = 7.5451 —16.9 ues R = 3.960 : R = 11.100 6 | July 16 (inj.') L = 5.105 | 128.9 @ || eer Pile oe be { L = 10.350/ — 7.2 ewe R = 1.665 7 | December 10 {| R = 0.235 29 1 : ae yee ny) \| L = 1.820 | + 9.3 (roup)..... \| L = 0.200] —17.5 8 | December 21 R= 0.590 (roup)ao.2 L = 0.635] + 7.6 Wild Duck 1 | (y’g) Novem- { | R = 0.023 2 | December 28 {| R = 0.040 ber 20.30... eit: = 0.027.) --17.4 (starved). . \ L = 0.040} 0.0 R = 8.3x3.2 | L=8.6x2).5 1 These cocks had been given a few injections of ovarian extract during the week preceding the days of killing and autopsy. 102 OSCAR RIDDLE SUMMARY The prevalence of atrophy of the right ovary in birds; the demonstrated differences in number of primordial germ cells in the two glands of the fowl; and the unequal—and opposite— size relations of the two adult gonads of the male, constitute a body of puzzling facts whose elucidation should contribute large- ly to our knowledge of the nature and basis of sexual difference. The right testis of the pigeon is normally larger than the left. In hybrid pigeons there are more exceptions to the normal size-relations of the two testes than in pure species. The num- ber of the exceptions seems to increase with the degree of hy- bridization (width of the cross); there being fewer in specific hybrids than in generic hybrids. The testes of pigeons suffer great reduction in size in disease— particularly in tuberculosis. It is probable that the right gland suffers greater reduction than the left. The left (persistent) gonad of the female does not suffer a similar reduction in tuber- culosis. Season is plainly not the cause of the differences and reductions noted in pigeons. The two testes of the pigeon are characteristically different in their dimensions. The left (like the left ovary) is thinner and more elongate. The right (represented in the female by atro- phied ovary) is shorter and thicker. In poultry the few data at hand fail to indicate a constant or decided predominance of size in either gland. Cold Spring Harbor, L. I., N. Y. June, 1916 LITERATURE CITED ALLEN, B. M. 1907 A statistical study of the sex cells in Chrysemys marginata. Anat. Ree., vol. 1, pp. 64-65. (Also Anat. Anz. vol. 30, pp. 391-399.) Erzouip, T. 1891 Die Entwicklung der Testikel von Fringilla domestica von der Winterruhe bis zum Eintritt der Briinst. Zeit. f. Wiss. Zool., vol. 52, pp. 46-84. FirKet, JeAN 1914 Recherches sur L’organogenese des glandes sexuelles chez les oiseaux. Arch. de. Biologie, T. 29. Harar, 8. 1915 On the influence of exercise on the growth of the organs in the Albino rat. Anat. Ree., vol. 9. Swirr, ©. H. 1915 Origin of the definitive sex-cells in the female chick and their relation to the primordial germ cells. Am. Jour. Anat., vol. 18. TANNENBERG 1789 Spielegium observationum circa partes genitales masculas avium. Gottingen. HYGIENIC CAGES FOR RATS AND MICE J. A. LONG Anatomical Laboratory, University of California TWO FIGURES The following is a brief account of cages recently constructed for the Department of Anatomy of the University of California for hous- ing colonies of rats and mice. In planning these cages the desirability was kept in mind of so designing them that not only might the animals be cared for conveni- ently, but the cages be easily cleaned and completely sterilized and the spread of infection prevented. Accordingly they were made entirely of metal: the sides and front of galvanized iron, the former preventing the direct passage of infection from cage to cage; and the lid, top, back, and floor of hardware cloth of }-inch mesh bound ‘with ‘strips of galvanized iron. They can be falcon apart, packed in a small space for boiling, and reassembled quickly without the use of any serews or bolts. All parts are interchangeable. The inside dimensions are: floor, 93 by 143 inches; height 11 inches; front 23 inches high. They are arranged in groups of 20 (4 rows of 5 each, fig. 1). Each group is supported by a rack made of iron pipe, and 4 pairs of angle irons on which the 4 rows of cages are hung. Below each row is placed a shallow, removable, galvanized iron pan intended to be filled with sawdust for receiving refuse falling through the bottoms of the cages. The racks measure 63 feet in height, 174 inches in depth, and 43 feet in width. There is a space of 94 inches between the lowest pan and the floor, and 3 inches between the floors of the cages and pans. If desired the racks can be continued upward to carry one or more addi- tional rows. Most of the details of construction can be seen in figure 2 which shows some of the cages taken down. It will be observed that the sides are suspended and in turn furnish support for the rest of the cage except the top. The sides are put into place by slipping the flanges on the upper edges into grooves (g gr fig. 2) for med by bending- under the edges of strips of galvanized iron (st). A projection (pr) prevents sliding the sides in too far. The ends of the strips forming the grooves are bent up and over the angle irons and are permanently fastened by means of bolts. These strips also have soldered to their upper sides grooves (lg) opening laterally formed by strips of metal bent in the form of a narrow trough. Into the latter slide the tops to which the lids are hinged by two rings. The backs when in place 103 LONG ate J. 104 HYGIENIC CAGES FOR RATS AND MICE 105 - a : oUBbeabadtesbnbagce z = 106 J. A. LONG rest on the flanges on the lower edges of the sides and against the front faces of the back flanges. The upper ends of the backs are held firmly because they pass behind the rear angle irons; at the lower ends pins (p) fit into holes in the bottom flanges (the pins can also be seen on the under side of the upper row of cages). The binding on the lower edge of each back is turned forward at a right angle and together with the flanges on the lower edges of the sides serves to support the floor. The latter are kept in place by two pins (p’) which fit into corre- sponding holes (fh) in the binding. The front is made of one piece of metal. One may be seen endwise resting on the edge of a tray. The ends are bent somewhat in the from of a letter S to form troughs which fit over the flanges on the front edges of the sides. In assembling the cages the sides are first put into place, then the backs, floors, front, seal top (with lid). It will be seen that the floors may be changed without disturbing the rest of the cage, or by remov- ing simply the front. A number of extra bottoms makes it possible to clean one set and have them ready to substitute for soiled ones every week. The other parts of the cages need cleaning only at longer intervals. For the cages used for rats, floors of 3 inch mesh are provided. It has been found in actual breeding that 4 and even 6 adult rats can be kept in one cage, and as many as 10 or 12 young rats raised to breeding age in single cages. The construction of this equipment was worked out by Prof. H. M. Evans and the writer with assistance from Mr. H. B. Foster, the University Engineer. THE GOLGI APPARATUS PERSONAL OBSERVATIONS AND A REVIEW OF THE LITERATURE ALWIN M. PAPPENHEIMER From the Department of Pathology, College of Physicians and Surgeons, Columbia University; and from the Marine Biological Laboratory, Woods Hole, Mass. TWENTY-TWO FIGURES CONTENTS |! +, JEMICUTROYe IOC) cert ee oa, er oe RI Reet = Oe A OS . eee 107 RIINOMENCLATUTE 5-15 dc ete es SN eRe nie ea ancl soe ene 109 LL 1 NEPETIESTIG Cy Caen Men het Bes eae Re ache. oor MUSA ge sf eV ee Pee 110 IV. The occurrence of the Golgi apparatus in various types of tissue cells: PeeNGly OUS WSSU C sa... (eee re cet eg Hr ok Seats. syste! c. ine 112 BN ON=meLVOUS LISSUCS “aren Se pire Men ee eee se nae Acne 114 at, Mpitheliial Gellscs see eee nar ilet Ode oat, Pee. ee 114 b. Connective tissue cells, cartilage, osteoblasts, odonto- blasts) striated, muscleyaellsy 22) oo oos00).s8 acs oo 127 CG ORS oe eres. pe AN RET ah ten hake yeti aoe 130 V2 Ehe Golgi apparatus in’ the cells of embryos................5%....-. 132 MitnbnerGolricapparatusin’ protozoa... esa bode leis ec lsc tl) See Te 133 VII. The Golgi apparatus under pathological conditions.................. 133 VIII. General considerations. Relation to centrosomes. Polarity. Physi- cal nature of the structures. Holmgren’s trophospongium theory.. 135 [2c ACHOENCLICE i031 NOIRE REE ys Se? A, ge eg 2 Ree SSR OME 138 I. INTRODUCTION It is rather extraordinary that there should be within every cell a structure as conspicuous as the nucleus, and sometimes surpassing it in size, the meaning of which is utterly obscure. One at least of the functions of the nucleus—its réle in heredity— is known to us. We have fairly definite ideas as to the rdéle of the centrosomes, and theories aplenty as to the part played by mitochondrial structures, and other types of granules. But as regards the structure to which Golgi has given the name ‘Apparato reticolare interno,’ we have learned only its appearance, its distribution in different types of cells, and its behavior dur- 107 THE ANATOMICAL RECORD, VOL. 11, No. 4 NOVEMBER, 1916 108 ALWIN M. PAPPENHEIMER ing cell division. One of the most recent papers on the subject— that of Kolster (102)—ends with the statement ‘‘These structures undoubtedly have a special significance, but we are ignorant of it.”’ The credit for the discovery of this intracellular organelle— if such it be—undoubtedly belongs to Golgi, and a large part of the work, including the working out of a fairly easy and satis- factory technique for its demonstration—has been done by Golgi himself, and by his pupils and co-workers, Veratti, Perroncito, Pensa, Negri, Gemelli, Brugnatelli and others. A number of papers dealing with the same structures have been published by Ramon-y-Cajal, who, indeed claims priority for their dis- covery over Golgi, and by his pupils, Sanchez, Fananas, Tello and others. Nussbaum, in Prague, has inspired a series of papers by his pupils (Weigl, Polyescynski, Bialschowska and Kulikowska) dealing chiefly with the appearance of the Golgi apparatus in the ganglion cells of invertebrates. Important papers have been published by v. Bergen, Deineke and by Kopsch, who discovered a new and very simple method for demonstrating the apparatus. The best and most exhaustive general review on the subject is that of Duesberg (48), before the XXVIII Meeting of the Anatomische Gesellschaft at Inns- bruck in 1914, and Cajal (31) in his most recent publication (15) which was not available at the time this study was begun, has contributed a most interesting critical survey of the entire field, and added many new observations. A whole chapter—largely controversial—is that contributed by Holmgren, whose views and their bearing I shall take up later. In this country only Bensley and Cowdry have made contributions to the subject. In reviewing the literature of the subject I found that but few workers, with the exception of Cajal and his pupils, had attempted to study the behavior of the Golgi apparatus under experimental conditions. I planned, therefore, to follow the modifications of the structure in the epithelial cells of the rat kidney, which might be produced by autolytic changes, secre- tory phases and toxie agents. The choice of material was un- fortunate. The Golgi apparatus of the cells of the renal tubules THE GOLGI APPARATUS 109 proved to be so atypical and variable in form that it was difficult to draw inferences from variations seen under experimental con- ditions. One was further handicapped by the difficulties and capriciousness of the impregnation method, as applied to this organ. In attempting to control the technique many other tissues were studied, and insofar as the observations made differ from those of previous workers, they are given below. Although I was unsuccessful in the main purpose of my study, it seemed that it might be useful at this time to collate the widely scattered and rather inaccessible literature, and to record my personal observations, insofar as they supplement or are at variance with those of other workers in this field. It. NOMENCLATURE Golgi (61), in his original communication before the Med. Chir. Society of Pavia in 1898, suggested the term ‘Apparato reticolare interno,’ and this term has naturally been adopted by all the Italian workers. Kopsch (103) proposed the term ‘Binnennetz’ as the German equivalent, but both of these desig- nations are open to the objection that the structures do not ap- pear in all types of cells, nor under all conditions, as a closed net. Ballowitz (4) in 1899 described a basket-like structure about the centrosomes of the cells of Descemet’s membrane, and sug- gested for it the name ‘Centrophormia.’ Later he recognized the homology of the ‘Centrophormia’ with the Golgi apparatus, and the term has not come into common use. The ‘Nebenkern’ of Platner (145) and la Valette St. George (106), and the ‘Zentral- kapsel’ of Heidenhain (68) in the sperm cells have been considered by some as related to or identical with the structures demon- strated by the Golgi technique. The terms, however, are not sufficiently inclusive to apply to the structures described by Golgi. Ramon-y-Cajal and his school who agreed with Holm- gren in regarding the apparatus as canalicular in nature—referred to it in their earlier publications as the Holmgren-Golgi apparatus. In his latest review, however, Cajal (31) recognizes the very doubtful identity of many of the structures described by Holm- 110 ALWIN M. PAPPENHEIMER eren with those brought out by the Silver methods, and there- fore refers to them more justly as the Golgi apparatus. Many of the German workers speak of the Golgi-Kopsch net or apparatus. Holmgren (90), who believes in the identity of the canaliculi described by him, with the structures put in evidence by the silver impregnation methods, uses the term ‘Trophospongium.’ Cowdry (42) and Bensley (14) speak of ‘canalicular apparatus.’ None of these terms appear to be entirely satisfactory. I shall, therefore, refer to the structures simply as the Golgi apparatus. Ill. TECHNIQUE The earliest studies of Golgi were made with a modification of his well-known silver chromate method. This gave capricious and inconstant results. Veratti introduced a modification, the essential feature of which was a fixation in osmium platinic chlorid mixture. Kopsch (103) in 1902 showed that prolonged immersion in 2 per cent osmic acid would demonstrate structures identical with those described by Golgi. The two methods now most commonly used are those of Golgi (66) and of Cajal (380), and, for the convenience of those to whom the original articles are not accessible, they are given here: The Golgi method is as follows: ii, TheiOmS LNomimelbin (CLOG aacke caso case deo sadsuwoucuhece sare 30 ce. Saturated solution arsenious acid (1%)........... 30 ce. Alcohola(QiOiiecaseeen ce aoe se eee 30 ce. 6 to 24 hours. LT Silver nitrate tore he we. woe terkre Gate, cee tes: 1 hour to several days III. Development: Hydroquinone.................. 20 gm. | Sodium Sul hitereees ae 1 gm. | b to abe Bornaalint hese eee 20 Lee wl Distilled water ad 1000 ce. j Wash in distilled water, dehydrate rapidly and embed in paraffin or celloidin. IV. Toning: Solution) Av Sodium hyposulip hitters see emeere 30 gm. Ammonium Sulphocyanate.................. 30 gm. Distillediwaters cs ..5 ees ee ee eee 1000 ce. Solution b.Goldrchloride: sana one eee eee 1% Use equal parts of ‘A’ and ‘B’. Tone to grey tone. THE GOLGI APPARATUS a | Veratti has devised the following procedure for ridding the preparation of silver precipitate after toning: ‘A’—Repeated washing in distilled water. ‘B’—Rapid passage through following solutions: (1) Potassium permanganate—0.5 gm. Sulphuric acid—l1.0 ce. Distilled water—1000 ee. (2) Oxalie acid—1% Wash in distilled water. Counterstain with alum carmine. The latest Cajal method differs from the Golgi chiefly in the use of uranium nitrate in place of the arsenious acid in fixa- tion. It is given as follows: I. Fixation: Uranium nitrate—1 gm. Formol—15 ce. > 9 to 11 hours Distilled water—100 ce. IT. Wash quickly IIT. Silver nitrate—1.5°%—30 to 40 hours IV. Wash quickly V. Reduce in Hydroquinone—2 gm. Formol—6 gm. Distilled water—100 ce. Add anhydrous sodium sulphite—0.15—0.25 gm. so that solution has a yellow color. VI. Dehydrate and embed in paraffin. VII. Toning: ‘A’ Sodium hyposulphite—3 gm. Ammonium sulphocyanate—3 gm. Distilled water—100 ce. ‘B’ Gold chloride—1% Use equal parts. The addition of 30 ee. of ethyl or methy] alcohol to the fixative is recommended by Cajal ('15), as advantageous in the case of nervous tissue. As counterstain I have found a dilute Giemsa solution to give the clearest pictures. A 1 per cent methyl-green solution may also be used, and it has been found possible to combine also the Altmann mitochondrial stain, as modified by Bensley.‘ The removal of the silver precipitate with permanganate and oxalic acid must be very carefully controlled, as it is easy to bring about a complete decolorization of the Golgi apparatus as well. 112 ALWIN M. PAPPENHEIMER Cajal and others have obtained the most constant results in the tissues of young animals. Because of the rapid occurrence of autolytic changes little confidence can be placed in the results obtained with tissues from human autopsies. Both the Golgi and Cajal methods are exceedingly capricious; the impregnation is rarely uniform throughout the entire block. The most delicate and important step in these photographic processes, according to several workers, is the initial time of fixation. Each type of cell has its optimum time of fixation, which must be determined experimentally. In many cells, however, as in the lymphocytes, spermatic cells, glomeruli of the kidney, this appears to-vary within wide limits. That, at least, has been my experience, and I have obtained identical pictures with fixation varying from 2 to 12 hours. The technique most recently advocated by Holmgren for demonstrating his Trophospongium is a fixation in trichlorlactic acid (6 per cent) and staining in a freshly prepared resorcin- fuchsin solution. The ‘canals’ take a purplish black color. Holmgren also gives methods which show the canals as colorless structures upon a stained background. IV. THE OCCURRENCE OF THE GOLGI APPARATUS IN VARIOUS TYPES OF TISSUE CELLS 1. Nervous tissue The first clear description of the structures is that of Golgi in 1898 (61, 62), in the spinal ganglion cells of Strix flammea (Barn-owl); in the same year, he made similar observa- tions upon the spinal ganglion cells of Mammalia; Veratti in 1898 found the same sort of structure in sympathetic ganglion cells. Since these early papers, the Golgi apparatus has been found to be present in many other types of nerve cells—the anterior horn cells (Golgi (66), Cajal (81)), the pyramidal cells of the cortex (Golgi (65), Legendre (107), Collin and Lucien (37), Soukhanoff (167), Cajal (31)), the Purkinje cells and other nerve cells of the cerebellar cortex (Golgi (66), Cajal (81)), of the olfactory lobe (Cajal (28) ), the ganglion cells of insects THE GOLGI APPARATUS LI3 (Bialkowska and Kulikowska (19) ), of the leech and earth- worm (Bialkowska and Kulikowska), Crustacea (Jawarowsky (98), Monti (127), Polenzynsky (146), of cephalopods (Weigl (180) ). The apparatus reaches its greatest complexity and size in the spinal ganglion cells of vertebrates, and these have, there- fore, been a favorite object of study. In adult vertebrates there is shown by the silver or osmic methods, a definite net- work of solid, tortuous varicose fibrils, which vary in thickness with different species. This network may completely or par- tially surround the nucleus and may be in contact with it in places. Where the threads cross or interlace, there are often nodular varicosities. In some species there is a sort of lobula- tion, into three or four partially separated skeins, and individual filaments may be given off from the main mass, and apparently end freely in the cytoplasm. In all cases the peripheral zone of cytoplasm is left free; at no point does the network, or any of its branches reach the surface. Monti (127), in the ganglion cells of invertebrates (crustacea, arthropods and cephalopods) found a simple apparatus in the form of curved filaments, often bifurcating or anastomosing, but not forming a closed reticulum. V. Bergen (10), working with the Kopsch osmium method, upon the spinal ganglion cells of the hedgehog, cat, rabbit, rat, mouse, and hen, found that not all the cells showed a complete reticulum as described by Golgi, some containing only short filaments, rows of granules or ring forms. Some of the filaments contained a central clear space, and these he interpreted as degeneration forms. This variation in the appearance of the apparatus in different cells in the same preparation v. Bergen interprets as indicating the transitory nature of these structures. He suggests that they are developed from granules, which range themselves into fila- ments, form more complex networks, and finally undergo central liquefaction with the formation of canaliculi. Other recent workers, however, using the newer methods of Golgi and Cajal have not confirmed v. Bergen’s theory, and ascribe the varia- tions to defective impregnation. 114 ALWIN M. PAPPENHEIMER Cajal (31), like v. Bergen, notes variation (or ‘modalities’) in the type of net occurring in ganglion cells of the same order and size. He strongly rejects the idea that these variations are due to irregularities in impregnation, since they may be found in adjoining cells at similar depths from the surface. The question has arisen as to whether the Golgi apparatus is identical with any of the other known cytoplasmic constituents of the nerve cell—namely, the neurofibrillae, the Nissl substance or the mitochondria. It seems quite certain, in spite of occa- sional statements to the contrary, that the Golgi net is unrelated to any of these structures. The net is not continued into the cell processes, as are the neurofibrillae, and the fibers of the net are much thicker and more varicose. By combining Kopsch’s method with Bensley’s aniline-fuchsin toluidin-blue stain, as Cowdry (42) has done, the Golgi net, Nissl bodies and mito- chondria may all be stained in the same cells, and their independ- ence of one another made obvious. It seems hardly worth while to go further into this discussion. 2. Tissue cells other than nerve cells a. Epithelial cells. The presence of a Golgi apparatus was first demonstrated in the squamous epithelial cells of Ammocoetes (Lamprey eel) by Marenghi (120) in 1903, and in Lumbricus by Ramon-y-Cajal in the same year. Since then it has been found in the corneal epithelium by Barinetti (6) and by Dein- ecke (47). The net is present in all layers. In the superficial cells the net becomes looser, and often almost entirely sur- rounds the nucleus, whereas in the rete mucosum, it lies at the superficial pole of the nucleus, and in the cells near the surface, only granular bodies are found. This change, therefore, accom- panies the aging of the cells, and is characteristic not only of corneal epithelium, but also of skin, oesophageal mucosa, the epidermis of the ducks bill (Deinecke, Kolmer (100) ).. Some of our preparations of the mucosa of the renal pelvis show a similar differentiation. A Golgi net has been found also in cells of the epidermal ap- pendages and glands—in the lachrymal gland by Ancona (3) THE GOLGI APPARATUS 115 and in the sweat and sebaceous glands by v. Bergen (10), by Bizzozero and Bottisella (21) (who picture a net surrounding the nucleus, and also, incidentally, could not demonstrate it in the epidermal cells), and by ‘Tello (172). Since the first paper of Ballowitz (4) in 1898, a net has been found in the single layered epithelial cells of Descemet’s mem- brane by Totsuka (174), Zawarzin (184), and by Deinecke (47). The net in these cells is of special interest because it very clearly hes in relation to the centrosomes, and because it was discovered independently of Golgi’s work by Ballowitz. Deinecke in these cells, also, made a careful study of the behaviour of the net dur- ing mitosis. Numerous observations confirm the presence of a Golgi ap- paratus in the glandular cells of the gastro-intestinal tract. Thus Ramon-y-Cajal (’03) found it in the intestinal epithelium of lumbricus, and of the guinea pig (27); v. Bergen (10) in the chief cells of the fungus region (04), Golgi (67) in the gastric and intestinal mucosa of frogs, birds and mammals, in the glands of Brunner and of Lieberkuhn (’09); d’Agata (48) in the gastric epithelium of triton (10) and in the gall-bladder epithelium of the guinea pig (44); Weigl (180) and Kolmer (100) in the gastric and intestinal mucosa of various vertebrates; Kolster (102) in the chief and parietal cells of the fundus, in the pylorus and in the cells of Brunner glands (713). Kolster has made several interesting observations on the behaviour of the Golgi apparatus in the gastric cells. He found that when the chief cells were successfully impregnated the parietal cells were not. He also showed that, by using the original Golgi silver chromate method, it was possible to im- pregnate a system of endocellular excretory canals in the chief cells of the fundus, and that these differed, both in their topog- raphy and in their form from the true Golgi apparatus. He noticed also in the pyloric gland cells that the appearance of the net varied with different phases of secretion. In the resting cells the net was quite dense, the meshes small, the form of the whole mass spherical; while in secreting cells, the net was rare- 116 ALWIN M. PAPPENHEIMER fied (aufgelockert), elongated and extended to the basal portion of the cell, in close contact with the flattened nucleus. Cajal (31) also describes in great detail a cycle of changes corresponding to different secretory phases in the goblet cells of the alimentary tract. The Golgi apparatus during the earlier phases undergoes an increase in size, later the argentophile substance becomes dispersed amongst the globules of secretion, and completely disintegrates—not as Kolster (102) believes, becoming merely compressed against the nucleus at the base of the cell. The inferences which Cajal draws as to the functional signi- ficance of these cyclical changes, will be discussed later. Before having access to Cajal’s paper, I had independently observed similar alterations in the mucous glands of the larynx (figs. 1, 2, 3). It seems to be quite clear that the net, which is more distinct and well-formed in cells during the inactive stage, becomes broken up and distributed amongst the globules of mucus in those cells which are actively secreting. In the course of this process, there appears to occur a real quantitative decrease in the amount of the argentophile substance not to be explained merely by its mechanical disruption, and implying some sort of regeneration of the apparatus, after the cell has discharged its secretion and returned to rest. A Golgi net has been demonstrated by numerous observers in the epithelial cells of various glandular organs, and I shall limit myself merely to giving a list of these. The Golgi net was described in the thyroid by Negri (180) and by Kolster (102); in the adrenal medulla by Pensa (135) and Kolmer (100), and in the cortex by Pilat (144), by Mulon (128) and by Kolmer (100); in the anterior lobe of the hypophysis by Gemelli (60), and in both glandular and nervous portions by Tello (172); in the pancreas by Negri (130), by v. Bergen (10) and by Kolster (102), Kolmer (100) and Cajal (31); in the dog’s prostate by v. Bergen (10) and in the hypertrophied human prostate by Verson (179) and by Taddei (171). Von Bergen claims to have been able to recognize the net in unstained scrapings of pros- tatic epithelium kept alive for a time in the prostatic secretion. THE GOLGI APPARATUS Piz This appears to be the only recorded attempt to observe the Golgi apparatus in the living cell. up iP @ ¥s eRe ‘ep fi Aan & oe ab ) ‘ ( BS tm “4 Fig. 1 Tracheal gland, non-secreting. Cajal. Figs. 2 and 3 Tracheal glands, showing fragmentation of Golgi apparatus during secretion of mucus. Fig. 4 Small group of thyroid epithelial cells, one in mitosis. Dittokinesis. Cajal. Fig. 5 Salivary gland of rat. Cajal, anilin-fuchsin-methyl-green. Note relation of Golgi net to Altmann granules. 1 We also have tried to observe it in growing chick embryo cells in vitro, both by direct light and using dark-field illumination, but without success. Lewis and Lewis (109) likewise report their inability to see structures corre- sponding to the Binnennetz in living chick embryo cells, nor could they be brought out by prolonged osmie acid fixation. 118 ALWIN M. PAPPENHEIMER The net has been found further in the epithelial cells of the epididymis by Negri (130), by Fusari (08), by Kolster (102) and by Kolmer (100); in the ciliated epithelium of the trachea by Kolster (102); in the choroid plexus by Biondi (20) and in the uterine mucosa and chorionic epithelium by Decio (46) and by Acconti (1). Negri (130), Kopsch (103), v. Bergen (10), Kolster (102) and Kolmer (100) and Cajal (31), record the presence of a net in the salivary gland epithelium. My preparations show one or two points which I do not find mentioned in their descriptions. I find in some acini remarkably large, coarse-meshed nets, enveloping the nucleus, joining by stout, varicose filaments with nets in adjacent cells, and not infrequently giving origin to trabeculae which loop about the lumen of the gland. They are thus not confined to a single cell, and anastomose freely one with another. By counterstaining the Golgi preparations by the anilin- fuchsin-methyl green modification of Altmann’s method, one can clearly recognize the independence of the net from the Altmann granules, which are evenly distributed through the en- tire cell. Indeed, it seems as if these were an inverse relation— that is, those cells in which the granules are poorly marked and absent, show the most conspicuous and clearly defined net, whereas the large cells which are replete with granules, may contain no net at all. Whether this is a constant relation or not, remains to be seen (fig. 5). It is surprising that the literature should contain but two references to the presence of a Golgi apparatus in the liver cells. Stropeni (169) is the only one who has succeeded, and he stated that in mammalian livers he obtained only a partial impregna- tion. With the livers of lower vertebrates, namely frogs and amphibians (Axolotl) he was more fortunate. He found the net to be definitely localized to the portion of cytoplasm between the nucleus and the bile-canaliculi, occasionally sending prolonga- tions into the rest of the cytoplasm; no continuity with the bile canaliculi could ever be observed, nor was there any striking difference in the appearance of the net in fasting or well-fed animals. THE GOLGI APPARATUS L19 Kolmer (100) says that he sueceeded but rarely in demonstrat- ing a net in the liver cells. In a new-born cat, the liver cells contained a simple juxta-nuclear net consisting only of several meshes or sometimes of single polygonal nets with one or two long processes. ‘They had no constant relation to the nucleus. We also have tried repeatedly to find a Golgi apparatus in the liver cells of rats, and have been almost uniformly unsuccess- ful with the Golgi or Cajal technique. In only one preparation were there found discontinuous curved filaments distributed through the cytoplasm, and bearing little resemblance to the complex reticulum found in other epithelial cells. By prolonged fixation in 2 per cent osmic acid, one may, however, demonstrate very clear-cut intracellular filaments and rows of granules, often eurved and occasionally branching, but never uniting to form a definite network (figs. 6, 7, 8, 9, 10, 11). These filaments may le against the nuclear membrane; in a few instances they appear to join filaments in neighboring cells; in no case do they connect with the bile canaliculi, nor do they appear to reach the surface of the cell. Whether these structures are the homo- logues of the Golgi apparatus in other cells, I am unable to say with certainty. Their resistance to impregnation by the usual methods implies some chemical variation. They disappear rapidly during autolysis and are absent in cells injured by chlo- roform poisoning. Surprisingly few workers also, have concerned themselves with the Golgi apparatus as it appears in the kidney. Brugnatelli (25), using the Golgi arsenic method has described a net in the cells of the tubuli contorti and of the tubuli recti of the guinea pig, which coincides perfectly with that of other epithelial cells, especially as regards its localization between the nucleus and the lumen of the tubule. In the collecting tubules the apparatus was much more complicated and definite than in the cells of the convoluted tubules, in which it invariably presented itself as small, very simple, almost simulated (..e., ‘aecenata’). It seemed, he says, as if the more complex structures here (basal-rods, granules) were harmful to a clear-cut demon- stration of the reticular apparatus. 120 ALWIN M. PAPPENHEIMER 4% 26 a a agereec go¢ * A! F Figs. 6, 7 and 8 Liver cells of rat. Kopsch, 2 per cent osmic acid, 12 days. Fig. 9 Liver cells of rat, autolysed 3 hours at 37°. Kopsch. Fig. 10 Liver cells, rat. Cajal. Fig. 11 Liver cell, rat, vacuolated with localized juxta-nuclear Golgi ap- paratus. Cajal. THE GOLGI APPARATUS 121 In the glomeruli, the apparatus is reduced to its lowest terms— sometimes appearing as a simple nodule, or as a small figure 8, and always lying adjacent to the nucleus. Brugnatelli gained the impression thai the net was restricted to the cells of epithelial origin forming the visceral layer of Bowman’s capsule. He is quite wrong in this, as the endothelial cells and the parietal cells of the capsular space also contain an easily demonstrable net (fig. 12). Barinetti® (12) describes and pictures a rather complex net in the renal epithelium, and shows its relation to the centro- somes by comparing it with impregnations in which the centro- some is stained by Benda’s method. He omits, however, to mention the portion of the renal tubule to which he refers. San Giorgi (156) studied the alterations of the Golgi apparatus during experimentally produced nephritis in guinea pigs. He used as toxic agents, uranium nitrate, cantharidin, ricin and diphtheria toxin. The modifications observed were a splitting up of the fila- ments or a granular fragmentation without complete loss of the reticular character. Such modifications were most clearly observed in the tubuli recti of the medulla, in which the net, as Brugnatelli showed, is normally more voluminous and com- plete than in the cells of the convoluted tubules. Close examina- tion showed relation between alterations of the cells as a whole and of the Golgi apparatus. The fragmentation of the net may be marked in some cells of the tubules, whereas others may contain a normal net. One may criticize San Giorgi’s work because of the fact that none of the poisons used produce obvious changes in the cells of the tubuli recti. Kolmer (100) briefly records the presence of a net in various elements of the kidney, but gives no detailed description. We have made numerous preparations of rats’ kidneys, both of normal animals and of animals in which a uranium nitrate nephritis had been produced. We have varied the time of fixa- tion from two hours (as recommended by Brugnatelli) to twelve hours, without obtaining striking differences. We have also 122 ALWIN M. PAPPENHEIMER AIT ee pel ye 5 THE GOLGI APPARATUS 123 studied, though incompletely, the effect of autolysis, and found that changes were discernable only after an hour at 37° when the kidney was removed from the body immediately after death. A kidney removed from an animal one hour after death, showed about the same type of structure as the freshly fixed tissue. The apparatus, therefore seems to be somewhat more resistant to autolytic change than the mitochondria, which after half an hour at 37° were broken up into coarse droplets. As regards the appearance of the Golgi apparatus in the cells of the convoluted tubules, my preparations do not coin- cide at all with those of Brugnatelli (25) or San Giorgi (156). The argentophile structures in these cells take on the most bizarre and varying forms. One finds smaller and larger drop- lets or granules, rings or signet forms often ending in a delicate filament, curved threads, uniform in ealibre, or with nodular thickenings and varicosities; and larger, more complicated skeins approaching the reticulum described in other types of epithel- lal cells (figs. 13, 14, 15, 16). The location of these structures with respect to the nucleus is as variable as their form. Their most frequent site is perhaps at either side of the nucleus, some- times in contact with it; the filaments in general tend to run at right angles to the basement membrane. Sometimes one finds a cluster of granules and short filaments in the supra-nuclear zone, very rarely between the nucleus and the basement mem- brane. The appearance varies also from one tubule to another in the same preparation; some tubules may show predominently irregular twisted threads and skeins, others only granulae of uniform or varying size. The appearances are so bewildering in their variety that it is difficult to draw any conclusions, and we are still experimenting with the technique in the hope of get- ting more constant pictures. We have about decided, however, that the Golgi apparatus in the convoluted tubules is not the Fig. 12 Rat kidney. Glomerulus. Cajal. Fig. 13 Rat kidney. Proximal convoluted tubule. Cajal. Figs. 14, 15 and 16 Rat kidney removed one hour after death. Convoluted tubules. Golgi. Fig. 17 Rat kidney. Large Henle tubule. Impregnation of basal fila- ments. Golgi. THE ANATOMICAL RECORD, VOL. 11, No. 4 124 ALWIN M. PAPPENHEIMER clear-cut definite structure which Brugnatelli depicts, but is normally fragmentary and dispersed. We have not been able to prove that these variations in form are correlated with dif- ferent phases of secretion. In the Henle tubules, the net is obscured by a very constant impregnation of the Stiibechen. Sometimes, however, we can distinguish a small, very dense, supra-nuclear network, and occasionally filaments are continued along the lateral aspects of the nuclear membrane (fig. 17). In the cells of the collecting tubules also, a net has been fre- quently seen, although in the rat’s kidney it is a looser and less complex structure than that. described by the Italian workers. In the glomerular cells, both epithelial and endothelial, the small juxta-nuclear net is constantly found, and very sharply defined and striking (fig. 12). In the uranium nitrate kidney I have made only a few observa- tions which seem worth mentioning at this time. In the first place, the more complicated filaments and skeins, usually evident in the normal tubules, tend to disappear entirely in the injured kidney. Even those cells, which in sections counterstained with Giemsa, show little or no obvious damage, rarely show structures other than granules or irregular black or grey-staining clumps. In the totally necrotic and desqua- mated cells, one often finds a single coarse black clump possibly representing the remains of the argentophile structure. In many of the injured cells I find oval or circular greyish bodies of varying size, many of which contain one or two ec- centrically placed black granules (fig. 19). Now starting with these, one can trace transitions both to small solid black gran- ules and to large droplets which have entirely lost their affinity for the silver-stain, taking the eosin of the Giemsa intensely, and resembling in every way the familiar hyaline droplets of degenerating renal cells. These largest droplets accept the acid fuchsin in the Altmann-Bensley stain, but I have gained the impression that they arise from the argentophile droplets rather than from a breaking-up and fusion of the mitochondrial structures. THE GOLGI APPARATUS 125 Fig. 18 Rat kidney. Two small collecting tubules with delicate supranu- clear filaments. Cajal. Fig. 19 Rat kidney. Uranium nitrate, Nephritis. Proximal convoluted tubule containing hyalin droplets with argentophile granule. Cajal. Fig. 20 Frog’s kidney. Tubules showing various types of argenthophile structures. Cajal. 126 ALWIN M. PAPPENHEIMER Fahr (52) has also recently made the observation that this type of ‘gross-tropfige’ degeneration may be produced experi- mentally in the rabbit’s kidney with uranium nitrate, and is disinclined to derive the droplets from the mitochondria of the cell. The appearances observed. in the epithelial cells of the frog’s kidney are also very puzzling and difficult to interpret. In the glomeruli, a concentrated juxta-nuclear mass is present in all the cells, identical with that described in the rat’s kidney. In the proximal portion of the convoluted tubule there is found to either side of the nucleus, but rather nearer the distal pole, a small irregular, granular filamentous or ring-shaped mass, which is quite definitely the homologue of the Golgi appara- tus of other cells. Such an epithelial cell, cut in a plane parallel to the basement membrane, shows the nucleus surrounded by a ring of discrete masses, which do not form a continuous skein, but are interrupted (fig. 22A). In frogs injected with trypan-blue this type of apparatus is present in the cells containing the granules of dye, which in general occupy the supra-nuclear zone of the cytoplasm. The dye granules and the argentophile bodies are quite unrelated. In some of my preparations, the cell boundaries are sharply impregnated by the silver, appearing as delicate blacklines. The striated border is also sharply brought out (fig. 22). In another part of the tubule, probably the distal portion of the convoluted tubule or Schaltstiick, which contains no blue staining granules, the argentophile bodies are in the form of rounded globules or granules, varying slightly in size and in- tensity of staining, and occupying a zone in the middle nuclear plane (fig. 22B). The homology of these granules to the Golgi apparatus is not clear, and it is possible that they represent ex- cretory substances of some sort, possibly chlorides or phosphates (Leschke (109) ). Finally, in still another portion of the tubules (corresponding to the Henle loop) there is obtained an excellent impregnation of the basal filaments and mitochondria (fig. 22 C). The cytoplasm in the supranuclear zone is somewhat more intensely stained, THE GOLGI APPARATUS 137 but no definite structures comparable to the Golgi apparatus of other cells is brought out. This deseription corresponds to the appearances usually observed in the frog’s kidney. Some of our preparations, how- ever, show curious structures of very different type, the nature of which is entirely obscure. These are limited to certain por- tions of the secretory tubules—probably the distal convoluted portion or Schaltstiick, although it is not possible to be sure of this. They consist of sheaves of filaments, often of great length, sometimes beaded or with nodular varicosities. They run either perpendicular to the basement membrane, along the lateral aspects of the nucleus, or in some instances, lie above the nucleus and have a course more or less parallel to the basement mem- brane (figs. 20-21). These bundles of fibrils do not appear to form closed skeins or to anastomose with one another, but overlie and cross. The individual fibrils are often irregularly fusiform, with tapering ends. They may be quite rigid, almost crystalline in appear- ance, or more wavy and filamentous. Scattered amongst them are small isolated clumps and granules of varying size. b. In ordinary connective tissue cells. A small juxta-nuclear apparatus has been described by v. Bergen (10), Cajal, Deinecke (47) and Kolster (102); in endothelial cells by v. Bergen (10) and by Cajal (28); in smooth muscle cells of blood vessels by v. Bergen (10); and in various types of wandering cells by v. Ber- gen (10), Verson (179) Maccabruni (115), Barinnetti (6) (Plasma- cells—relation to centrosome, 712) and Fananas (55). In cartilage cells, Pensa (136) first described a distributed apparatus which he considered to resemble the diffuse net found by Golgi in the ganglion cell. Later v. Bergen showed that the Golgi apparatus in cartilage cells, as in most other non-nervous elements, was limited to the juxta-nuclear region, and inter- preted Pensa’s diffuse apparatus as a chondriom. Pensa (137) has accepted this correction, and v. Smirnow (165), Barinetti (6) and Kolster (102) are in agreement with v. Bergen on this 128 ALWIN M. PAPPENHEIMER Fig. 21 Frog’s kidney. Tubules showing argentophile filaments. Golgi. Fig. 22 Frog’s kidney. A—Proximal convoluted tubule. Golgi apparatus in the form of discrete peri-nuclear filaments, rings and loops. B—Tubule containing argentophile droplets. C—tubule showing impregnation of basal- rods. THE GOLGI APPARATUS 129 point. Comes (40, 41) still claims that Golgi net and mito- chondria in cartilage cells are identical structures. The controversy is interesting, because it has brought out the point that the silver method is not always specific, and that under certain conditions the mitochondria may be impregnated. This occurs regularly, as I have mentioned, in the Henle tubules of the kidney. Both Pensa (136) and Cajal (31) have deseribed an interesting series of changes in the zone of growing cartilage adjoining the line of ossification. Following the enlargement of the cells the net loses its localized character, hypertrophies and finally, with the degeneration of the cartilage cell, undergoes granular disintegration. Cajal (81) seems to have been the only one to study the be- havior of the Golgi apparatus in osteoblasts. During the period of functional activity, the net is very well developed, and large, usually occupying that part of the cell directed towards the osteoid tissue. In the finished bone corpuscle, the net shrinks to a small compact mass. Teeth. Although Massenti (123), using pig embryos, had previously described a Golgi apparatus in the form of a large skein occupying almost the entire cytoplasm of the pulp cells and odontoblasts Cajal (31) depicts structures of a more typical character and localization. As the odontoblast becomes differ- entiated from the connective tissue elements of the pulp, the net increases in size, and comes to form a large oval rather granu- lar mass, occupying the supra-nuclear portion of the cytoplasm. The further fate of the structure could not be followed, since decalcification interferes with the reaction, but Cajal regards the hypertrophy during the secretory phase of the odontoblast as another example of the cyclical changes seen in goblet cells, growing cartilage cells, ete. Muscle fibers. There are a number of papers dealing with the endo-cellular reticulum of striated muscle fibers. The earliest is that of Cajal (26), in which he found in the wing mus- cle of certain insects a network continuous with the ramifica- tions of the tracheal tubes, and therefore probably a true cana- 130 ALWIN M. PAPPENHEIMER licular system. This was confirmed by Fusari in 1894 for mam- malian muscle and by Veratti (178) in 1902. Veratti, however, denied that the endocellular apparatus in insects represented a continuation of the tracheal tubes, and regarded it as com- posed of solid filaments. Sanchez (155), a pupil of Cajal, con- firmed the tracheal origin of the reticulum in insects, looking upon it as a tubular apparatus, probably of importance for the nutrition of the fibers. The system is composed of transverse meshes on either side of the ‘bandes claires,’ united by longitudi- nal connections. Two other recent papers, one by Martinotti (122), the other by Fananas (54) have not been available. Quite different is the apparatus described by Luna (113) in the cardiac muscle fibers. Here he finds granules, rods, curved filaments or more complete nets lying at one or both poles of the nucleus. He believes that the granules and rods might possibly be mitochondrial. c. Gonads. The studies upon the gonads are of much more theoretical interest, because of their bearing upon the question as to whether the Golgi apparatus is a permanent structure, and a heritable constituent of the cytoplasm, or whether it is more ephemeral in character, and related to the vegetative activities of the cell. It is also in the sperm cells that the topographical relation of the net to the centrosphere is most evident, so that the structure during spermatogensis might be expected to show interesting modifications. Platner (145), a number of years before the discovery of the Golgi apparatus, had described a structure surrounding the cen- trosomes of the spermatogonia, which he called a Nebenkern. Heidenhain (68) in 1900, in the sperm cells of Proteus, found with the iron hematoxylin stain, an incomplete basket-work surrounding the centriole. Heidenhain used the term Zentral- kapsel or pseudo-chromosomes for these bodies, and believed that they were formed by special differentiation from the Benda mitochondria. This view finds support in the recent observations of Chambers (33) upon spermatogenesis in the grasshopper, in THE GOLGI APPARATUS 131 which the mitochondria, vitally stained with Janus green, ap- peared to enter directly into the formation of the Nebenkern. Sjovall (164), using a modification of the Kopsch method, demonstrated these structures in the spermatocytes, spermato- goma and spermatids of the white mouse, and concluded that Heidenhain’s view of their mitochondrial origin was erroneous. Benda, and especially Weig! (180), have also taken this stand. SjOvall also found a net in the Sertoli cells. Perroncito (140, 141), using the Golgi method, carried the observations of Sjévall a step further, by describing the altera- tions of the net during the maturation divisions. He finds in Paludina, that in the prophase, the net breaks up into granu- lar fragments, which he calls dittosomes, and which are dis- tributed equally to. the two daughter cells. The skeins are then reformed from the granules, and in the spermatids come to occupy their usual juxta-nuclear position. This process of fragmenta- tion and distribution during mitosis he calls dittokinesis. The fate of the Golgi apparatus in the adult spermatozoa is unknown, nor has it been established that the substance of which it is composed enters the egg during fertilization. The phenomenon of dittokinesis, however, is established, not only for the sperm cells, but also for the somatic cells (fig. 4). Dein- ecke (47) (12) has given a very clear description of this process in the flat cells of Descemet’s membrane, where the net forms in the resting cell a thick skein of interwoven and anastomosing threads. During karyokinesis the skein becomes looser and gradually surrounds the nucleus, the individual threads grow thicker, loose their connection and break up into unequal bent fragments which are heaped up at the poles of the nucleus. Dur- ing anaphase they change into short thick rods and granules surrounding the nuclei, and lying chiefly in the equatorial plane. The size of these dittosomes is only slightly smaller than that of the chromosomes, their number somewhat greater. With the formation of the diaster, the dittosomes surround the daughter chromosomes, being more densely aggregated at both poles. The region of the spindle remains free of them. 132 ALWIN M. PAPPENHEIMER The new nets are formed by a fusion or sticking together of the granules or rods, but may remain discreet for a time. In this way, it is possible to recognize a recent mitosis, even after the nuclei have reformed. During the monaster stage one sees often a pairing of the gran- ules and double rods. Whether this indicates a splittmg of the dittosomes comparable to that of the chromosomes, Deineke leaves undecided. At any rate, the above series of changes unquestionably leads to an even distribution of the mass to the two daughter cells. Studies of the Golgi apparatus in the female gonads have been made by Sjévall (164), Weigl (180), Cattaneo (32), and Kulesch (104), and Weigl (180) and Hirschler (70) in the ovo- cytes of invertebrates; in the primitive germ-cells of 3-4 day chick embryos by v. Behrenberg-Gossler (16). All these writers are in substantial agreement as to the main facts namely, that in the young ovocytes and in the follicle cells, there is a circum- scribed net at one pole of the nucleus, which in the ripe ovum breaks up into filaments and granules (or, according to Kulesch, small irregularly angular rings, bent threads and dises) which are with difficulty distinguished from the mitochondria and other cytoplasmic granulations. V. THE GOLGI APPARATUS IN EMBRYONAL CELLS The recognition by Golgi (64), Fananas (54) and Cajal (81) that the Golgi apparatus is present in all types of cells, even at a very early stage of development (chick embryos of 30-40 hours—Cajal) seems to establish firmly the principle that the structure is an important and constant component of the cell. In many of these fetal cells—the mesenchyme—the endothelium of the pericardium and of the primitive blood spaces—the cells of the Wollfian ducts and the entoderm of the intestinal tract, the neuroblasts, and even the erythroblasts and wandering cells, the apparatus is highly typical and constant in its relation to the centrosphere. The attempt has been made by Fananas to trace the develop- ment of the net from granules and batonnets in the cytoplasm. THE GOLGI APPARATUS 133 As Cajal (81) points out, however, the not infrequent impregna- tion of the mitochondria and of skeletal and sustentacular struc- tures in early embryonic cells makes such an interpretation doubtful. One general principle can be deduced from a study of the Golgi apparatus in embryonic tissue, and that is the definite polarity of the structure in all fixed non-mobile cells. The location of the net in every case, as Cajal has pointed out, is such that it occupies the ‘external’ part of the cell,—that is, the portion above the nucleus directed originally towards the free surface, and opposite that pole which is towards the interstitial tissue and the nutrient supply. This polarity is preserved in adult life in the case of epithelial cells lining ducts and cavities, but may be lost in the case of solid glandular organ or tissues which undergo profound modification and derangement during development. The significance of this polarity which would seem to be bound up with the relation of the Golgi net to the centrosphere, is by no means clear, but it seems to be one of the most fundamental and most striking characteristics. VI. THE GOLGI APPARATUS IN PROTOZOA The literature contains but one reference to the occurrence of the Golgi apparatus in Protozoa. Hirschler (70) describes in the cytoplasm of Monocystis ascidiae, a Gregarine parasite of the ascidian Ciona intestinalis, diffusely scattered ring and half ring forms, demonstrable by prolonged exposure to 2 per cent osmic acid and resistant to turpentine (Sjévall’s modifica- tion of the Kopsch method). Whether these structures are the homologues of the Golgi apparatus of the metazoan cell, needs further study. VII. THE GOLGI APPARATUS UNDER PATHOLOGICAL CONDITIONS The modifications which the Golgi apparatus undergo under pathological conditions have been little studied, and, so far, have not added any new suggestions as to the real nature of the structure. 134 ALWIN M. PAPPENHEIMER In the cells of malignant growths, Golgi nets, often atypical, have been found by Moriani (125) in a human breast carcinoma, by Veratti (177) in a transplantable mouse cancer; by Lucioni (112) in a naevus; by Savagnone (157) in carcinoma of the breast, in a sarcoma of the jaw and in a giant-celled sarcoma; by Tello (172) in carcinoma and adenoma, in epithelioma and in experi- mental granuloma caused by Kieselguhr injections. Tello studied especially the distribution of the net in foreign body giant cells, where there are multiple nets—one usually in rela-. tion to each nucleus. In tuberculous giant cells, on the other hand, as shown by Fananas (55), the net is usually centrally placed, in relation to the multiple centrosomes. Regressive changes (fragmentation, pulverization, etc.) in the Golgi net have been described by Fananas (55) in caseating giant cells; by Marcora (117) in the ganglion cells of the hypo- glossal nucleus, following avulsion or section of the nerve; by Battistessa (8) in the ganglion cells of animals poisoned by lead or strychnin; by San Giorgi (156) in toxie nephritis, and by Del Rio Hortega (97) in the ganglion cells of a case of paralytic rabies. Very interesting are the recent experimental studies of Cajal (31) dealing with the effect of traumatic injury of the nerves upon the Golgi apparatus. An incision of the central nervous tissue brings about com- plete destruction of the net only in those ganglion cells most severely injured by the trauma. The apparatus of cells near the line of incision, though perhaps slightly compressed or de- formed, shows no grave disorganization. This would indicate a considerable fixity of structure, and firmness of texture, since, were the impregnated substance of fluid consistence, one would expect to find it dispersed through the cytoplasm or confluescing into larger droplets. Cajal has further established the fact that section of a periph- eral motor nerve had no effect upon the structure of the Golgi apparatus in the central ganglion cell. There does occur a de- generation of the apparatus in the cell of the sheath of Schwann, distal to the section. THE GOLGI APPARATUS 135 There are thus very few controlled studies of the alterations of the net under experimental conditions, and if seems that something further should be added to our knowledge in that way. The great difficulty had been, and will be, the capricious behavior of impregnation methods; until simpler and more relia- ble methods shall have been discovered, the interpretation of slight variation in the morphology of the structures will always be open to considerable suspicion. VIII. GENERAL CONSIDERATIONS This completes the list of cells and tissues in which structures of this type have been demonstrated. They may be considered as universally present in every type of cell, although the variety of form which they assume at once brings up the query as to whether they are all homologous structures. That, I think, is almost impossible to answer until we know something of their function and significance. Morphologically there seems to be no single character by which we can group them altogether. The staining reactions are probably not entirely specific; we have found instances—as for example in the Henle tubules and in cartilage cells—in which mitochondrial structures are more or less regularly impregnated by the silver methods. In many types of cells in which the location of the centrosome is known, there is, as Barinetti (6) insists, a topographical rela- tion between Golgi net and cytocentrum. But such a relation cannot be established for the ganglion cells, in which the net completely encircles the nucleus, nor for the muscle cell, nor for the cells of the choroid plexus, still less for the cells of the con- voluted tubules, or for the liver cells. So that this criterion is not universally applicable, at least to fully differentiated and highly specialized types of cells. Much of the discussion about the nature and homologies of these things has hinged about the question as to whether they are solid, that is fibrillary; or canals filled with fluid, and made to appear as solid filaments by the metallic impregnation meth- ods. With the canalicular theory the name of Holmgren is associated, and though there are many shades of opinion in 136 ALWIN M. PAPPENHEIMER regard to detail the general idea that these nets and filaments represent canals with or without definite Walls, has had the sup- port of such expert histologists as Studnicka (170), Retztus (149), Cajal (31), and, in this country, of Bensley (14) and Cowdry (42). Holmgren was not the first to observe ‘endocellular canals’ or ‘Saftkandlehen.’ As far back as 1887 Nansen (129) described in the protoplasm of nerve cells of Homarus and in the spinal ganglion cells of Myxina glutinosa, primitive tubes consisting of hyalin contents enveloped in sheathes of spongioplasm. These. were probably identical with the Saftkanélchen. Nelis (132) also described an ‘état spirémateux’ in the cytoplasm of certain mammalian nerve cells. He speaks of ‘bandes incolores’ of about the same diameter, sometimes straight, sometimes con- voluted which did not branch, and therefore formed no reticulum. This spireme was not constantly found, and does not seem to be the same thing as the Golgi net. Holmgren (73) made his first contribution to this subject in 1899, a year after Golgi’s first paper, and without, apparently, knowing of the work of Nansen and Nelis. In the spinal gan- glion cells of rabbits and of Lophius piscatorius he found an endo- cellular system of canals communicating with the pericellular lymph spaces; and in the following year he published a large monograph on the ganglion cells of Lophius (72). His earlier views, expressed in these papers, that the endocellular Saftkanél- chen are to be regarded as lymph channels, and are thus continuous with structures of connective tissue origin, were later abandoned by him. Morphologically Holmgren considered his canaliculi to be identical with the Golgi net, basing his opinion upon a study of his own preparations, and those of Retzius, prepared accord- ing to the earlier Golgi technique. In 1900 (76), in a study of the ganglion cells of Helix pomatia Holmgren described a penetration of the nerve cells by cell- processes from surrounding cells. This view was developed in subsequent studies. The penetrating cells of neuroglial ori- gin he called trophoeytes, and to the network formed by the penetrating cell processes, he applied the term trophospongium. . THE GOLGI APPARATUS 137 These prolongations of the trophoeytes, he believed, became canal- ized by a vacuolization of their cytoplasm, the confluescence of the vacuoles giving origin to the canal. This was an irreversible process, but the cell prolongation was capable of amoeboid mo- tion within the host cell. Studies on various tissue cells, which I shall not review in detail, confirmed him in his view, and lead him to the following generalization. The cells of the body are of two orders of physio- logical dignity—high and low. Those of exalted function are the nerve cells, muscle cells, sex cells, certain glandular cells. The lower order of cells, which are the trophocytes, function as servitors, looking after the wants of their more specialized neighbours by means of their trophospongia. Although this hypothesis is vaguely expressed, and open to obvious criticism, Holmgren has maintained it in a long series of papers (77-96), many of them controversial, and adding no new evidence. The arguments against any such generalized con- ception are apparent enough. To what order shall we assign the leucocytes and other wandering cells? Where are the trophocytes of cartilage cells? Why have the trophocytes about the gan- glion cells, as well as all the other types of cells classed with the lower order, endocellular nets, and what cells look after their lowly wants? Holmgren has always insisted vigorously upon the identity of his trophospongium with the Golgi apparatus; on the other hand, all who have worked with the Golgi or Cajal methods deny that the structures which they bring out reach the surface of the cells or communicate with other cells. Even Cajal, who regards the Golgi structures as canaliculi, believes them to be wholly en- docellular, except perhaps in the special case of the insect mus- cles, in which the homology with the Golgi net is not very clear at best. Ross (153) in a recent paper on the trophospongium of the ganglion cells of the crayfish, describes the penetration of the cytoplasm by partitions and fibrils from the surrounding neurog- ha, but rejects the idea that these have any relation to the in- ternal reticular apparatus. 138 ALWIN M. PAPPENHEIMER This view, I think, may be accepted without reserve; nor does it seem that Holmgren’s generalizations are based on sound evi- dence, nor that they have added much of value to the subject. Leaving aside, then, this controversial phase of the subject, one may ask what can be said as to the more intimate physical structure of the Golgi apparatus. It seems to the writer, that the conceptions of Cajal (31) best meet the observed and es- tablished facts. Cajal’s view is that the apparatus represents a canalicular system, filled with a lpoid-containing substance which reacts to the specific impregnation methods employed. The walls of this system are presumably fairly fixed and rigid in cells of a sedentery habitus, and permanent form, but more plas- tic in secretory cells and in young cells frequently undergoing mitosis. It seems probable that the quantitative changes observed dur- ing activity indicate a using up of a store of stainable material within these canaliculi, and that the re-appearance of the net during the quiescent stage is due to the re-accumulation of the substance within more or less preformed and permanent chan- nels. What purpose this material serves in the cell metabolism, and what is its more intimate chemical structure, are questions unanswerable with the data at hand. Ix. SUMMARY AND CONCLUSIONS There is present in the somatic and sex cells of all metazoa, and possibly also, of protozoa, a cytoplasmic structure of con- siderable complexity and size. demonstrable by prolonged fixa- tion in osmic acid, or by silver impregnation and reduction. The reaction of this structure to osmic acid indicates, of course, a lipoid component, but there are no other data bearmg upon its chemical composition. Nor is anything certain known of its physical characters. Its invisibility in the living cell would in- dicate a low refractive index. The fundamental question as to whether the impregnated structures are canalicular or filamen- tous remains unsolved. The constant topography in many types of cells, particularly the definite relation to the cytocentrum would favor the idea that the structures are at least in part THE GOLGI APPARATUS 139 solid, rather than casual rifts or fluid-filled canals in the eyto- plasm. ‘The fragmentation or dispersion of the net which oc- curs during certain secretory phases, or accompanying patho- logical changes in the cell, and particularly during cell division, would also suggest a solid or semi-solid consistence. The Golgi apparatus in the secretory portion of the renal tubules does not conform to the usual closed skein found in many types of glandular epithelial cells, but is dispersed and assumes complex and varying forms. In the glomerular cells, on the other hand, and in the epithelium of the collecting tubule and of the pelvis the structure is more typical both in form and location. Injury to the epithelial cells of the convoluted tubules (ura- nium nitrate poisoning) is followed by complete disintegration and disappearance of the Golgi apparatus. This appears to precede the complete necrosis of the cell. The large hyalin droplets found during the degeneration of the cells contain an argentophile component possibly derived from the remains of the Golgi apparatus. The structures brought out by the Golgi or Cajal technique are more resistant to autolysis than are the mitochondria. There occurs regularly in the rat’s kidney an elective impreg- nation of the mitochondrial filaments of certain portions of the Henle loops. This illustrates the fact that the method is not absolutely specific. The writer wishes to express his thanks to the Marine Bio- logical Laboratory of Woods Hole for according him the privileges of the Laboratory during the summer of 1915-1916; and to Dr. E. V. Cowdrey for helpful suggestions and criticism. THE ANATOMICAL RECORD, VOL. 11, No. 4 140 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) ALWIN M. PAPPENHEIMER BIBLIOGRAPHY Acconti, 1912 Di aleuni fini particolarita di struttura della mucosa uterina della decidue e dell’ uovo. Boll. Soc. Med. Chir. di Pavia, 25, 125. ARNOLD, J. 1908 Haben die Labzellen Membranen u. Binnennetz. Anat. Anz., 32, 257. Ancona, 1909 Apparato reticolare nelle cellule della ghiandola lachry- male. Diss. di laurea (unedita) Pavia. Batitowitz, 1900 Ueber d. Epithel. d. membrana elastica posterioris des Auges, seine Kerne u. eine merkwerdige Struktur seine grosse Zell-sphaeren, u.s.w. Arch. Mikr. Anat., 56, 230. 1900 Eine Bemerkung zu den v. Golgi und seine Schiilern beschrieb- enen ‘apparato reticolare intorno’ der Ganglien u. Driisenzellen. Anat. ANZ AS, hid: Barinetti, 1912 L’apparato reticolare intorno e. la centrospera nelle cellule di aleuni tessuti. Boll. Soc. Med. Chir. di Pavia, 25, 289. 1911 Di una fina particolaritaé di struttura nelle cellule del epitelio della cornea. Boll. Soc. Med. Chir. di Pavia, 5. Batistessa, 1911 Sulle alterazioni dell apparato reticolare interno delle cellule nervose dei gangli spinali in seguito ad advelenamento da piombo e stricnino. Riv. ital. di Neuropatologia, 4, 345. Benpa, 1902 Weitere Mittheilungen tiber die Mitochondrien. Ergebn. d. Anat. u. Entw., 12, 743. v. Bercen, 1904 Zur Kentniss gewisser Strukturbilder (Netzapparate Saftkinalchen, Trophospongien) im Protoplasm verschiedener Zel- larten. Arch. f. Mikr. Anat., 64, 498. Besta 1910 Sull apparato reticolare intorno (apparate di Golgi) della cellula nervosa. Anat. Anz., 36, 476: 1911 Ricerche sull reticolo endocellulare degli elementi nervosie nuovi metodi di dimonstrazione. Riv. di patol. nerv. e. ment., 16, 341. BIALKOWSKA AND KuLtkowsKa, 1911 Ueber d. Golgi-Kopsch’en Ap- parat der Nervenzellen bei den Hirudineen u. Lumbricus. Anat. Anz., 38, 193. Benstey, R. R. 1910: On the nature of the canalicular apparatus of animalcells. Biol. Bull. Mar. Biol. Lab. of Woods Hole, 19, 179. 1911 Studies on the pancreas of the guinea pig. Am. Jour. Anat., 12, 297. BERENBERG-GOSSLER, 1912 Ueber gitterkapselartige Bildungen in den Uhrgeschlechtszellen v. Vogelembryonen. Vorl. Mitth. Anat. Anz., 40, 587. 1912 Verh. d. Anat. Ges. Muenchen., 26, 263. 1912 Die Uhrgeschlechtszellen der Hiithnerembryonen am 3ten u. 4ten Bebriitungstage, mit. besonder Beriicksichtigung der Kern- plasma Strukturen. Arch. f. Mikr. Anat., 81, Abteilung, ii, p. 24. BIALKOWSKA u. KuLtkowska, 1911 Ueber den feineren Bau der Ner- venzellen bei verschiedenen Insekten. Bull. Acad. Se. Cracovie. Cl. Se. math. et nat. (29) (30) THE GOLGI APPARATUS 141 Bronpi, 1911 Sulla fina struttura dell’ epitelie dei plessi coroidei. Arch. f. Zellforsch., 6, 387. . Bizzozpro © Borresetita, 1909 Sull’ apparato interno nelle cellule delle ghiandole sudoripare e sebacee. Arch. per le Sc. med., 33, 279. BcoORKENHEIM, 1913 Golgi’s apparato reticolare intorno in den Pla- centar-epithelien. Arch. f. Gynek., 100, 446. Bourn er Ancen, 1905 Apropos du ‘trophospongium’ et des ‘canali- cules du suc.’ Comptes Rend. de la Soc. de Biol., 2, 221. Browicz, 1902 Meine Ansicht iiber den Bau der Leberzelle. Virch. Arch., 168, 1. Bruenareci, 1908 Di una fina particolarita di strutturi degli epitelii dei tubuli renali. Boll. Soc. Med. Chir. di Pavia, 22, 96. Cagat, Ramon y, 1890 Coloration par la méthode de Golgi des ter- minaisons des trachées et des nerfs dans les muscles des ailes des in- sectes. Zeitschr. f. Wiss. Mikr., 7, 332. 1904 El apparato tubuliforme del epitelio intestinal de los mami- feros. Trab. del Lab. Invest. Biol. Madrid, 3, 35. 1908 Les conduits de Golgi-Holmgren du protoplasm nerveux at le reseau pericellulaire de la membrane. Trab. del Lab. Invest. Biol. Madrid, 6, 123. 1912 El apparato endocellular de Golgi de la celula de Schwann y algunas observaciones sobre la estructura de los tubos nervosa. rab. del Lab. Invest. Biol. Madrid, 10. 1912 Férmula de fijacién para la demonstracién facil del apparato reticolar de Golgi y. apuntes dobre la disposicién de diche apparato en la retina, en las nervios y algunos estados patolégicos. Trab. del Lab. Invest. Biol. Madrid, 12, 209. 1914 Algunas variaciones fisiolédgicas y patolégicas del aparato reti- cular de Golgi. Trab. del Lab. de Invest. Biol., 12, 127. CatraNeo, 1914 Ricerche sulla struttura dell’ ovario dei mammiferi. Arch. ital. de anat. e di embriol., 12, 1. CHAMBERS, 1915 Microdissection studies on the germ cells. Science, NS., 41, 290. Craccro, 1903 Communicazione sopra i canaliculi di secrezions nella capsula soprarenali. Anat. Anz., 22, 493. Coun, 1903 Zur Histologie u. Histogenese des Corpus luteum u. des interstitiellen Ovarialgewebes. Arch. f. Mikr. Anat., 62, 745. Cotson, 1910 Histogenese et structure de la capsule surrénale adulte. Arch. de biol., 25, 535. Coxirn et Lucien, 1909 Observations sur le réseau interne de Golgi dans les cellules nerveuses de mamiferes. Comptes rend. de la Soe. Anat., Nancy. 1914 Algunas variaciones fisiolégicas y patolégicas del aparato reti- cular de Golgi. Trab. Lab. de Invest. Biol., 12, 127. 1909 Sur les rapportes du réseau interne de Golgi et des corps de Niss!] dans la cellule nerveuse. Bibl. Anat., 19, 123. Comes, 1909 Sulla natura mitocondriale dell’ apparato reticolare delle cellule cartilagine. Boll. Acad. giolinica di sc. nat. Siena, Ser. II, fasc. 6. ALWIN M. PAPPENHEIMER 1913 Apparato reticolare 0 condrioma? Condrocinesi o dittocinesi? Anat. Ant., 43, 422. - Cowprey, 1912 The relations of mitochondria and other cytoplasmic constituents in spinal ganglion cells of the pigeon. Int. Monatsch. fs Anaty uh Rhys. 9295 1. D’Acata, 1910 Sulle modificacazioni dell’ apparato reticolare interno nell’ epitelia della mucosa gastrica. Boll. Soc. Med. Chir. di Pavia, 25, 517. : 1910 Di una fina particolarita di struttura delle cellule epiteliali della cistifellea. Boll. Soc. Med. Chir. di Pavia, 25, 531. 1911 Ueber eine feine Struktureigenthiimlichkeit der Epithelzellen der Gallenblase. Arch. f. Mikr. Anat., 77, 78. Decro, 1910 Sulla minuta struttura dell’ epitelio uterino. Boll. Soc. Med. Chir. di Pavia, 25, 476. DEINEKE, 1912 Das Netzapparat von Golgi in einigen Epithel- u. Bindegewebszellen wihrend der Ruhe und Teilung derselben. Anat. Anz., 41, 289. DuesBerG, 1914 Trophospongien und Golgi’schen Binnenapparat. Verh. d. Anat. Ges., 46, 11. 1912 Plastosomen, ‘Apparato reticolare interno,’ und Chromidal- apparat. Erg. d. Anat. u. Entw., 20, 567. ELLERMANN, 1899 Ueber die Struktur der Darmepithelzellen von Helix. Anat. Anz., 16, 590. Eruarp, 1910 Studien iiber Trophospongien. Zugleich ein Beitrag. z. Kenntniss der Sekretion. Festschr. f. R. Hertwig, 50, 133. Faur, 1914 Zur Frage der Hyalintropfige Zelldegeneration. Verh. d. deutschen Path. Ges., 17, 110. Fananas, 1912 El apparato endocellular de Golgi de la mucosa y bulbo olfactorios. Trab. Lab. Invest. Biol. Madrid, 10, 253. 1912 Nota preventiva sobre el apparato reticolar de Golgi en el em- brién de pollo. Trab. Lab. Invest. Biol. Madrid, 10, 247. 1913 Alteraciones del aparato reticular de Golgi en los cellulas gi- gantes y altros elementos del tuberculo. Trab. Lab. Invest. Biol. Madrid, 11, 119. Faure, Fremiet, 1910 Apropos d’une note de M. Perroncito sur le réseau de Golgi des cellules spermatiques. Bull. Soc. Zool. de France. 1910 Un appareil de Golgi dans l’oeuf de Ascaris megalocephala. Response a M. Perroncito. Bull. Soc. Zool. de France. Fepeui, 1912 Apparati retocolari e sarcolemma nella fibra muscolare cardiaca. Rend. d. R. Accad. d. Se. fis. e mat. di Napoli. FraGnito, 1901 Le développement de la cellule nerveuse et les canali- cules de Holmgren. Bibl. Anat., 9, 72. ‘ GEMELLI, 1900 Ricerche sperimentali sulla struttura della ghiandola pituitaria nei mammiferi. Boll. Soc. Med. Chir. di Pavia, quoted by Golgi-Arch. per le Se. Med., 1909, 33, 5. Goucr, 1898 Intorno alla struttura delle cellule nervose. Boll. Soe. Med. Chir. di Pavia. 1898 Arch. ital. de biol., 30, 60. (62) (63) (64) (65) (66) (67) (68) (69) (70) (71) (72) (73) (74) (75) (76) (77) (78) (79) (80) (81) (82) THE GOLGI APPARATUS 143 1898 Sulla struttura delle cellule nervose dei gangli spinali. Boll. Soc. Med. Chir. di Pavia. Arch. ital. de biol., 30, 278. 1900 Di nuova sulla struttura delle cellule nervose dei gangli spinali. Boll. Soc. Med. Chir. di Pavia. 1899 Arch. ital. de biol., 31, 273. 1900 Intorno alla struttura delle cellule della corteccia cerebrale. Verh. Anat. Ges. Pavia, 164. 1909 Sulla struttura delle cellule nervose della corteccia del cervello. Boll. Soc. Med. Chir. di Pavia, 23, 341. 1908 Une méthode pour la prompte et facile démonstration de l’ap- pareil reticulaire interne des cellules nerveuses. Arch. ital. de biol., 49, 269. 1909 Di una minuta particolarité di struttura dell’ epitelio della mu- cosa gastrica ed intestinale di alecuni vertebrati. Arch. per le Sc. Med., 33, 1. 1909 Boll. della Soe. Med. Chir. di Pavia, 1. Heipennarin, M. 1900 Ueber die Centralkapseln u. Pseudochromoso- men in den Samenzellen. Anat. Anz., 18, 513. Henscuen, 1904 Ueber Trophospongienkanilchen sympatischer Gan- glienzellen beim Menschen. Anat. Anz., 24, 385. Hrirscuuer, 1912 Ueber die Plasmastrukturen (Mitochondrien, Golgi’- scher Apparat u. A.) in den Geschlechtszellen der Ascariden (Sper- mato- u. Ovogenese). Arch. d. Zellforschung, 9, 351. 1914 Ueber Plasmastrukturen (Golgi’sche Apparat, Mitochondrien, u. A.) in den Tunikaten, Spongien und Protozoenzellen. Anat. Anz. 47, 289. HotmGren, 1899 Zur Kenntniss d. Spinalganglienzellen v. Lophius piscatorius. Anat. Hefte, 12, 71. 1899 Zur Kenntniss der Spinalganglienzellen des Kaninchen’s u. des Frosches. Anat. Anz., 16, 161. 1899 Weitere Mittheilungen tiber den Bau der Nervenzellen. Anat. Anz., 16, 388. 1900 Noch weitere Mittheilungen iiber den Bau der Nervernzellen verschiedener Tiere. Anat. Anz., 17, 113. 1900 Studien in d. feineren Anatomie d. Nervenzellen. Anat. Hefte, 15, 7. 1900 Von den Ovocyten der Katze. Anat. Anz., 18, 63. 1902 Beitrige z. Morphologie d. Zelle. I. Nervenzellen. Anat. Hefte, 18, 269. 1902 Einige Worte iiber das ‘Trophospongium’ verschiedene Zellar- ten. Anat. Anz., 20, 4383. 1902 Weiteres tiber das Trophospongium d. Nervenzellen u. der Drii- senzellen des Salamanderpankreas. Arch. f. Mikr. Anat., 60, 669. 1902 Ueber die Trophospongien d. Darmepithelzellen, nebst. eine Bemerkung in Betriff seiner v. Prof. Browicz neulich publizierten Abhandlung iiber die Leberzelle. Anat. Anz., 21, 477. 1903 Ueber die Saftkanilchen der Leberzellen u. der Epithelzellen der Nebenniere. Anat. Anz., 22, p. 9. 144 (83) (84) (85) (86) (87) (88) (89) (90) (91) (92) (93) (94) (95) (96) (97) (98) (99) (100) (101) (102) (103) ALWIN M. PAPPENHEIMER 1903 Ueber die ‘Trophospongien’ d. Nebenhodenzellen und die Le- bergangzellen v. Helix pomatia. Anat. Anz., 20, 83. 1902 Beitrige'zur Morphologie d. Zelle. Ergebn. d. Anat. u. Entw., 1; 274: 1903 Weiteres iiber die ‘Trophospongien’ der Leberzellen u. der Darm- epithelzellen. Anat. Anz., 22, 313. 1903 Einige Worter zu der Mittheilung v. Kopsch: ‘‘Die Darstellung des Binnennetzes in spinalen Ganglienzellen u. andere K6rperzellen mittelst Osmiumsaure.’’ Anat. Anz., 22, 374. 1903 Weitere Mittheilungen iiber die ‘Trophospongienkaniilchen’ d. Nebennieren vom. Igel. Anat. Anz., 22, 476. 1903 Ueber die sogenannten ‘intrazellularen Faden’ der N ervenzellen von Lophius piseatorius. Anat. Anz., 23, 37. 1903 Weiteres iiber die Trophospongien verschiedene Driisenzellen. Anat. Anz., 23, 289. 1904 Ueber die Trophospongien der Nervenzellen. Anat. Anz., 24, 225. 1904 Beitrige z. Morphologie der Zelle. Il. Verschiedene Zellarten. Anat. Hefte, 25, 99. 1904 Ueber die Testes ee ae Centraler Nervenzellen. Arch. f. Anat. u. Phys., 15, Anat. Abt. 1905 Zur Kenntniss der Zylindrischen Epithelzellen. Arch. f. Mikr. Anat., 65, 280. 1907 Ueber die Trophospongien der Quergestreiften Muskelfasern nebst. Bemerkungen iiber den allgemeinen Bau dieser Fasern. Arch. f. Mikr. Anat., 71, 165. 1910 Untersuchungen iiber die morphologische nachweisbaren stof- flichen Umsetzungen der quergestreiften Muskelfasern. Arch. f. Mikr. Anat., 75, 240. 1915 Die Trophospongien spinaler ganglienzellen. Arch. f. Zool.. (Scandinavian), 9 (Hft. 2, Art. 15). Horteca, 1914 Alteraciones del sistema nervioso central en un caso de moquillo de forma paralitica. Trab. Lab. Invest. Biol., 12, 97. JAworowsky, 1902 ‘Apparato reticolare interno’ von Golgi in Spinal- ganglienzellen d. niederen Wirbeltiere. Bull. Acad. Sc. Cracovie, 403. Korransky, 1904 Ueber eigentiimliche Gebilde in d. Leberzellen d. Amphibien. Anat. Anz., 25, 435. Koimer, 1916 Ueber einige mit der Ramon-y-Cajal’sche Uransilber- methode darstellbaren Strukturen u. ume Bedeutung. Anat. Anaz., 48, 506, 529. Konossow, 1902 Zur Anatomie und Pi dele pie d. Driisenepithelzellen. Anat. Anz., 21, 226. Kouster, 1913 Ueber die durch Golgi’s Arsenik- u. Cajal’s Urannitrat- silber Methode darstellbaren Zellstrukturen. Verh. d. Anat. Ges. Greifswald. Korscu, 1902 Die Darstellung des Binnennetzes in spinalen Ganglien- zellen u. anderen Koérpenzellen mittelst Osmiumsaure. Sitzungsber. d. k. preuss. Akad. d. Wiss., 40, 1. (104) (105) (106) (107) (108) (109) (110) (111) (112) (113) (114) (115) (116) (117) (118) THE GOLGI APPARATUS 145 Kuxescu, 1914 Der Netzapparat v. Golgiin den Zellen des Kierstockes. Arch. f. Mikr. Anat., 84, 142. Kuxikowska, 1911 Ueber d. Golgi-Kopschen Apparat. in den Nerven- zellen der Insekten. Festschr. f. J. Nussbaum. LA VaLerre Sr. Georar, 1886 Spermatologische Beitrage. Arch. f. mikr. Anat., 27, 1. LeGeNpre, 1910 Recherches sur le réseau interne de Golgi des cellules nerveuses des ganglions spineaux. Anat. Anz., 36, 207. 1905 De la nature pathologique des canalicules de Holmgren des cellules nerveuses. Comptes rendues Soc. Biol., 687. Lescuke, 1914 Untersuchungen iiber den Mechanismus der Harnab- sonderung in der Niere. D. Arch. f. Klin. Med., 81, 14. Lewis, M. R., ann Lewis, W. H., 1915 Mitochondria (and other cy- toplasmic constituents) in tissue cultures. Am. Jour. Anat., 18, 339. Lucaro, 1900 Sulla patologie delle celluli dei gangli sensitivi. Riv. di Pat. Nerv. e ment., 5, 145. Luctonr, 1909 Contributo allo studio dei nevi molli. Arch de Se. Med., 33, 21. Luna, 1911 Sulla fina struttura della fibre muscolare cardiaca. Arch. f. Zellforschung, 6, 383. 1914 Sulla fina struttura delle cellule endotheliali dell’ endocardio e delle cellule que tappezano le fendituri di Henle. Arch f. Zellfor- schung, 12, 518 MaccaBruntI, 1909 Sulla fina struttura dei Megacariociti. Boll. Soc. Med. Chir. di Pavia, 57. : 1910 I Megaecariociti. Intern. Monatschr., 27, 477. Marcora, 1908 Di una fina alterazione delle cellule nervose de nucleo d’origine del grande ipoglosso, consecutiva allo strappamento ed al taglio del nervo. Boll. Soc. Med. Chir. di Pavia, 22, 134. 1910 Sull’ alterazione dell’ apparato reticolare interno delle cellule nervose motrici consecutivi 4 lesioni dei nervi. Riv. di Pat. nerv. e ment., 15, 393. 1910 Arch. ital. de biol., 53, 346. 1909 Sui rapporti tra apparato reticolare interno e corpi di Nissl negli elementi nervosi. Boll. Soc. Med. Chir. di Pavia. Mareneur, 1903 Aleune particolarita di struttura e di innervazione delle cute dell’ ammocoetes branchialis. Ztschr. f. wiss. Zool., 75, 421. Martinottri, 1899 Sur quelques particularités de structure des cel- lules nerveuses. Arch. ital de biol., 32, 293. 1904 Contributo allo studio dell’ apparato reticolare nei muscoli striati di aleuni mammiferi. Giorn. Accad. med. Torino, 67, 639. MassentI, 1914 L’apparato reticolare interno del Golgi nel germe dentale. Monit. zool. Ital., 25, 107. Misco, 1903 Das Binnennetz der spinalen Ganglienzellen bei verschied- enen Wirbeltieren. Intern. Monatsch. f. Anat. u. Phys., 20, 329. Mortant, 1901 Di un apparato reticolare entre alcune cellule cancer- ique. Atti della R. Accad. dei Fisiocr. di Siena fasc. 6. 146 (126) (127) (128) (129) (130) (131) (132) (133) (134) (135) (136) (137) (138) (139) (140) (141) (142) (143) (144) (145) ALWIN M. PAPPENHEIMER 1904 Ueber ein Binnennetz der Krebszellen. Ziegler’s Beitrige, 35, 629. Monti, 1915 Icondriosomi e gli apparati di Golgi nelle cellule nervose. Arch. ital. di anat. e di embr., 14, 1. Mutton, 1912 Apparato reticolare et mitochondries dans la surrenale du hérisson. Comptes rend. Soc. Biol. Paris, 268. NaANSEN, 1886 The structure and combinations of the histological elements of the central nervous system. Bergens Mus. Aarsberetning. (Quoted by v. Bergen). Neari, 1900 Ueber die feinere Struktur der Zellen mancher Driisen bei den Siugetieren. Verh. Anat. Ges. Pavia, 178. 1900 Di una fina particolarité di struttura delle cellule di alcune ghiandole dei mammiferi Boll. Soe. Med. Chir. di Pavia, 1, 61. Neuis, 1899 Un nouveau détail de structure du protoplasme des cel- lules nerveuses (état spirémateux du protoplasme). Bull. acad. R. Belgique, Cl. Sc., Ser. 3, T. 37. Nusssaum, 1918 Ueber die sogenannten inneren Golgi’schen Netz- apparat u. seine Verhiltnisse zu den Mitochondrien, Chromidien, u. andere Strukturen im Tierreiche. Arch. f. Zellforschung, 10, 359. OPPENHEIM, 1912 Die Nervenzelle, ihr feinerer Bau u. seine Bedeutung. Anat. Anz. 41, 271. Prensa, 1899 Sopra una fina particolaritaé di struttura di alcuni cellule delle capsule soprarenali. Boll. Soc. Med. Chir. di Pavia, No. 2. 1901 Osservazione sulla struttura della cellule cartilagines. Boll. Soc. Med. Chir. di Pavia, No. 3/4, 119. 1901 Rend. d. R. Inst. Lomb. de Se. e Lett., Ser. i1., 34, 448. 1913 La struttura della cellula cartilaginea. Arch. f. Zellforschung., 11, 557. PerRonciro, 1908 Condriosomi, cromidi. ed apparato reticolare in- terno delle cellule spermatiche (Nota preventiva), Rendiconti R. Inst. Lombardi., Ser. 2., 41. 1909 Contributo allo studio della biologia cellulare. Il fenomeno dello dictiocinesi. Atti Soc. ital. di patologia, 6. 1910 Mitocondrii, cromidi ed apparato reticolare interno dello cel- lule spermatiche. Atti. Accad. dei Lincei., Ser. 5, 7. 1911 Beitrige zur Biologie der Zelle (Mitochondrien, Chromidien, Golgi’schen Binnennetz in den Samenzellen (Autoreferat). Arch. f. Mikr. Anat. 257, 311. 1913. Mitochondries et appareil reticulaire interne. (Apropos d’une publication de J. Duesberg.) Anat. Anz., 44, 69. 1913 A proposito di un articolo di S. Comes sulla dittocinesi. Anat. Anz., 44, 78. Pinat, 1912 Der intracellulare Netzapparat in den Epithelzellen der Nebenniere vom Igel Erinaceus europeus. Arch. Mikr. Anat., 80, 157. PLATNER, 1889 Beitrige zur Kenntniss der Zelle u. ihrer Teilungser- scheinungen. Arch. f. mikr. Anat., 33, 125, 108. (146) (147) (148) (149) (150) (151) (152) (153) (154) (155) (156) (157) (158) (159) (160) (161) (162) (163) (164) (165) (166) THE GOLGI APPARATUS 147 PoLNEZYNSKI, 1911 Untersuchungen iiber dem Golgi-Kopsch’en Appar- at u. einige andere Strukturen in den Ganglienzellen d. Crustaceen. Bull. Acad. Se. Gracovie, 104. Poporr, 1906 Zur Frage der Homologisierung des Binnennetzes d. Ganglienzellen mit. den Chromidien. Anat. Anz., 29, 249. Reinke, 1906 Ueber die Beziehungen d. Wanderzellen zu d. Zell- briicken, Zell-liicken u. Trophospongien. Anat. Anz., 28, 369. Rerzius, 1901 Ueber Kanilchenbildung in den Riesenzellen des Knoch- en-markes. Verh. Anat. Ges. Bonn., 92. Riquier, 1910 L’involuzione dell’ apparato reticolare interno nelle cellule dello corpo luteo. Boll. Soc. Med. Chir. di Pavia, 24, 185. 1913 L’apparato reticolare interno. Rivista critico-sintetica. Riv di patologia nerv. e ment., 18, 314. 1910 Der innere Netzapparat in den Zellen des Corpus luteum. Arch. Pp Makrs Anatis 7a iis Ross 1915 The trophospongium of the nerve-cell of the crayfish (Cam- barus), Jour. Comp. Neur., 25, 523. Rosst 1912 L’apparato reticolare endocellulare di Golgi. Perugia, 8vo. (Abstracted in Anat. Anz., 1912, 44.) SancHEZ, 1907 L’appareil reticulaire de Ramon-y-Cajal-Fusari des muscles strieés. Trab. Lab. Invest. Biol., Madrid, 5, 154. San Giorer, 1909 Sull’ apparato reticolare interno di Golgi nel epitelio renale in condizioni patologico-sperimentali. Giorn. della R. Accad. di Med. di Torino, 15, 340. SAVAGNONE, 1910 Sur le réseau interne de Golgi dans les cellules des tumeurs. Arch. ital. de biol., 53, 1. 1909 Also Lo Sperimentale, 63, 574. 1910 Das Golgi’sche Binnennetz in Geschwulstzellen. Virch. Arch. 201, 275. ScHMINCKE, 1903 Zur Kenntniss d. Driisen der menschlichen Regio respiratoria. Arch. Mikr. Anat., 61, 233. Scumaus vu. Boum, 1898 Ueber einige Befunde in d. Leber bei experi- menteller Phosphorvergiftung u. Strukturbilder von Leberzellen. Virch. Arch., 152, 261. SINIGAGLIA, 1910 Osservazioni sulla struttura dei globuli rossi. Arch. pi Sei. Med., 34. Ss6vaLt, 1901 Ueber die Spinalganglienzellen des Igels. Anat. Hefte., 18, 239. 1906 Ueber Spinalganglienzellen u. Markscheiden. Zugleich ein Versuch die Wirkungsweise der Osmiumsaure zu analysieren. Anat. Hefte, 30, 259. 1906 Ein Versuch das Binnenetz von Golgi-Kopsch bei der Spermato- und Ovogenese zu homologisieren. Anat. Anz., 28, 561. vy. Smrrnow, 1901 Ejinige Beobachtungen itiber d. Bau der Spinal- ganglienzellen bei einem 4-monatlichen menschlichen Embryo. Arch. f. Mikr. Anat., 59, 459. 1906 Ueber die Mitochondrien u. den Golgi’schen Bildungen analogen Strukturen in einigen Zellen v. Hyacinthus orientalis. Anat. Hefte, 32, 143. 148 (167) (168) (169) (170) (171) (172) (173) (174) (175) (176) (177) (178) (179) (180) (181) (182) (183) (184) ALWIN M. PAPPENHEIMER SouKHANorr, 1901 Réseau endocellulaire de Golgi dans les éléments nerveaux des ganglions spinaux. Rev. neurol., 1228. 1903 Sur le réseau endocellulaire de Golgi dans les éléments nerveaux de l’écorce cérébrale. Le Névraxe, 4. Srropenr, 1908 Sopra una fina particolarité di struttura delle cellule epatiche. Boll. Soc. Med. Chir. di Pavia, 22, 146. StupNIGKA, 1899 Ueber das Vorkommen vy. Kaniilchen u. Alveolen im Koérper der Ganglienzellen. Anat. Anz., 16, 397. Tapper, 1910 Sull’ apparato reticolare interno di Golgi negli elementi epiteliale della prostata ipertrofica. Lo Sperimentale, 64, 434. TeLLo, 1913 El reticulo de Golgi en las celulas de algunos tumores y en las del granuloma experimental prodicios por el Kiesel guhr. Trab. Lab. Invest. Bio!l., Madrid, 11, 146. TeRNI, 1914 Condriosomi, idiozoma e formazioni peri-idiosomiche nella spermatogenessi degli amfibii (Ricerche sul Geotriton fuscus). Arch. f. Zellforschung, 12, 1. TotsuKA, 1902 Ueber die Centrophormien in dem Descemets’schen Epithel des Rindes. Intern. Monatschr. f. Anat. u. Phys., 19, 1/2; 68. VASTARINI-CrEsI, 1903 Trophospongium e canalini di Holmgren nelle cellule luteiniche die mammiferl. Anat. Anz., 24, 203. Veccut, 1909 Di una fina paricolarita di struttura della cellula cartil- deciduale. Anat. Anz., 34, 224. Veratti, 1909 Sulla fina struttura delle celluli di aleuni tumori. Boll. Soc. Med. Chir. di Pavia, 34. 1902 Recherche sulla fina struttura della fibra muscolare striata. Rend. 1st. Lomb. di Scienze e Letter, 19, 6. Verson, 1908 Contributo allo studio delle cellulo giganti tubercolare e di altri elementi cellulare normali e pathologici. Arch. per le Se. Med., 32, 489. Wercu, 1912 Vergleichende-zytologische Untersuchungen itiber den Golgi-Kopsch’en Apparat u. dessen Verhiltniss zu anderen Struk- turen in d. Somatischenzellen, Geschlechtszellen verschiedenen Tieren. Bull. Acad. Se. Cracovie, 417. 1910 Studien itiber den Golgi-Kopsch’en Apparat u. die Tropho- spongien Holmgrens in d. Nervenzellen d. Wirbeltiere. Wiss. Arch. 1. WIGERT UND Exserc, 1903 Ueber binnenzellige Kanilchenbildungen ge- wisser Epithelzellen d. Froschnieren. Anat. Anz., 22, 364. 1903 Studien itiber das Epithel gewisser Teile der Nierenkaniile von Rana esculenta. Arch. Mikr. Anat., 62. ZAWARZIN, 1909 Beobachtung an dem Epithel der Descemet’schen Membran. Arch. f. Mikr. Anat., 74, 116. ON THE FORM AND ARRANGEMENT IN FASCICULI OF STRIATED VOLUNTARY MUSCLE FIBERS A PRELIMINARY REPORT G. CARL HUBER Department of Anatomy, University of Michigan FOUR FIGURES In the last few decades, relatively little special attention has been given to the form of striated voluntary muscle fibers and to their arrangement in the fasciculi. In the anatomic litera- ture of this period, consideration is given mainly to the struc- ture of the myofibrils, to the relation of the connective tissue of the muscle to the fibers, to the development of muscle fibers and the development of the musculature as a whole. One studying current texts is impressed with the unanimity of ex- pressed views concerning the form of muscle fibers and the question is treated as having received satisfactory solution. Heidanhain! in ‘Plasma und Zelle’ treats of the form and length of voluntary muscle fibers as follows: Da der Gegenstand allgemein bekannt ist, kénnen wir uns kurz fassen. Es handelt sich um lange faserf6rmige Gebilde, welche in kleinen Muskeln von einem Sehnenende bis zum anderen hindurchlaufen und an diesem immer abgestumpft enden, im Inneren sehr grosser Muskeln hingegen auch frei und zwar unter allmihlicher Verschmale- rung mit spitzen Enden auslaufen. Sie werden bei geringer Breite (9-60 uw) bis zu 12 cm. lang und daher findet man nur in Muskeln, welche, parallel der Faserung gemessen, die Ausdehnung von 12 cm. uberschreiten, die erwihnten freien Endigungen. This quotation expresses fairly well, I believe, the current views of the form and mode of ending of striated voluntary muscle fibers. 1 Heidanhain, M., Plasma und Zelle. Fischer, Jean, 1911, p. 529. 149 150 G. CARL HUBER Bardeen,? as a result of a study of teased preparations made from the external oblique of certain mammals, deviates from the current views and gives a much more correct statement as concerns the form and relations of striated voluntary muscle fibers; indeed his brief statement is one of the most accurate I have found and is here given in full. His words read as follows: The individual muscle-fibres either run from one tendon to another or they may end at one extremity or at both within the muscle fasciculi which extend from tendon to tendon. We may therefore distinguish two modes of ending of individual muscle-fibres: the ‘intratendinous’, where the tip of the fibre terminates within a definite extension of a well marked tendon; and the ‘“intrafascicular,’ where the muscle-fibre terminates in the midst of a bundle of other muscle fibres which have a different region of termination. In the former case the muscle- fibre has a rounded or cone-shaped termination, often swollen in iso- lated specimens. In the intrafascicular mode of ending the muscle- fibre gradually becomes more and more narrow until it terminates in a thread-like extremity. In Bardeen’s figures 2 and 4, types of these modes of ending and of the form of muscle-fibers are given, figure 4 including spindle-shaped fibers. Bardeen’s statement of the form and mode of ending of muscle fibers, however, is antedated by the account given by v. K6lliker in his Handbuch der Gewebelehre des Menschen, which may be added to the references here given. Kolliker’s? account reads as follows: Ueber die Gestalt der Muskelfasern haben besonders die Unter- suchungen von Herzig und Biesiadecki, dann von mir, W. Krause, Weismann, Aeby und Kiihne Aufschluss gegeben. Nach diesen Erfah- rungen kann es wohl als Regel bezeichnet werden, dass die Muskel- fasern im Innern grésserer Muskeln spindelformig sind, die an den Enden dagegen ein inneres spitzes und ein in die Sehne itibergehendes breites Ende besitzen, welches entweder abgerundet ist oder in einige stumpfe Spitzen ausliuft oder auch wie treppenformige Absitze dar- bietet. Ausser spindelformigen Fasern kommen ein Innern der Muskel noch manche andere Formen vor, am gewohnlichsten an dem einen oder an beiden Enden stumpfe Fasern. 2 Bardeen, C. R., Variations in the internal architecture of the m. Obliquus Abdominis Externus in certain mammals. Anat. Anz., vol. 23, 1903. 3. Kélliker, A., Handbuch der Gewebelehre des Menschen. Erster Band. Engelmann, Leipzig, 1889, p. 371. FORM AND ARRANGEMENT OF MUSCLE FIBERS THE Koélliker quotes Ek. H. Weber (the original I have been unable to find) as regarding spindle-shaped fibers as the prevalent form of striated voluntary muscle fibers. It is not my purpose at the present time to enter upon a more extended discussion of the literature dealing with the form and arrangement in fasciculi of striated voluntary muscle fibers. It is hoped that the quotations given may suffice to orient the results here to be presented. In the course of this brief report other pertinent literature will be considered as occasion demands. My own studies on the form of striated muscle fibers have been made largely on teased preparations. The muscular tis- sue was obtained largely from adult rabbits. The maceration preparatory to teasing was by the hydrochloric acid method developed in this laboratory... The method as used for the maceration of muscular tissue may be given here in detail in the hope that other workers may feel tempted to make use of it in a further analysis of this tissue, since a correct understand- ing of the form and arrangement of muscle fibers in fasciculi, as also their length, is of importance in valuating certain fun- damental conceptions concerning the functions of muscles. For instance, according to E. Weber’s law of the working capacity of a muscle we are taught that the lifting power of a muscle is proportionate to the cross section of its fibers or fasciculi when arranged parallel, while the extent of elevation is proportionate to the length of its fibers. The method as used is as follows: After freely bleeding an adult rabbit a cannula was inserted into one of the iliacs central to the inguinal ligament or into the subclavian before it passes under the clavicle and firmly secured by ligature. A 75 per cent solution of hy- drochloric acid was then quickly injected at a pressure of 25 to 30 pounds. The apparatus used in obtaining and maintaining pressure was described by the author in the Am. Jour. Anat., vol. 6.5 It is desirable to have the acid injected enter the tissues as quickly and as freely as possible. The pressure is main- tained for several minutes. Some 15 to 20 minutes after the injection is com- 4 Huber, G. Carl, A method for isolating the renal tubules of mammals. Anat. Rece., vol. 5, 1911. 6 Huber, G. Carl, The arteriolae rectae of the mammalian kidney. Am. Jour. Anat., vol. 6, 1907. 152 G. CARL HUBER pleted, the muscles are exposed and separated and removed and placed in a 75 per cent solution of hydrochloric acid. In removing a muscle care should be taken to remove the entire muscle, at least portions extending from tendon to tendon and great care should be taken not to crush the muscle during removal. The muscle pieces or entire muscles remain in the hydrochloric acid for about 3 hours, the period varying a little, depending on the thoroughness of the pre- liminary injection. After thorough maceration is obtained, the acid is care- fully poured off and distilled water slowly added. The water is renewed at fre- quent intervals until it is practically free from acid. In the distilled water the muscle pieces remain about 24 hours, though a stay of 4 to 6 days is not harmful. After thorough washing in distilled water the larger pieces are usually readily broken up into smaller bundles of fasciculi. Small bundles of fasciculi are now transferred to a hemalum solution diluted to one half with distilled water. The transfer from the distilled water to the hemalum solution should be executed with eare if one wishes to obtain fasciculithrough their entire length. The trans- fer is best made with a glass rod, lifting the small bundle carefully as it leaves the water. In the hematoxylin solution the small bundles remain about 24 hours. This interrupts the maceration and stains the fibers. They may be kept indefinitely in the hematoxylin solution and are best stored in this solution for future use. In this solution the muscle bundles become quite hard and brittle and contract to about two-thirds or even to one-half of their former length. The preliminary teasing I have carried on in Esmarch dishes under the stereoscopic binocular and in a 0.5 per cent solution of ammonia water. In the ammonia water the stain attains a purple-blue color and the hard and brittle bundles become soft and pliable. A stay of one half hour to one hour in the ammonia water pre- pares the bundles for preliminary teasing. Inthe Esmarch dishes the bundles of fasciculi are with care separated into separate fasciculi. It should be stated, however, that a fasciculus is not a unit of muscle structure. For a distance all of the fasciculi of a bundle are readily separated. However, at one or sev- eral points small bundles of fibers or single fibers pass from one fasciculus to contiguous fasciculi. Great care is thus necessary and very careful teasing to completely separate what is known as a fasciculus. Bardeen? has noted the fact that muscle fasciculi are joined by fibers. After the preliminary teasing re- sulting in the separation of a single fasciculus, the final teasing is undertaken on a large slide or lantern slide cover prepared as follows. The slide is thoroughly cleaned in acids and alcohol and wiped dry. Narrow strips of wax plates (the plates used in wax reconstructions) 2 mm. thick are cut and placed near the bor- ders of the slide in the form of an oblong and pressed to the slide. The slide is then gently heated until the wax strips adhere. The slide on cooling is ready for use. The shallow well thus formed is filled with ammonia water and an isolated muscle fasciculus transferred to it. The final teasing may then be un- dertaken. It has repeatedly been possible to separate completely all or nearly all of the muscle fibers of a given fasciculus, even with fasciculi having a length of about6cm. The teased fibers may then be arranged in their approximate posi- tions. Only when this has been accomplished can a muscle fasciculus be considered as having been teased. A worker should not attempt this unless he has at his disposal some 3 to 4 uninterrupted hours, and ought to bear in mind that the best results are obtained by ‘making haste slowly.’ The mounting of such preparations presents many difficulties and discouragements. My procedure FORM AND ARRANGEMENT OF MUSCLE FIBERS 153 is as follows: A fasciculus is teased until nearly all its fibers have been separated. The ammonia water is then very slowly and carefully withdrawn by means of a small dropper with point drawn to a capillary tube. This is undertaken under the binocular, observing the effects of currents. The water is withdrawn until only a thin layer remains, only sufficient to enable moving the fibers on the slide. The final teasing and arranging of fibers may now be undertaken. As the am- monia water evaporates, the muscle fibers begin to adhere to the slide. The wax wall may now be removed and a large cover glass, on the under side of which a thin layer of glycerin has been spread, is gently lowered over the preparation from one edge. It is necessary to obtain the right degree of drying in order to gain successfully mounted preparations. If not sufficiently adherent to the slide the muscle fibers will move, float and break. If allowed to dry too much the muscle fibers, although fixed in place, will appear fragmented. Such prepara- tions are not valueless since the fragments of the fibers are not displaced laterally. Thus a single fiber may readily be traced throughout its whole length. By this method of preparation the muscle fibers show only faint cross stria- tions, though they present a blue color. The nuclei are not evident. Neither has it been possible to locate the place of entrance of the nerve fibers. The sar- colemma seems very resistant to the acid, the neurolemma less so. In the am- monia water the muscle fasciculi appear to regain the length they had prior to staining in the hematoxylin solution. The exact relation existing between the lengths as presented by teased fibers and living muscle fibers I am unable to de- termine definitely. The drawings here presented were made from preparations of muscle fasciculi teased completely, and arranged on the slide in their approximate positions, approximate with reference to the ends of the fasciculus teased. The drawings were made with the aid of the camera lucida at a magnification of 50 diame- ters, and are reduced 10 times in reproduction. The length of the respective fibers is accurately given. The thickness of the fibers is correctly given as pertains to the thicker portions of the fibers. At the attenuated ends the ink lines follow the outer border of the pencil outlines. The results of these observations may be briefly recorded as follows: It is usually stated that in muscles having relatively short fasciculi the muscle fibers extend from tendon to tendon. ‘This is of course not determined by the size and length of the muscle as a whole since in semipinnate, pinnate and compound pinnate muscles and in muscles where distal and proximal tendons over- lap the lengths of the respective fasciculi of a given muscle are much shorter than the length of the muscle as a whole. 154 G. CARL HUBER In figure 1 are presented some muscle fibers teased from fas- ciculi taken from the gastrocnemius of an adult rabbit. The fibers in group A, drawn from a completely teased fasciculus taken from the proximal portion of this muscle have an actual length of 1 em. (these and all measurements given are obtained by dividing the length of the respective fiber as measured in vn | \ | | | H | | IM Fig. 1 Muscle fibers from the gastrocnemius of an adult rabbit. Group A, actual length 1 em., from fasciculus taken from the more proximal portion of the muscle. Group B, actual length 1.5 cm., from fasciculus taken from the more distal portion of the muscle. X 5. the drawing by 50, the drawing having been made at a magnifica- tion of 50 diameters). The fibers in group B have an actual length of 1.5 em. and are from a completely teased fasciculus taken from the more distal part of the same muscle. In all of the fasciculi from this muscle completely teased, the great ma- jority of the muscle fibers extend from end to end or from tendi- nous insertion to tendinous insertion. In material prepared as above described, at the place of termination of a muscle fiber FORM AND ARRANGEMENT OF MUSCLE FIBERS 155 in tendon, the end of the muscle fiber stains more deeply in hematoxylin than does the same fiber in close proximity. The true end of the fiber differs in appearance from the end of a broken fiber. The tendon ends of various fibers vary slightly in shape. ‘They may appear as if cut at right angle to the fiber, as slightly beveled, as slightly rounded, tapering a little or hav- ing the form of a blunt cone and now and then as slightly ex- panded, though this may be due to a slight flattening of the end of the fiber. Now and then tendon ends of muscle fibers are met with that give the impression as though the sarcolemma did not enclose the end but terminated ring-shaped at the ex- treme tendon end of the respective fiber, but the limitations of the method used are such that this question could not be con- clusively decided. In this connection it is of interest to note the observations of O. Schultze,* who believes that muscle fibrils and tendon fibrils are parts of a single structure but this observer adds that the behavior of the sarcolemma at the ends of the fibers deserves further study. Also the studies of Bald- win,’ who regards the sarcolemma as covering the tendon ends of muscle fibers and denies the continuity of muscle fibrillae and tendon fibrillae, and discusses two types of terminations of muscle fibers in tendon; one type in which the long axis of muscle and tendon fibers coincide, the other type in which they meet at an angle. In the former the tendon fibrils are attached to cone shaped processes of the sarcolemma dovetailed into the tendon ends; in the latter type the sarcolemma end is con- siderably thickened and presents a number of projections into the muscle substance. Digitations or branchings of muscle ends or step formations have not been observed by me in my teased preparations. It should be understood, however, that in successfully macerated preparations the collagenous connective tissue is so completely removed that it is not evident on teasing. Out of quite a number of fasciculi with fibers of type B, of figure 1, § Schultze, O., Uber den direkten Zusammenhang von Muskelfibrillen und Sehnenfibrillen. Arch. f. Mik. Anat., vol. 79, 1912. 7 Baldwin, W. M., The relation of muscle fibrillae to tendon fibrillae in volun- tary striped muscle of vertebrates. Morph. Jahrb., vol. 45, 1913. THE ANATOMICAL RECORD, VOL. 11, No. 4 156 G. CARL HUBER successfully and completely teased, in .only two and in each only one fiber was found which did not extend from tendon end to tendon end. In both of these fibers one end reached the ten- don, terminating as adjacent fibers, while the other end reached to about the middle of the fasciculus ending in a fine tapering filament. The fibers in a number of fasciculi having muscle fibers of the length of type B were counted and averaged about 20 fibers to a fasciculus. What length a muscle fasciculus of an adult rabbit may attain and still have the great majority of its fibers reach from end to end is a question I am at present unable to answer definitely. Of the muscles teased, none in which the contained fasciculi reached a length of a little over 2.5 em. did I find such in which the majority of the muscle fibers reached from end to end. However, samples have not been taken from nearly all of the muscles and it may be that in certain of them fasciculi having a length of over 2.5 em. in which the majority of the fibers extend from end to end, may be found. In figure 2 are presented type fibers obtained from a completely teased and successfully mounted fasciculus, taken from one of the adductor muscles of the thigh of an adult rabbit. In this fasciculus a single muscle fiber (A) extends from end to end or from tendon insertion to tendon insertion; both extremities showing the characteristic staining and appearance of the tendi- nous end of a muscle fiber. This fiber has an actual length of 3.64 em., a length which is regarded as the length of the fasciculus. After final teasing and after withdrawing the ammonia water Fig. 2 Muscle fibers from the thigh adductor of an adult rabbit. Teased fasciculus had a length of about 3.5 cm. The completely teased fibers are in the drawing placed with reference to the ends of the fasciculus. Fiber A, has actual length of 3.64 cm.; a, 2.1 cm.; b, 1.8 cm.; c, 1.5 em.; d, 1.3 cm.; e, 2.1 cm.; f, 1.9 CMe po, lascimen eco: Fig. 3. Types of muscle fibers teased from a single muscle fasciculus, having a length of a little over 4 cm., taken from one of the larger thigh muscles of a rabbit. This fasciculus contained 37 fibers. The fibers are arranged with ref- erence to an imaginary line, bottom of figure. The tendon ends of fibers ending intrafascicular are brought to this line. Certain of the fibers sketched have an actual length as follows: a, 2.9 em.; b, 2.4 cm.; ¢, 1.92 cm.; d, 0.9 em.; e, 1.4 cm.; f, 0.14 cm.; g, 2.9 cm., ; h, 3.04 cm.;iandj,2.9cm. xX 5. 158 ° G. CARL HUBER from the well on the slide, as explained in the detailing of the method used, I was able to arrange the teased fibers so as to have the tendon ends of the teased fibers reach imaginary lines drawn at right angles to the ends of the single muscle fiber which extends from end to end in this fasciculus. The spindle- shaped fibers hold approximately the same relative position with reference to the ends of the fasciculus as before teasing as was determined at the time of teasing. This fasciculus, com- pletely teased, contains 26 muscle fibers, of which as stated one passes from end to end, 10 others reach one tendon end, 12 the other tendon end and 3 are spindle shaped fibers reaching neither tendon end. Of the 26 fibers, 15 type fibers are given in figure 2. This bundle of fibers completely teased is here spoken of as a fasciculus. I have above stated that fasciculi are not units of structure, but that from each small bundles of fibers or single fibers pass from one fasciculus to contiguous fasciculi. A single ‘fasciculus’ completely separated constitutes thus an artificially separated bundle of muscle fibers. Thoma’ has also appreciated the fact that a muscle fasciculus is not a unit of structure. In serial cross sections of the gastrocnemius of the frog, in which, with the aid of the camera lucida, the out- lines of the muscle fibers were sketched serially he noted single muscle fibers passing from one muscle fasciculus to another, concluding as follows: “‘Die einzelnen Muskelfaserbiindel hingen somit vielfach durch Muskelfasern zusammen, welche bald mit dem einen, bald mit dem anderen Biindel sehr innig verbund- en sind, und die ganze Muskelmasse bildet ein stark in die Linge gezogenes Netzwerk von Muskelfasern.”’ The fasciculus above referred to as containing 26 fibers is to be considered in this light. In the figure as drawn, at each end 5 fibers begin with blunt ends showing by form, structure and staining that they are muscle fibers ending in tendon. Hach of the fibers extends into the fasciculus for a distance which varies for the several fibers, becoming attenutated and finally terminates in a thread like filament having a thickness of 3 u to 4 u. It requires very ‘Thoma, R., Uber die netzformige Anordnung der quergestreiften Muskel- fasern. Virchow’s Archiv, vol. 191, 1908. FORM AND ARRANGEMENT OF MUSCLE FIBERS 159 thorough maceration to enable one to separate completely these fine, intrafascicular terminations of the muscle fibers. The length of the muscle fibers having one tendon end at the end of the fasciculus, the other ending in an intrafascicular filamen- tous termination varies as follows; fiber a, 2.1 em.; fiber b, 1.8 em.; fiber c, 1.5 em.; fiber d, 1.8 em. The other fibers of this type sketched are intermediate in length between fibers a and d. The three spindle shaped intrafascicular fibers with both ex- tremities attenuated and neither end reaching the tendon ends of the fasciculus measure as follows, fiber e, 2.1 em.; fiber f, 1.9 cm.; and fiber g, 1.7 cm. .The extent of overlapping of fibers beginning at the tendon end of the fasciculus and ending intrafascicular in fine, attenuated ends may be noted in this figure (2). Their exact relation cannot be readily seen in a com- pletely teased preparation, with fibers separated and arranged on the slide. ‘To gain their relationship actual teasing is neces- sary. While teasing the details of the arrangement of the mus- cle fibers becomes evident and it is observed that the fine fila- mentous intrafascicular ends are applied usually to the thicker: portions of other fibers, usually not near a filamentous end of another fiber. The same is true of the ends of the spindle- shaped fibers reaching neither fascicular end. This figure (2) I regard as representative of the form and arrangement of the striated voluntary muscle fibers in the fasciculi of rabbit muscles having a fascicular length of from about 3 em. to about 5 em. Probably the same is true of voluntary muscle of other verte- brates, though my observations have not been extensive out- side of rabbits and birds (rooster). In muscle with longer fasciculi the length of the muscle fibers having blunt tendon ends and filamentous intrafascicular ter- minations varies more than indicated by the measurements above given, and the spindle shaped fibers with intrafascicular position may lie nearer one end or the other of the respective fasciculus or occupy a more middle position. This variation in the length of the muscle fibers I have indicated in figure 3, giving type fibers from a fasciculus having a length of some- what over 4 em., and taken from one of the thigh muscles. Un- 160 G. CARL HUBER fortunately the specific muscle could not be determined after the maceration. This fasciculus was also completely teased and successfully mounted. It contains 37 fibers of which 8 are spindle shaped and have an intrafascicular position. In it one fiber extends from end to end, through the length of the fascic- ulus. The fibers could readily have been sketched in approxi- mate relative position with reference to the ends of the fasciculus, but the resulting figure, at the magnification used, would have been too long to admit of publication in the pages of this Jour- nal. The arrangement of the fibers, however, is not unlike that presented in figure 2. For figure 3, type fibers were selected. The single fiber extending from end to end could not be included by reason of its length. The fibers having a tendon end are arranged with reference to an imaginary line, at the bottom of the figure; the tendon end being brought to this line. Of cer- tain of the fibers with tendon ends and intrafascicular filamen- tous terminations the actual lengths are as follows, fiber a, 2.9 em.; b, 2.4 cm.; c, 1.92 em.; d, 0.9 cm.; e, 1.4 cm.; f, 0.14 em. The spindle shaped fibers sketched with both ends ter- minating intrafascicular with filamentous endings present the following measurements, fiber g. 2.9 em.; h, 3.04 em.; i and j, 2.9 em. The single fiber extending through the entire fasciculus presents a length of almost 4.5 em. For rabbit muscle fasciculi having a length of more than 4.5 cm. to about 5 cm., so far as my observations go, there are no muscle fibers that extend the whole length of a respective fascic- ulus. In some of the longer fasciculi taken from the latissi- mus dorsi, the pectoralis major and the extensor cruris almost complete teasing was obtained. Many muscle fibers were completely isolated, though never all of the fibers of a given fasciculus. In some of the most successfully macerated fascic- vli, their distal ends were slightly crushed during removal, so that not all of the fibers could be traced to their tendinous ends. For final teasing of these longer fasciculi, lantern slide covers answer the purpose of slides very well. In the longer fasciculi, having a length of 6 em. to about 6.5 em., in which many fibers were completely isolated, no fibers were found FORM AND ARRANGEMENT OF MUSCLE FIBERS 161 reaching from end to end. Fibers with blunt tendon ends and filamentous intrafascicular terminations, these, severally of varying lengths, and spindle shaped fibers with intrafascicular position, with ends terminating in hair like processes, consti- stuted the types of fibers isolated. In these longer fasciculi one end of certain of the spindle shaped fibers reaches nearly to one or the other tendinous end of the respective fasciculus while others of the spindle shaped fibers have a more nearly central position, with reference to the length of the fasciculus. In the longer and longest fasciculi teased, no muscle fibers hav- ing a length of more than about 3.5 em. were observed. Felix’ is quoted as having isolated striated muscle fibers ap- proaching a length of about 12 em. In his account stress is laid on the fact that in the macerating fluids used, acids mainly, the muscular tissue contracts by one-third to two-thirds of the original length. In his own material he sought to obviate this contraction by maintaining the original length through tension. I have noted the fact that in the method used, hydrochloric acid is injected into the living muscle while under extension, that during immersion in the hydrochloric acid and in the hema- toxylin stain, a contraction of the muscle fasciculi to about two- thirds to one-half of their original length is obtained, but also that in the ammonia water fasciculi of muscle taken from the hematoxylin solution extend in length so as to approach very nearly their length in fresh muscle. Exact measurements I am unable to give since, obviously, it would be necessary to isolate at least small bundles of fasciculi from fresh muscle, and trace them through the various steps, making measurements at various stages. Of the longest fibers isolated by Felix, one from the gracilis of man measured 11.5 em. and one from the sartorius of man 12.3 cm.; the latter fiber having a broken end. Division of fibers was not seldom found. A figure of a single fiber with branchings is reproduced natural size. This fiber in the figure measures approximately 12 cm. Concerning this fiber the text speaks as follows: ‘‘Die Faser theilt sich, lasst 2 Felix, W., Die lange der Muskelfaser bei dem Menschen und einigen Sauge- thieren. Festschrift, Albert von Kélliker, Englemann, Leipzig, 1887. 162 G. CARL HUBER Spaltriiume erkennen, steht mit anderen Fasern in Verbindung, kurz um, das Bild wird durch veilfach abgehende Fasern ein so complicirtes, dass’ man ein Gewirr von mehreren Fasern vor sich zu haben glaubt, bis eine genaue mikroskopische Unter- suchung ihre Zusammengehorigkeit nachweist.’’ Felix teased unstained tissue. I have not teased human muscle. However, the figure presented by Felix is not unfamiliar to me. In in- completely macerated tissue such ‘fibers’ are now and then obtained. However, they are interpreted by me as represent- ing an incompletely teased fiber complex. The fine hair like intrafascicular ends of muscle fibers are so closely applied to the sides of other fibers that the cross diameter of the thicker fiber is scarcely increased. Such a misinterpretation, I can conceive, may readily be made in incompletely macerated and teased muscle tissue. Felix gives data concerning the length of muscle fibers in the rabbit, a tissue with which I am familiar. This observer isolated fibers from the pectoralis, sartorius, latis- simus dorsi and extensor cruris of the rabbit. His own words concerning them read as follows: “Hier waren fast simmtliche Fasern mindestens 5 cm. lang, doch waren unter 6 cm. nur wenige zu erzielen. Die meisten Fasern schwankten zwischen 6.0 und 7.5 em. Die Fasern zeichneten sich simmtlich durch ihre Stiirke aus. Die lingste Faser isolierte ich aus dem extensor cruris, der am Thiere selbst nur 8 cm. mass, von 8 cm. Linge. Die Dicke war ungemein schwankend, dickere und diinnere Stellen wechselten ab, die diinnste Stelle mass nur 0.0109 mm., wiihrend dickere Stellen 0.111 mm. gemessen wurden. Offenbar sind hier verschiedene Wirkungen der Salpetersiure zur Geltung gekommen. Theilung konnte ich hiiufig beobachten.” An analysis of this statement from Felix in the light of my own investigations leads me to conclude that this observer did not obtain completely teased muscle fibers. Many hundreds of muscle fibers of the rabbit have been completely isolated and in no instance have I observed branching of fibers. Often have I seen apparent branching, but on careful teasing such structures have been separated into several fibers. The variation in thick- ness of the long fibers referred to in the above quotation, I be- FORM AND ARRANGEMENT OF MUSCLE FIBERS 163 lieve, is explained by a linking in chain of several fibers. Even granting that the fasciculi teased by me after a stay in ammonia water, some attaining a length of about 6.5 em., had not attained their full, original length, the difference in the length of muscle fibers of the rabbit teased by Felix and by myself is not accounted for. Felix found few attenuated ends of fibers with intra- fascicular terminations, while, as my own figures show, these are numerous. In the light of these studies I am inclined to regard the measurements of the length of striated voluntary muscle fibers as given by Felix as inaccurate and as made on incompletely teased muscle tissue, and to regard the figures given by earlier observers as more accurate. These, to quote freely from Felix, are for the medium length of muscle fibers of man 2 cm. to 3.5 em., Krause giving as the longest of the fibers of the sartorius 4 cm. Striated voluntary muscle fibers of other mammals and other vertebrates have thus far been only incidentally teased by me. Bardeen’s? figure 4, b, gives a flat band of fibers dissected from the external oblique of a dog, having a length of approximately 15 em. (figure one-half natural size) with figures of completely isolated fibers; spindle shaped fibers having a length of approxi- mately 8 em. and fibers with blunt tendon ends and attentuated intrafascicular terminations, varying in length from approxi- mately 4cem. to6cm. The general shape of these fibers appears to me as correctly drawn. Since I have not teased muscle fasciculi of the dog I am unable to verify the accuracy of the measurements given. For the dogs muscle fibers Felix gives 3 cm. to 4.5 em. as common measurements and 5.5 em. to 6.5 em. as long fibers. Opportunity presented itself to tease muscle fibers of an adult rooster (Gallus domestica), injected with hydrochloric acid for other purposes. In one specimen, the thigh muscles were well macerated. In figure 4 are shown four completely teased spindle shaped fibers taken from these muscles. These fibers, some of which are among the longest completely teased, present the following measurements: fiber a, 3.2 em.; b, 3 em.; ¢, 3.2 em. and d, 2 cm. Several spindle shaped fibers with intra- 164 a mat “ - i | - rete Vc j Pe. } i , em p AALS a1 +. i cy ) Be! he a3 A a 4 a ; air Me ; = let a, a) A . : i a eae gin 3s Cio keri UBER, hei Si as * i 4 Tin tk “ > gal : . t : P 2 ae | t- ab ’ $2 4 fFo ‘ : Ay ad oe = od =~ */ ' iy 2 5 . . 4 Fig. 4 Spindle shaped muscle fibers teased from the thigh muscles of an adi rooster (Gallus domestica). Actual length of fibers, a, 3.2 em.; b, 3 — c, 3. ult. cth.; d, 2m. “Xo. oy | PON ge) eae ew =) a Paw FORM AND ARRANGEMENT OF MUSCLE FIBERS 165 fascicular position, with undoubted branching were observed. The division extended to about the middle of the respective fibers, the two parts terminated in attenuated, hair like fibers. Muscle fibers with blunt tendon ends and filamentous intra- fascicular terminations were also observed. It is the purpose, as oportunity presents, to include in this study fibers from different types of muscles from the different classes of vertebrates and to extend the investigation so as to include several different mammals with types of muscle from each. Schiefferdecker!? and certain of his pupils have spent infinite pains in determining, among other things, the relative thick- ness of muscle fibers. The thickness and form of muscle fibers these workers have determined largely in cross sections of va- rious muscles. Each muscle is said to be composed of muscle fibers having specific size and form (cross section) with specific arrangement of connective tissue and elastic fibers. It is recog- nized that in each muscle, muscle fibers of varying sizes are found. In many muscles this difference in size of fibers is said to be considerable, in others less so. This difference in size of fibers may be ascribed, according to Schiefferdecker, to two pos- sibilities: 1, the muscle may be composed of fibers which in reality differ in size; 2, the smaller and smallest cross cut fibers of a given cross section may represent cross sections of the ends of fibers terminating in the muscle. In considering the structure of muscle, he adds, the second possibility plays only an unim- portant rdle, and only as concerns the smallest fibers. The fibers sketched in figures 2 and 3 may serve to show that such conten- tion is difficult to support in the light of this work. Except for muscles in which the fibers of the respective fasciculi extend from end to end, or in which the majority of the fibers do this, the variation in the size of the fibers in a given cross section is largely dependent on the fact that many of the fibers of a given fasciculus do terminate intrafascicularly. In order to make the numerous measurements of Schiefferdecker and his pupils of real value, or of similar investigations, it would be necessary to ascertain by means of teasing and complete isola- 10 Schiefferdecker, P., Muskeln und Muskelkerne. Barth, Leipzig, 1909. 166 G. CARL HUBER tion of fibers, the arrangement of the fibers in the fasciculi of muscles, the fibers of which are measured in cross sections. MacCallum’s" investigations led him to conclude, as a result of counting the fibers of the sartorius muscle in man at various ages that the muscle fibers cease to multiply in the fetuses from 13 em. to 17 em. in length, and that after that period muscles increase in size by increase in size of individual fibers. This statement, it would seem to me, needs verification and could only be verified by study of muscles in which all of the fibers of the fasciculi extended from end to end or by very careful and painstaking teasing, of fasciculi, covering the several periods in which the muscle fibers are counted. Myofibrils are usually regarded as extending from end to end in a given muscle fiber. In muscle fibers having filamentous intrafascicular terminations, and this includes the majority in the longer fasciculi, this is obviously not the case. Concerning the relations of the ends of myofibrils not reaching the ends of the respective muscle fibers, my teased preparations give no evidence. The festooning of the sarcolemma, described by certain authors, may perhaps be brought in relation with the ends of myofibrils which do not extend the entire length of the muscle fiber. In this communication the expression “completely teased and isolated muscle fibers” has been repeatedly used. There- fore it will no doubt seem paradoxical, for me to express in this concluding paragraph, even tentatively, the view that striated voluntary muscle is syncytial in character. From the arrangement of muscle fibers in the fasciculi of striated voluntary muscle; from the fact that muscle fasciculi are not units of structure; from the further fact that in teasing muscle fibers there are always found points of contact where the fibers are ultimately separated with great difficulty, I am led to tentatively express the view that striated voluntary muscle tissue presents syncytial character even in its fully developed state, as does involuntary muscle and cardiac muscle, though ' MacCallum, J. B., On the histogenesis of striated muscle fibers and the growth of the human sartorius muscle. Johns Hopkins Bull., 1898. FORM AND ARRANGEMENT OF MUSCLE FIBERS 167 not to the same degree as the last named. ‘This question can- not be finally decided by teasing. It is not my purpose at the present time to enter upon the mooted question of the histo- genesis of voluntary muscle tissue, nor to consider the extensive literature involved. The problem of the syncytial character of voluntary muscle is one of histogenesis. Embryological evidence at hand indicates that the histogenesis of voluntary muscle lends support to the view that striated voluntary muscle is syneytial in origin. Material is being collected to determine this question if possible. One of Schiefferdecker’s!® general conclusions reads as follows: ‘““Muskelnetze fanden sich in den untersuchten Muskeln so vielfach, dass man sie wohl als eine allgemein verbreitete Erscheinung ansehen kann.” Thoma’ finds frequent anastomoses between fibers. Reference, however, is not had to anastomoses between fibers such as described by Thoma. This observer finds intimate contact between adjacent fibers, so that for a distance only a single layer of sarcolemma appears to separate them. Myofibrils are not thought to pass from one fiber to another. It has seemed to me that this may be verified in teased preparations. Now and then two fibers adhere together, for a short distance, so closely, that separation, even in well macerated tissue, is impossible; this very generally in thicker portions of fibers. Involuntary muscle, if successfully macerated in potassium hydrate or by the hydrochloric acid method here detailed is readily teased so as to present spindle shaped cells, although as shown by McGill” this muscle develops from mesenchyme, retaining its syncytial character. The mere arrangement of striated, voluntary muscle fibers in a fasciculus possessing fibers with attenuated intrafascicular terminations, is such as to suggest the syncytial character of this tissue. In partially teased, though well macerated tissue, a mesh work of fibers, with long meshes is now and then evident. It is usually possible to tease the fibers having intrafascicular termination, quite readily, so far as concerns the thicker portions of these fibers and to isolate them to near their thread like terminations 12 McGill, Caroline, The histogenesis of smooth muscle in the alimentary and respiratory tract of the pig. Monatschrift Anat. u. Phys., vol. 24, 1907. 168 G. CARL HUBER on other fibers. Near their intrafascicular ends they adhere very tenaciously to adjacent fibers. In ammonia water the macerated and stained fibers become quite pliable and present an elasticity and a tensile strength which is often surprising. Yet, often the finer ends are broken before they can be detached from adjacent fibers. It is evident that the relations of the intrafascicular ends of muscle fibers to adjacent fibers is dif- ferent at their attenuated terminations than in course. Their exact relation I am unable to determine in teased preparations, though even the finest ends often present the appearance of a torn sarcolemma which does not extend to the extreme tip. I am unable to state whether the myofibrils extend from the at- tenuated ends to fibers on which they appear to end. In a number of preparations of rabbit embryos of the tenth day, cut serially in the sagittal plane, sections having a thickness of 2u and 3 u, stained in iron-lac-hematoxylin, the syncytial char- acter of the cells from which the voluntary muscle tissue is developing is evident. Conclusive preparations, from embryos varying in ages, have thus far not been obtained. This question shall form the subject of a further study now under way. It may be recalled here that Godlewski" considers striated muscle as presenting a syncytial character, basing his deductions on a study of the histogenesis of skeletal and heart muscle. It is impossible at the present time to do more than suggest that striated voluntary muscle, like involuntary and cardiac muscle, presents a syncytial character, evidence of which is seen in its full development. 13 Godlewski, E., Die Entwicklung des Skelet- und Herzmuskelgewebes der Sdugethiere. Arch. f. Mik. Anat., vol. 60, 1902. A NOTE ON THE STRUCTURE OF THE ELASTICA INTERNA OF ARTERIES G. CARL HUBER Department of Anatomy, University of Michigan ONE FIGURE A comparison of a number of texts, descriptive of the struc- ture of the elastic intima—the fenestrated membrane of Henle— of arteries, reveals the fact that the views concerning the struc- ture of this layer are byno means unanimous. Schafer! speaks of the elastic intima as follows: ‘‘The elastic tissue is represented by one distinct lamina, which is separated from the endothelium by the subendothelial layer. It is, on its outside, in direct contact with the non-striped muscle of the middle layer.” The ‘internal elastic lamina’ is spoken of as membranous in char- acter, the membrane is not, however, a continuous one, but is perforated by apertures. In figure 517, of Schiifer’s text, is shown a portion of the fenestrated membrane from the femoral artery as figured by Henle. Mall? in his study of connective tissue fibrils states that ‘‘elastic fibers are composed of two dis- tinct substances—the interior, which stains intensely with ma- genta, and the membrane, which does not.” who studied the finer structure of the brain arteries of several mammals, gives especial consideration to the internal elastic membrane, and gives emphasis to closely arranged longitudinal ledges, which have a course parallel to the long axis of the vessels. He recognizes a fibrillar structure in the elastic intima as expressed in these words, ‘‘Vielfach zeigt sich auch, dass die Membrana flava interna keine homogene Platte ist, sondern wie die Betrach- tung von Schragschnitten bei 1000facher Vergrésserung lehrt, aus, sehr feinen elastischen Fiaserchen besteht. Die erwahnten Leistchen werden durch Ausbildung stiarkerer nach dem Lumen vorspringender Fasern bedingt.” His figure 6, which shows an oblique longitudinal section of a basilar artery presents the fibrillar character of the elastic intima clearly. Diirek® records observations made on connective tissues studied by means of Weigert’s iron-hematoxylin myelin sheath staining method.. In tissues fixed in formalin and Miiller’s fluid or in formalin, 3y. Ebner, Victor, Koélliker’s Handbuch der Gewebelehre des Menschen, Dritter Band, Zweite Halfte. Engelmann, Leipzig, 1902, p. 643. 4 Triepel, H. Das elastische Gewebe in der Wand der Arterien der Schidel- hohle. Anat. Hefte, vol. 7, 1897. 5 Schéppler, H. Ueber die feinere Strukture der Hirnarterien einiger Siuge- tiere. Anat. Hefte, vol. 15, 1900. ° Diirck, H. Ueber eine neue Art von Fasern im Bindegewebe und in der Blutgefiisswand. Virchow’s Archiv, vol. 189, 1907. STRUCTURE OF ELASTICA INTERNA-ARTERIES 171 mordanted in a copper salt and stained in iron-hematoxylin, following the Weigert method for staining myelin’ sheaths, certain connective tissue fibrils were stained blue-black. Cer- tain of these differentially stained fibrils were regarded by Dirck as a special type of connective tissue fibrils, others, as yellow elastic fibers. This method as used by this observer, gave, in successful preparations, unusually distinet staining of the elastic intima of vessels. His words read as follows, ‘“Untersucht man zuniichst kleine Arterien auf dem Lingsschnitt oder auf Schriigschnitten, welche das Rohr in langer Ausdehnung tref- fen, so erkennt man an den durch die Intima fallenden Schnitten, dass die Elastica interna hier nicht dureh zirkulire Fasern, Faserbiindel oder Lamellen dargestellt wird, wie man dies ge- wohnlich abgebildet und beschrieben findet, sondern unmittel- bar tber dem Endothelrohr legt wie eine Basthille unter einer Baumrinde eine einfache Schicht von straffen Léingsfasern, welche unter sich allerdings durch kurze quere Zwiechenstiicke verbunden sind und so ein Netz mit sehr langgestreckten und langs verlaufenden Maschen darstellen.’”’ In cross-sections such fibers appear as points. The method used in staining the sections on which this study was based and from one of which the figure accompanying this note was drawn, was presented by Dr. De Witt’ at the Wiscon- sin meeting of the American Association of Anatomists in 1907. This differential elastic tissue staining method consists of a modification of Weigert’s’ iron-hematoxylin van Gieson method. According to Weigert’s method two stock solutions are prepared. Solution I EVERIO RAL TTC CEVSER IS rer 8 ap heya eti te ster GeFals soe a kee Bs 1 gram VG lige GLE! COMiber it sonra aoe meee eter gus AGRIRE oe te 100 ce. Solution IT cc, hiquor termi sesqurchlorati (WS: Poe s fey. at. Caden ne» cee ete 40) Livdrochloncractd (sp. sera ie)... Mea. esaea5. ten esos Seo. ee Sere 7 PCPA CLAS Geer: ORs it Be tS PIE I ae ela ate eee a 959 * DeWitt, Lydia M. Abstracts of papers presented at the 22nd Session Amer. Ass. Anat. Anat. Record, vol. 1, p. 74. 3Weigert, K. Eine kleine Verbesserung der Himatoxylin-van Gieson- Methode. Zeitsch. f. wissensch. Mik., vol. 21, 1904. THE ANATOMICAL RECORD, VOL. 11, No. 4 172 G. CARL HUBER Solutions I and II are mixed in equal proportions just before using. Differentiation is obtained by means of van Gieson’s picric acid fuchsin solution, prepared after Weigert as follows: PICHIGMGIONSacUraedsaG MeOUs:SOlUtlO N= sees eee 100 Acid Huchsin (Weivert), I 600. network of elastic fibers, the larger fibers of the network having in the main a direction which is parallel to the long axis of the respective vessel. A well stained and well differentiated longi- tudinal or longitudinal oblique section of an artery including the elastic intima, appears not unlike a successfully teased prepa- ration of yellow elastic tissue from the ligamentum nuchae. In figure 1 is presented a drawing of a portion of the elastic intima of one of the larger deep plantar arteries of a human foot. During fixation the artery had collapsed in such a way that on one side, for a distance, its wall was nearly in a plane. Several sections of a series thus included long stretches of the elastic intima. In this figure only the elastic tissue, which is 174 G. CARL HUBER stained deeply blue-black, is reproduced as drawn with the aid of the camera lucida, using a ; inch oil immersion objective and a No. 4 Zeiss compensation ocular with paper at table level. The network character of the coarser elastic fibers with frequent anastomoses and numerous cross-bridges is faithfully reproduced. It was not possible to draw accurately all of the finest fibrils throughout their entire extent. However, the figure as a whole gives a correct impression of the appearance presented by the section. At both ends (above and below the figure), the intima leaves the plane of section and the elastic fibers, shown as a network in the figure, appear as cross cut or obliquely cut fibers. In numerous other sections of vessels of varying sizes, longitudi- nally or obliquely cut, including the elastic intima, similar ap- pearances are found. The character of the network varies but slightly, dependent on the degree of extension or distension of the respective vessel. Oblique sections approaching cross sections of vessels are especially instructive. In such sections a side view of the elastic network of the elastic intima with end view of the fibers as seen in cross-cut, is obtained by moving the micrometer screw of the microscope. In cross sections of ves- sels, in place of the usual line representing the elastic intima as seen after the usual staining, there is observed a row of deeply stained blue-black dots, varying in size with the size of the ves- sel, with here and there a longer or shorter blue-black dash where a cross anastomosis between fibers is included in the section. Sections of areolar connective tissue, differentially stained for elastic tissue by means of the iron hematoxylin picric acid, acid fuchsin method, present no evidence of an ‘outer mem- brane’ for elastic fibers as described by Mall. However, the existence of such a membrane is in no sense denied, since a slight tinging with picric acid would not be evident against the deep blue-black stain of what is probably the ‘inner substance,’ stained readily in magenta. In certain of the longitudinal sections of vessels including the elastic intima, as for mstance in the section from which the figure here presented was drawn, a delicate grey-blue color overlies the elastic network. This is represented in the figure by a light wash of neutral tint. If a | t ~ STRUCTURE OF ELASTICA INTERNA-ARTERIES | this be expressive of structure it reveals a homogeneous structure and may possibly indicate the presence of a homogeneous mem- brane. Such a membrane, however, I have not detected in cross sections of vessels. From this study of the elastic intima of arteries the conclusion seems warranted that the stainable substance of this layer consists of a network of yellow elastic fibers, with coarser fibers having in the main a course which is parallel to the long axis of the respective vessel, these fibers presenting frequent anasto- moses and cross bridges, and with numerous finer fibrils which pervade the network. Here and there certain of the fibers of the elastic intima may in cross or oblique sections be traced in anastomosis with elastic fibers of the media. It would thus appear desirable to discard the term ‘fenestrated membrane,’ since this term does not express the structure of this layer. Of previous descriptions, that given by Dirck appears to me the most nearly conforming with observed facts. Ay ay A NOTE ON THE MORPHOLOGY OF THE SEMINIFEROUS TUBULES OF BIRDS G. CARL HUBER Department of Anatomy, University of Michigan ONE FIGURE In the course of a study of the renal tubules of birds, by means of the maceration method devised by Huber,! in which full grown roosters (Gallus domestica) were used as material, the injection of the 75 per cent solution of hydrochloric acid was through the aorta central to the branches supplying the kid- neys and sex glands. In a number of the cases the testes were found to be well injected and were removed and placed in 75 per cent hydrochloric acid with a view of obtaining thorough maceration preparatory to teasing. Following the method as described, the macerated pieces were washed thoroughly in distilled water, stained in hemalum, softened and cleared in 0.25 per cent to 0.5 per cent ammonia water, in which they were teased. Even during the preliminary teasing of the larger pieces it was noted that the testis of the rooster was not separable into lobular masses, as is the case in mammalian testes, so that it was found impossible to isolate structural units with which the final teasing could be carried out. Huber and Curtis? found that in the mammalian testis the seminiferous tubules presented no blind ends, diverticuli or nodular enlargements but were arranged in the form of an arch or a variable number of linked arches, all of the ends terminating 1 Huber, G. Carl. A method for isolating the renal tubules of mammalia. Anat. Ree., vol. 5, 1911. 2 Huber, G. Carl, and Curtis, George Morris. The morphology of the semi- niferous tubules of mammalia. Anat. Rec., vol. 7, 1913. LG 178 G. CARL HUBER in tubuli recti attached to the rete testis. Wax reconstructions made by Curtis confirm the observations made on teased prepara- tions. Repeated teasings have convinced me that in the adult bird no such arrangement of tubules pertains, but that the seminiferous tubules of the bird are arranged in the form of a network, presenting a varying number of anastomoses found at different levels in the gland substance. For this reason the teasing of these tubules is exceedingly difficult in that it is im- possible without breaking or tearing tubules to separate favor- able pieces preparatory to final teasing. All of my teased prepa- rations present an endless net, with broken tubular ends as a boundary. Fig. 1 Teased preparation showing a portion of the tubular system of the testis of the bird (Gallus domestica). x _ 5. In the accompanying figure is presented one of the most suc- cessful preparations obtained. The figure was traced and sketched with the aid of the camera lucida at a magnification of 50 diameters, reduced to a magnification of 5 diameters in the reproduction. The portion of the tubular net reproduced presents in all some forty broken ends, and at least three closed rings. Such closed rings I have found in nearly all of my prepa- rations. Their complete separation requires great care and patience since uniting tubular portions are very easily broken. The clearness of the figure, it is thought, obviates the necessity SEMINIFEROUS TUBULES 179 of extended deseription of the character of the network formed by the seminiferous tubules of birds. The figure, however, should be studied with the understanding that in the mount from which the figure was drawn the teased tubules were spread out as much as possible. The figure, therefore, does not give spatial relations of the tubules. In the gland, as is well known, these tubules form compact coils, evident somewhat from the extended kinks and bends seen in the figure. In a rather careful search of the literature -I have been un- able to find any description of the form of the seminiferous tubule of birds. This note would thus seem justified. However, the observations here recorded would seem to me to have a bearing on previous work emanating from this and other labora- tories, relating to the form of the seminiferous tubules of mam- malia. The results here recorded seem to me to confirm the observations made on teased preparations of the. seminiferous tubules of mammals. The fact that complex anastomoses resulting in closed ring structures have been teased in the bird’s testis argues for the possibility of teasing such structures in the adult mammalian testis, did they exist. Bremer,’ as a result of careful wax reconstructions of the tubular system of the human testis, working on embryonic and fetal tissue, the oldest stage studied being that of a human fetus of seven months, reached the conclusion that The testis cords, growing from the germinal epithelium of the genital ridge, form a network with three sets of anastomosing branches. After completion, this network breaks down partially, leaving certain cords as persistent stems. The tubules of the adult show, in their course, connection, and position in the testis, traces of this network. Testis tubules may be single, ending blindly, may branch, or may anastomose. In the adult mammalian testis tubules, completely teased, no blind endings, buds, nor ring formations were observed, while in the teased preparations of the seminiferous tubules of birds, the remains of the network of tubules as observed in the embryonic and fetal stages and well figured by Bremer, may be 3 Bremer, John Lewis. The morphology of the tubules of the human testis and epididymis. Amer. Jour. Anat., vol. 11, 1911. 180 G. CARL HUBER noted. In a eryptorchid of the rabbit, as described by Huber and Curtis, extended anastomoses of testis tubules were ob- served in two regions of the tubule complex, and in two regions, near the periphery of the gland, tubules were joined so as to form two folded rings. The preparations from the cryptorchid of the rabbit present appearances not unlike those shown in teased preparations of the seminiferous tubules of the bird. The presence of the remains of the embryonic network of the seminiferous tubules in the cryptorehid of the rabbit and in the bird’s testis, postulates a relatively late, complete morpho- genesis of the seminiferous tubules of the mammal. Phylogeny and ontogeny indicate this. In the light of these observations Iam of the opinion that Bremer’s careful study of the morphology of the seminiferous tubules of the human testis are of value as concerns embryonic and late fetal stages, but may not be trans- mitted to the adult gland in that in the oldest stage studied, a human fetus of the seventh month, the seminiferous tubules, in all probability, had not completed their morphogenesis. The question is one deserving further study and will form the subject of a future, more comprehensive communication, based on especially prepared and ‘timed’ material from the rabbit. This form is chosen since the morphology of the seminiferous tubule of the adult rabbit has received special consideration in this laboratory, both by means of teased preparations and re- constructions. THE HISTOLOGY OF BLOOD AND LYMPHATIC VESSELS DURING THE PASSAGE OF FOREIGN FLUIDS THROUGH THEIR WALLS II. STUDIES ON ABSORPTION FROM SEROUS CAVITIES P. G. SHIPLEY AND R. 8S. CUNNINGHAM From the Anatomical Laboratory of the Johns Hopkins University, Baltimore It is at once evident to any one who studies the enormous literature which has been published ‘during the last century, on the absorption into the body of foreign material from the serous cavities, that the problems presented to the present day investigators in this field may be roughly grouped under four heads, as follows: 1) the localization of absorbing surfaces; 2), the definite establishment of the channels of removal; 3) the determination of the forces concerned in the passage of matter through the walls of the serous cavities, and its entrance into and exit from the vessels which distribute it about the body for storage and digestion, or for destruction and excretion; and 4) the location and identification of the organs concerned in the storage or destruction of substances or fluids absorbed. Up to the present time not even the questions which are in- cluded under the first and fourth of these heads can be said to have been sufficiently answered; the second has been settled definitely only for a single area and with a limited number of substances, and about the third practically nothing is known. Although a vast amount of work has been done on the locali- zation of absorbing surfaces and their related organs of storage and excretion, until quite recently but little was known regard- ing them; and this is so in spite of their immense clinical im- portance in connection with the postural treatment of the various serousitides. It is owing to the work of MacCallum (1), who made a care- ful and productive study of the removal of foreign granules from 181 THE ANATOMICAL RECORD, VOL. 11, No. 5 DECEMBER, 1916 182 P. G. SHIPLEY AND :R. S. CUNNINGHAM the peritoneal cavity through the lymphatic lacunae of the dia- phragm, that practically all who have made a study of serous absorption accept it as a fact that a great deal of any solution or foreign body introduced into the peritoneal cavity passes through the peritoneal surface of the diaphragm, and enters the system of the experimental animal or the patient via its vessels. The position of other foci of drainage has remained unknown, and the tendency to deny or ignore their existence has been and is very strong. A few observers have suggested the broad surface of the greater omentum as a possible agent in the removal of foreign fluid from the peritoneum, but their assertions have rested upon probability rather than definite proof. Rubin (2), who attacked the problem from an experimental basis, showed, however, that less fluid was absorbed from the peritoneal cavities of animals whose omenta he had amputated, than from the peritoneal cavities of normal controls; and Crouse (3) after careful study concluded that the omentum is an impor- tant factor in the mechanism of peritoneal drainage, and hypoth- ecates a protective lymphatic drainage to account for the phenomena which he has observed. The authors (4) have been able to show experimentally that beyond doubt the omentum is a very efficient agent in the drainage of the peritoneal cavity. By drawing the omentum out of the animal’s body through a mid- line incision, and keeping it immersed in a fluid medium under physiological conditions, it was possible not only to isolate the organ and to prevent the experimental fluid from reaching other surfaces, but also to eliminate any influence on absorption which might be exerted by the increased abdominal tension which follows the intra-peritoneal injection of large amounts of the fluid. In spite of conditions which might be supposed to make for secretion rather than absorption, we found that a large amount of the fluid in which these omenta were immersed passed into the omental vessels, and reaching the general circulation, was carried at once by the blood stream to the organs of excre- tion, from which the test fluid could then be recovered. ABSORPTION FROM SEROUS CAVITIES 183 As to the second question, the establishment of the vascular system concerned in the drainage, for example, of the peri- toneum, Meltzer (5), Muscatello (6), and others, held that drainage is accomplished through the lymphatics, while Heid- enhain (7), Cohnstein (8), Dandy and Rowntree (9), and others, have shown that much of the fluid absorbed from the peritoneal cavity leaves it through the blood vessels. We have never been able to demonstrate the presence of lymphatics in the omental tissue of the adult cat, and Ranvier (10) claimed that while there are lymphatics in abundance in the omenta of young kittens many of them are obliterated by degenerative changes at the age of three months. If lymphatics exist in the cat’s omentum they must necessarily drain in the same direction as those of the gastric system; that is, an omental lymphatic stream, if such a thing exists, must eventually become tributary to the lymph content of the thoracic duct. In the experiments mentioned above with the omentum of the cat, the influence of lymphatic vessels was entirely eliminated in many of our experiments by the ligation of the duct. Hence we were able to prove not only that the omentum furnishes a surface where absorption takes place, but, by varying the fluid in which the omenta were immersed, we have shown that the removal of molecular solutions and colloidal solutions and of fine particulate matter in true suspensions may be accomplished through the blood vascular system to a large extent; though we do not by any means deny the probability of drainage through the lym- phatic channels in the localities where these vessels exist. But when any attempt is made to ascertain, through the medium of existing literature, the forces concerned in the absorption of foreign matter from serous surfaces one enters at once into a region of guess and hazard, where only a few isolated facts ex- ist as a guide to certain knowledge. We have only just ceased to argue for and against the presence of preformed ‘stomata’ and ‘stigmata,’ and to indulge in surmises as to their physiological significance. Students of the physiology of absorption are still discussing whether absorbed material passes through or between the lining endothelium of blood and lymphatic vessels and the 184 P. G. SHIPLEY AND R. S. CUNNINGHAM mesothelial cells of serous cavities. In our own experiments we found that a great deal of fluid may enter the blood stream when the influence of intra-abdominal pressure is removed; and, since our fluids were isotonic with the blood serum of the experi- mental animal, osmosis as it is generally understood, could have had only a negligible amount of influence upon the phenomena observed. Indeed if osmotic pressure had any influence at all, it would seem that it would have been exerted against rather than for the passage of the experimental fluid into the blood vessels, since even the small amount of fluid lost from the solu- tion by evaporation from exposure to the air, must have changed an originally isotonic to a slightly hypertonic fluid, to which one might expect water to pass from the serum through the vascular wall. If such a passage occurred it in no way interfered with the imbibition of the experimental solution. We know very lit- tle about the part played by fluid pressure, the movement of the blood and lymph in their respective vessels, or the influence on serous absorption of the movement of contractile somatic organs, like the diaphragm, or the contraction of the muscula- ture of the vessels themselves. We cannot say what chemical changes accompany or influence the transport of material from cavity to vessel; or whether the cytoplasm of the cells of the serous cavities, or of the blood and lymphatic vessels play any part in the transmission of matter through the vascular or serous walls. And does a disturbed balance of intra- and extra-cellular equilibrium militate for or against absorption? We do not know. It will readily be seen that the examination of histological preparations made from the omentum during active drainage, may be of great value in strengthening the positive evidence for absorption through the blood vessels, and in aiding us to under- stand the mechanism of the removal of foreign matter through the vascular wall. With this end in view, sections have been made and studied of omenta which,,up to the time of fixation, had been exposed to and were absorbing all sorts of material from true solutions to mechanical suspensions. A report of the findings in this ma- terial is the purpose of the present paper. ABSORPTION FROM SEROUS CAVITIES 185 By far the most valuable preparations were yielded by omental tissue which had been absorbing an isotonic solution of potas- sium ferrocyanide and iron ammonium citrate, and which was fixed immediately upon removal from that fluid in hydrochloric acid formalin with a resulting precipitation of prussian blue— the method used by Weed (11) to study the drainage of the cerebro spinal fluid. An omentum so treated appears in gross to be stained a uni- form pale blue except for the fat, and is patterned by an irreg- ular nerwork of an intense dark blue color. It is only necessary to examine the spread preparations with a binocular microscope to be convinced that the network is made up of the omental blood vessels whose lumina are filled with precipitated prus- sian blue; the picture is strikingly suggestive of a complete blood vascular injection of the omentum with a somewhat dilute prus- sian blue gelatine mass, and, in fact, we are dealing with much the same thing, since the coagulation of the colloidal proteids during fixation of the blood serum causes the same comminution of the nascent dye stuff which follows its precipitation in pectizing gela- tin. In contrast to the general tissue which fills the meshes of the vascular net’ and which is very pale blue, or uncolored, a wide deeply stained zone of thickly precipitated dye surrounds each vessel. The capi laries are all filled with prussian blue, even those supplying the perivascular fat being crowded with the dye and the capillary knots or glomeruli which form the support of many of the taiches laiteuse are completely injected. Here and there a capillary may be seen empty or nearly so, perhaps because contraction of its walls during fixation forced the absorbed dye from its lumen. Of the larger vessels all have a greater or less amount of dye precipitate within the lumen. All are completely filled, but in some the blue color is perceptibly paler than in others. In general the arteries show much less absorption than the cor- responding vein, but the depth of the color may not be the same throughout the length of a given vessel. There are often light and dark blue areas present. These preparations show that ac- 186 P. G. SHIPLEY AND R. S. CUNNINGHAM tive absorption is going on through the walls of the arteries as well as through the veins, even arteries with thick muscular walls taking part in the general process as will be shown below: The larger vessels are paler than the smaller, probably be- cause less fluid is taken in through their walls. In other words as the vascular size increases there is a gradually decreasing concentration of the intra-vascular dye solution, the signifi- cance of which will be discussed below. From the point where the vessels begin to be surrounded by a perivascular sheath of fat the pallor of the precipitated dye in the vascular lumen markedly increases, evidently because the advent of the perivascular fat is accompanied by an in- creased thickness of the vascular wall and a diminished absorp- tion. That the fat itself can have no effect in. the decrease is shown by the intense color of the injection mass in the capillaries supplying the fat; and examination of sections shows that the ferrocyanide solution penetrates easily between the fat cells themselves. Sections of the same material confirm the evidence of toial spread preparations. The capillaries, even those imbedded in and sulpplying the fat, are distended with the blue color, and the veins are full of blue precipitate of varying depth of color. It is however not so easy to see the blue color in the larger arteries in sections. It is possible even in thin sections to distinguish the deep blue perivascular area described above, and to trace its exist- ence to precipitated dye in the intrafibrillar tissue spaces. In- dividual fat cells are outlined by dye precipitated from the fluid which has worked its way between the cells that it has never penetrated. Coarse precipitates of dye may be seen along the surface of the omentum and adherent to the sur- faces of the elastic fibres. Im some places the tissue is diffusely stained, and throughout the omentum, cells, probably of the clasmatocyte type are found, like those described by Weed in the meninges, whose cytoplasm is filled with fine granules of prussian blue, their nuclei however remaining uncolored. This intracellular precipitate is the result of imbibition of fluid by the ABSORPTION FROM SEROUS CAVITIES 187 connective tisue cells, an adsorption phenomenon of the same nature as the drinking in of solutions of high molecular vital dye stuffs which is responsible for the diffuse cytoplasmic colo- ration seen early in a course of staining. The blood vessels contain many leucocytes, mostly of the mononuclear type, embedded in the precipitated blue of the injection mass. ‘This prussian blue precipitate in the blood ves- sels is not the coarse amorphous mass in which that color is usually seen under the microscope, but because the dye was thrown down in the presence of the colloidal serum proteids it is so finely divided as to appear homogeneous except when ex- amined with the highest power immersion lenses with which its finely granular nature can be ascertained. It is in the same phys- ical condition of finely divided suspension as the silver in silver gelatine mixtures of photographers, or the dye granules in injec- tion masses made by precipitating colors in the presence of solidifying gelatin. The intracellular precipitates and those adherent to the surface of the omentum and its component fibres are much more coarsely granular. The endothelial walls of both capillaries and larger vessels are stained a dark blue. The cytoplasm of the endothelial and mesothelial cells is entirely filled with a fine granular precipitate of the prussian blue, and in some places dye particles are found apparently between the cells, though the walls of the cells are in such close apposition that it is difficult to say with certainty that such is the cause. The cell nuclei are uncolored, and the cytoplasm of the serous mesothelium covering the omental sur- face is filled with the fine granules of dye which show the track of fluid which has passed through their bodies. By the time that the dye bearing serum has reached the vis- ceral blood vessels—liver, lung, ete.,—it has become so diluted with blood from non-absorbing parts of the body that it is not possible to follow the course of the chromogen through the body by the examination of sections. Large quantities are present in the kidney tubules, and the presence of the dye in the urine of the animal can easily be demonstrated. 188 P. G. SHIPLEY AND R. S. CUNNINGHAM The same conditions prevail, though. they are much more difficult to demonstrate, in preparations made from omenta which have been immersed in strong solutions of trypan blue and col- largol and colloidal solutions of other metals. In animals which have been injected intraperitoneally with the chromogen solution—or where certain isolated portions of the peritoneal surface (small intestine or bladder)—have been immersed in, or covered with cyanide-citrate solution, and fixed in hydrochloric acid formalin, the blood vessels and lym- phatics directly beneath the peritoneal surface, are found on section to be filled with dye precipitate and to have the same appearance as the omental vessels. Moreover it was possible in the gross to trace the stained lymphatic vessels directly to the lymph nodes into which they drained, and to obtain definite macroscopic evidence of the presence of prussian blue in the lymph gland. This feature of peritoneal absorption will be taken up separately in a later communication. It is evident then from these histological preparations that there is very active absorption of foreign fluids through the peritoneal blood vessels, not only those in the omentum, but also through those beneath the peritoneum over the gut and bladder. In all probability fluids may be removed from the peritoneal cavity through any area in which blood or lymphatic vessels lie just beneath the peritoneal surface. Furthermore absorption of fluid obtains not only through capillaries, but through vessels of quite large caliber, and through arteries as well as veins, though not to as great an extent probably, because of the greater obstacles to fluid passage offered by the tissues which go to make up the thicker, denser arterial wall. This is probably the reason for the pallor of the dye mass within the larger vessels, since it would seem reasonable to suppose that their thicker walls would hinder the passage of fluid and make it slower and of less amount. That fluids do pass through is shown by the fact that the wall and its lining endothelium contain granules of stain precipitated from the fluid during its passage into the vessels. That surrounding tissues have no significance in preventing fluid from coming in ABSORPTION FROM SEROUS CAVITIES 189 contact with these Jarge vessels is shown, as we have pointed out above, by the ease with which it penetrated between the cells in the perivascular fat and filled the capillaries by which the fat is supplied. There is of course the possibility of dilution of the chromogen fluid in the larger vessels as a result of their receiving blood from vessels through which absorption was not going on, but this is unlikely, since the vessels themselves and their entire tributary area were immersed in the test fluid. The significance of the dark stained areas about the blood vessels is not quite clear. Apparently the solutions are drawn forcibly from the general tissue towards the blood vessels taster than they can be forced through the blood vessel wall, and then, removal bemg delayed (perhaps by the condensation of the connective tissue in the vascular margin) they are concentrated there. What the forces are which are exerted on the fluid, and what part is played by the movements of the omentum as a whole, the contraction of the blood vessels and the movement of the blood within them, it is impossible yet to say. The material demonstrates also that while some fluid may pass between the lining cells of vessels on its way to their lumen, by far the larger part goes through the cytoplasm of the cells themselves. The sections are also of interest in that they show how little, if at all, the omentum was damaged during the op- erative procedure which preceded its immersion in the test fluid. Nowhere is there any sign of exudation or haemorrhage; there is no cellular death, as may be seen by the uncolored nuclei of the various cells; and, moreover, the mesothelial cells of the serous surfaces show no sign of disturbance or desquamation. 190 P. G. SHIPLEY AND R. S. CUNNINGHAM BIBLIOGRAPHY (1) MacCatium, W. G. . 1903 Johns Hopkins Hospital Bull., 14, 105. (2) Rupin, I. C. 1911 Surgery, Gynecology and Obstetrics, 12, 117. (3) CrousE, H. 1912 Bulletin of the El Paso Med. Soc. April. (4) Surptey, P. G. and CunnineHam, R.S. 1916 Am. Jour. of Physiol., 40,1, 75. (5) Apter, I. and Meutzer, S. J. 1896 Jour. of Exper. Med., 1, 482. (6) MuscaTeLLo, G. 1895 Arch. f. Path. Anat., 142, 327. (7) Herpennain, R. 1891 Pfliiger’s Arch. f. d. gesammt. Physiol., 49, 209. (8) CounstEIn, W. 1895 Zentralbl. f. Physiol., 60, 484. (9) Danpy, W. E. and Rowntrer, L.G. 1914. Annals of Surgery, 59, 587. (10) Ranvier, L. 1896 C. R. Ac. des Se., 122, 578. (11) Weep, L. H. 1914 Jour. Med. Research, N. S., 26, 1, 21. A CASE OF A LEFT SUPERIOR VENA. CAVA WITHOUT A CORRESPONDING VESSEL ON THE RIGHT SIDE WILBUR C. SMITH The Anatomical Laboratory of the Wake Forest School of Medicine’ TWO FIGURES Many instances are recorded in the literature of the presence in human fetuses and adults of two venae cavae superiores, with or without a transverse inter-jugular anastomosis. The presence, however, of a left vena cava superior persisting with- out a right (the viscera not being transposed) is comparatively rare. I have studied the original descriptions of all the cases of this nature occurring in the bibliographies by Ancel, P. et Villemin (’08),! Boyd (93), Halbertsma (’62), McCotter (’16), Nitzel (14), and Weigert (81) with the exception of the case of Mausert (99) which was not available, and find that thirteen such cases have been previously recorded. The subject of the anomaly here recorded is a middle aged, well developed male. The right internal jugular and subclavian veins unite to form a comparatively long innominate vein (re- ferred to in this description as the right innominate) which ex- tends obliquely downward and to the left, ventral to the roots of the arteries arising from the aortic arch and unites with the short left innominate vein to form the left vena cava superior. The left vena cava superior crosses ventral to the arch of the aorta, to the left pulmonary artery, and ventral to the root of the lung as it approaches the dorsal surface of the heart; here 1 The case of Ancel, P. et Villemin (’08) is usually cited in the literature as being one of simple left vena cava superior, is one of a double vena cava. The case of Cheselden (1713) (the correct reference to which is given in the accom- panying bibliography), has been difficult to find on account of the frequence with which an erroneous reference has been given. 191 192 WILBUR C. SMITH it reaches the sulcus coronarius and becomes continuous with a large coronary sinus which opens into the right atrium in the usual situation. | A careful dissection was made for a vein representing the right vena cava superior, but no trace was found excepting the ter- minal part of the azygos which represents that part of the vena cava superior developed from the anterior cardinal. The highest right superior intercostal vein (draining the first space) is a tribu- tary of the right vertebral. The azygos vein is somewhat smaller than normal, but receives the usual tributaries. It opens by means of the persisting caudal part of the anterior cardinal into the right innominate vein about one inch from its right extremity. On the left side the highest intercostal is a tributary of the left vertebral vein. The uninterrupted hemiazygos system isa large vein (representing the left superior intercostal, hemiazygos and aecessory hemiazygos) which opens into the left vena cava superior. Its caliber is nearly as large as that of the normal internal jugular vein. ‘There are two inferior thyreoid veins (one for each lobe), each of which opens into the right innomi- nate vein. The right internal mammary vein empties into the right innominate vein ventral to the termination of the azygos; the left is represented by two veins, the larger empties into the left vena cava superior, the smaller (representing the pericardio- phrenic tributary) into the left innominate vein. The cardiac velns are normal in position and termination. The great car- diac is smaller than usual. ; The heart is normal in size and position. In the upper dor- sal part of the right atrium at the site of the ostium of the vena cava superior, the atrial wall is covered within by musculi pectinati. Below this point the inner surface of the dorsal wall is smooth. The ostium of the vena cava inferior at the lower and dorsal part of the atrium is normal, there is a faint trace of the inferior caval valve. The fossa ovalis and its limbus are normal, a fora- men ovale being absent. Between the large coronary ostium and that of the vena cava inferior there is no intervening space, nor is there a coronary valve. Both from the exterior and in- LEFT SUPERIOR VENA CAVA WITHOUT THE RIGHT 193 terior of the atrium, the two veins appear to communicate with the atrium by « common opening. ‘The tricuspid valve is nor- mal in position and arrangement. ‘The remaining chambers of the heart are normal. Before offering an explanation for this anomaly, the normal method of development of the veins in question may be briefly recalled. It is well known that the early embryological condition is one in which the veins are symmetrical on the two sides. On either side the anterior cardinal vein unites with the posterior cardinal to form the common cardinal vein (duct of Cuvier), and each common cardinal opens into the lateral part of the sinus venosus (sinus horn) of its own side. ‘The sinus venosus, which is a transversely widened chamber, at first- communicates with the common atrium by a large foramen, but during the for- mation of the interatrial septum, the sinus venosus comes to open into the right portion of the dividing atrium. Somewhat later in development the terminal part of the subclavian veins (which at first open into the posterior car- dinals) migrate cephalad to become tributary to the anterior cardinals. The large trunk on either side caudad of the con- fluence of the subclavian and anterior cardinal veins becomes the primitive vena cava superior. Each primitive superior — cava consists of two regions, a cephalic part originally derived ' from the anterior cardinal and a caudal part derived from the common cardinal. The smaller trunks lying cephalad of the anterior cardinal-subclavian junction of either side represent the internal jugular of the adult. Subsequently there is formed upon the medial side of each internal jugular vein, in close proximity to its junction with the subclavian, a vein (Vena thymico-thyreoidea, Thyng 714) which drains a venous plexus about the developing thyreoid and thymic glands. An anastomosis between these thymico-thyreoid veins evidently forms a transverse anastomosis, connecting the right and left jugulars, which normally becomes the vena anonyma sinistra (Szawlowsky ’91, Anikew ’09, and Thyng 714). This interpretation is substantiated by the fact that the inferior thy- reoid and thymic veins of the adult are usually tributary to the vena anonyma sinistra. 194 WILBUR C. SMITH V. jug. int.d. — ‘ LEG Vinterc. supr.d. =P << inf. d. V- subcl.d. V. anom. d. V. mam. int.d. V. thy m. ey 3 —} V. ag. any v. interc, su.d. V. cav. inf. = V. jug. int.s. V. thyr. med.s. yr iy WV. trans. Scap. ees V. yerteb.s. “a N. subcl.s. = V. anom.s. 5 V: peric. phr, = Vi mam. ints. 2 D2 V. Cav. Sup. Soa ay hemiaz D 4 4 V interc. SWS, Fig. 1 Ventral aspect of heart and thoracic veins ABBREVIATIONS USED IN FIGURES 1 AND 2 A.pulm., A. pulmonalis Aor.asc., Aorta ascendens Atr.d., Atrium dextrum Au.s., Auricula sinistra Gl.thyr., Glandula thyreoidea V.anom.d., V. anonyma dextra V.anom.s., V. anonyma sinistra V.az., V. azygos V.cav.inf., V. cava inferior V.cav.sup., V. cava superior V .hemiaz., V. hemiazygos V.interc.supr.d., V. intercostalis su- prema dextra V interc.su.d., V. intercostalis superior dextra V .interc.su.s., V. intercostalis superior sinistra V.jug.int.d., V. jugularis interna dex- tra V.jug.int.s., V. jugularis interna sinis- tra LEFT SUPERIOR VENA CAVA WITHOUT THE RIGHT Vv. jug. int. d. | a ar V. subcl. d. ly, = QQ \infad én i, 195 V. thyr. med. S. —— ee, jUD.INTS, V. verteb.s. V trans. Scap.S. V. subcl.s. V.anom.s. : V. anom.d \ D2 V. mam. int. d. ~ a V. peric.phr. V. thym. aise) Nv; mam. ints Aor. asc. ee V. hemiaz Ala) sy i NV. interc. Sus. vicov. inf: Vicav. Sup Z Owing s- EE, Fig. 2. Ventral aspect of thoracic veins. The apex of the heart has been turned toward the right to expose the left vena cava superior. ABBREVIATIONS (CONTINUED) V.mam.int.d.. VY. mammaria interna dextra V.mam.int.s., V. sinistra V.peric.phr., V. pericardiaco phrenica V.subel.d., V. subclavia dextra V.subcl.s., V. subclavia sinistra V.thym., V. thymica V.thyr.inf.d., V. thyreoidea inferior dextra mammaria interna V.thyr.inf.s., V. thyreoidea inferior sinistra V.thyr.med.s., V. thyreoidea media sinistra V.trans.scap.s., V. transversa scapulae simstra V.verteb.s., V. vertebralis sinistra Vent.d., Ventriculus dexter Vent.s., Ventriculus sinister 196 WILBUR C. SMITH Normally the blood which reaches the left side of the neck now presumably finds a more favorable course through the trans- verse inter-jugular anastomosis into the primitive right vena cava superior and thence into the right atrium, the greater part of the sinus venosus by this time having been absorbed into the latter. At any rate, the portion of the primitive left vena cava superior, representing the part of the common cardinal immediately caudal of the termination to the hemiazygos sys- tem (posterior cardinal), either atrophies or becomes fibrous. The transverse inter-jugular anastomosis then becomes the left innominate vein of the adult; the terminal part of the left ante- rior cardinal forms the proximal part of the left superior inter- costal, and the caudal portion of the left common cardinal per- sists as the oblique vein which is tributary of the coronary sinus. The adult vena cava superior formed by the confluence of the right left innominate veins, represents the terminal part of the right anterior cardinal together with the entire right common . cardinal] vein. EXPLANATION OF ANOMALY The anomaly above described is apparently due to the fact that subsequent to the formation of the transverse inter-jugular anastomosis, the right common cardinal was obliterated instead of the cephalic portion of the left common cardinal, which here remained intact. This condition may be explained by assuming either that the left thymico-thyreoid vein had a more caudal origin than normally occurs, or that it migrated early in develop- ment to a more caudal position than is usual. In either case when the transverse inter-jugular anastomosis was formed, the blood, by following the course of least resistance, must have flowed from right to left instead of vice versa as usually occurs. I wish to take this opportunity to extend my appreciation to Profs. H. D. Senior and F. W. Thyng of the University and Bellevue Hospital Medical College for their kind suggestions during the preparation of this paper. LEFT SUPERIOR VENA CAVA WITHOUT THE RIGHT 197 BIBLIOGRAPHY Antkew, A. 1909 Zur Frage tiber die Entwickelung der Vena anonyma sinis- tra. Anat. Anz., Bd, 34, 8. 24-29. ANCEL, P. ET VILLEMIN 1908 Jour. de l’Anat., vol. 44, pp. 46-62. Béparp, 1892 Vena Cava supérieure située a gauche. Bulletins de la société d’ anthropologie de Paris, T. 30, p. 379. Boyp, 8S. 1893 A case of left superior cava without transposition of viscera. Jour. Anat. and Physiol., vol. 27, N. S., vol. 7, p. 20. Cuares, J. J. 1889 Note of a case of persistent left superior vena cava, being in great part a fibrous cord. Jour. Anat. and Physiol., vol. 33, N.S., vol. 3, p. 649. CHESELDEN, W. 1713 Some anatomical observations. Philos. Trans., vol. 27-28, p. 281. Derrricu, A. 1913 Uber ein Fibrioanthrosarkom mit eigenartiger Ausbreitung und iiber eine Vena Cava superior sinistra bei dem gleiden Fall. Ar- chiv f. Path. Anat., Bd. 212, S. 119-139. GREENFIELD, W. 8. 1876 Persistence of left superior vena cava with absence of the right. Trans. Pathol. Soc. of London, vol. 27, p. 120. Gruser, W. 1880 Vorkommen einer Vena cava superior sinistra (bei Abwe- senheit der V. cava superior der Norm.) (3. der im Verlaufe von 167 Jahren zur Kenntnis gekommenen Fille.) Virchows Archiv, Bd. 81, S. 458. HaLBertsMA 1862 De Afwyking van het Tusschenschot der kammers en der primitive aorta naar links, met hare gevolgen; spiter deutsch im Archiv fiir die hollandischen Beitrige zur Natur-u. Heilkunde. Bd. 3; 5S. 387. Linpes, Geora 1865 Ein Beitrag zur Entwicklungsgeschichte des Herzens. Diss. Dorpat. MarsHALu, J. 1850 On the development of the great anterior veins in man and mammalia. Philos. Trans. Royal Soc. vol. 140, part 1, pp. 133-170. Mia&usert, A. 1899 Zue Casuistik der vena cava superior sinistra und der einen Spitzenlappen der rechten lung abschniirenden anomalie der vena azygos. Diss. Giessen. McCorrer, Rotito E. Three cases of the persistence of the left superior vena cava. Anat. Rec., vol. 10, pp. 371-383. MerkeL, H. E. 1912 a Missbildungim Bereich der oberen Hohlvene. Muen- chener Medizinische Wochenschrift, Bd. 59, S. 615. 1912b Missbildung der oberen Hohlvene. Muenchener Medizin- ische Wochenschrift, Bd. 59, S. 110. NurzeL, H. 1914 Beitrag zur Kenntnis der Missbildungen im Bereiche der oberen Hohlvene. Zeitschr. f. Path., Bd. 15, S. 1-19. Scuréeper, R. 1911 Uber anomalien der pulmonalvene Zugleich im beitrag zum Cor biloculare. Archiv f. Path. Anat., Bd. 205, S. 122. SzawLowskI, J. 1891 Zur Morphologie der Venen der oberen Extremitat und des Halses. Dockt.-Diss., St. Petersburg. THE ANATOMICAL RECORD, VOL. 11, No. 5 198 WILBUR C. SMITH Tuyne, F. W. 1914 The anatomy of a 17.8 mm. human embryo. Am. Jour. Anat. vol. 1@.pps!-09. WEIGERT, C. 1881 Ueber einen Fall von links cenlnabemion Vena cava superior, mutmaaslich bedingt durch frihzeitige Synostose der Sutura mastoi- dea dextra. Virchows Archiv, Bd. 84, 5. 184. THE INNERVATION OF THE MUSCLE RETRACTOR OCULI G. S. HOPKINS Cornell University, Ithaca, New York ONE FIGURE In view of the great number of dissections of the cranial nerves of the horse that presumably have been made in the veterinary colleges of this country and of Europe and in view of the probably still greater number of similar dissections of cer- tain of our domestic animals, especially the dog, the cat and the rabbit that have been made in the numerous laboratories of comparative anatomy and physiology, it would seem that nothing further remained to be said concerning the gross anatomy of these nerves. However, after many dissections of the cranial nerves of the horse and certain other of the domestic animals I am convinced that the descriptions of two of these nerves, viz., the N. oculo- motorius and the N. abducens as given in many of the standard veterinary and comparative anatomies, are incorrect. The error referred to consists in attributing two sources of nerve supply to the M. retractor oculi (retractor bulbi, suspensor oculi, posterior rectus, choanoid) namely, the N. oculomotorius and the N. abducens whereas the muscle is innervated exclu- sively by branches from the latter nerve. The most common statement as to the distribution of the Nn. oculomotorius and abducens, in quadripeds, is essentially that given by Chauveau as long ago as 1857. According to this author the N. oculomotorius is distributed to the following eye muscles—the dorsal, medial and ventral recti, the obliquus ventralis (or externus), the levator palpebrae dorsalis and the retractor oculi with the exception of its lateral portion; it also supplies one or more motor roots to the ciliary ganglion. The 199 200 G. S. HOPKINS N. abducens, according to Chauveau, supplies the M. rectus lateralis and the lateral portion of the retractor oculi. (Foltz states that Chauveau subsequently found that the M. retractor oculi was innervated exclusively by the N. abducens). This distribution of the two cranial nerves under discussion is, in the main, correct; the nerve supply to the M. retractor oculi however, as given by Chauveau and a number of other writers is without doubt incorrect. A brief review of the innervation of the M. retractor oculi as given by several writers, in mammals and in some other animals will first be noted. M’Fadyean gives precisely the same distribution as Just quoted. Bradley’s description is the same as the above with this shght difference, viz., the medial part only of the M. retractor oculi is mentioned as receiving a branch from the N. oculomotorius; the lateral portion of the muscle, according to Bradley, is sup- plied by a branch of the N. abducens, as described by Chauveau and M’Fadyean. In the latest American edition of Strange- way’s Veterinary Anatomy no mention whatever is made of any portion of the M. retractor oculi being supplied by the N. oculo- motorius. But taken in connection with what is said of the muscle ‘‘that it completely envelopes and forms a sheath round the extra cranial portion of the optic nerve’ and also in con- nection with what is said concerning the distribution of the N. abducens “‘it is distributed to the lateral rectus and the lateral portion of the retractor oculi”” one may fairly infer that the M. retractor oculi with the exception of its lateral portion, is sup- plied by some other nerve than the abducens, presumably by the N. oculomotorius. In the first edition of his Veterinary Anatomy Sisson men- tions both the oculomotorius and abducens as supplying branches to the M. retractor oculi; one portion of the muscle being sup- plied by a branch from the dorsal portion of the oculomotorius while the dorsal and lateral parts of the muscle are supplied by the N. abducens. In a subsequent edition, however, this error is corrected the M. retractor oculi being described as innervated by the N. abducens only. INNERVATION OF MUSCLE RETRACTOR OCULI 20) According to Share-Jones the M. retractor oculi receives branches from three different sources—from the oculomotorius, the trochlearis and the abducens. Martin and Franck state that the M. retractor oculi receives its nerve supply from both the dorsal and ventral branches of the oculo-motorius and from the abducens. In contrast with this statement of Martin and Franck is that of Ellenberger and Baum, also Gurlt, who mention the dorsal branch only of the oculomotorius and the abducens as supply- ing branches to the M. retractor oculi. Struska agrees in all respects with Ellenberger, Baum and Gurlt. Leisering says that the lateral portion of the retractor oculi is supplied by the abducens and the remaining portions of the muscle by branches from the oculomotorius. According to Zimmer! the M. retractor oculi is supplied from the dorsal branch of the oculomotorius and from the abducens but he does not mention the particular portions of the muscle supplied from each of these two sources. Varaldi gives practically the same distribution as Zimmer] only he does not state from which branch, dorsal or ventral, of the oculomotorius the filaments to the retractor oculi are given off. In an article on the development of the eye muscles of the pig, Reuter describes the M. retractor oculi as receiving its nerve supply from both the oculomotorius and the abducens. In the dog Bradley, Ellenberger and Baum describe both the dorsal and ventral portions of the N. oculomotorius as supplying branches to the M. retractor oculi; they make no mention of any branch from the N. abducens to this muscle. In the second edition of Anatomie des Kaninchen, Krause describes and figures the ventral ramus of the N. oculomotorius as giving off a branch tothe M. retractor oculi. Concerning the N. abducens he says that it gives branches to the M. rectus oculi posticus (rectus lateralis). In many mammals, he says the M. retractor oculi is supplied, as in the rabbit, by the N. oculo- motorius; in the cat and the calf, however, the N. abducens is the source of the nerve fibers for the same. 202 G. S. HOPKINS Bensley gives precisely the same distribution of these two nerves in the rabbit as does Krause. ; Reighard and Jennings say that in the cat the M. retractor oculi receives its nerve supply from the N. oculomotorius and that the N. abducens is distributed to the M. rectus lateralis; there is no intimation whatever that the latter nerve gives any branches to the M. retractor ocull. Mivart, Wilder and Gage on the other hand describe the M. retractor oculi of the cat as supplied wholly by branches from the N. abducens. Concerning the innervation of this muscle, Brinton says ‘‘the muscle which sweeps the broad nictitating membrane across the bird’s eye and the funnel shaped or choanoid muscle (retractor oeuli) which surrounds the optic nerve and eyeball of many mammatlia are both supplied from this nerve (N. abducens). Wiedersheim also describes the N. abducens as supplying the lateral rectus, the retractor oculi and the muscular apparatus of the membrana nictitans in sauropsida, thus agreeing in all respects with Brinton’s account of the nerve. From experimental evidence on the live horse and rabbit Foltz asserts that the N. oculomotorius supplies nothing to the M. retractor cculi but that the N. abducens alone supplies this muscle and the lateral rectus. In a note he further states ‘it is stated in the treaties of Veterinary Anatomy that this muscle (retractor oculi) in the domestic animals is animated chiefly by the common oculomotor.” Chauveau has found re- cently and our experience confirms it, that this muscle is ani- mated exclusively by the oculomotor externus (N. abducens). According to Owen “in lower Quadrumana a few fibers seem to be detached from the inner part of the origin of the recti to be inserted into the sclerotic nearer the entry of the optic nerve. This is the remnant of a stronger muscle which in other mam- mals, with few exceptions, surrounds the optic nerve, expanding funnelwise, as it approaches the back of the eyeball; it is called the choanoid, muscle, or suspensor oculi, and is supplied by a branch of the sixth cerebral nerve.”’ INNERVATION OF MUSCLE RETRACTOR OCULI 203 Montane and Bourdelle describe and figure the N. oculomo- torius as supplying all the muscles of the eye except the external rectus, the posterior rectus (retractor oculi) and the great oblique. The N. abducens they state supplies the external rectus and the posterior rectus. Confirmatory evidence of the error of those who described the N. oculomotorius as supplying branches to one or more portions of the M. retractor oculi are found in the distribution of these two nerves in some of the reptiles. In a paper on the development of the musculature of the head and extremities of reptiles, Corning says that in the lizard (Lacerta vivipera) the abducens muscle mass which gives rise to the Mm. retractor oculi and rectus lateralis is supplied by the N. abducens. Pre- cisely the same distribution of these two nerves is given by Hoff- man for another lizard (Lacerta agilis) and for the turtle. In the Crocodile also, according to Fischer, the N. abducens is distributed as in the lizards. In an investigation on the development of the prootic head somites and eye muscles in Chelydra serpentina, Johnson found that the N. abducens supplies the Mm. rectus lateralis and re- tractor oculi. No portion of the latter muscle is innervated by the N. oculomotorius. The writer’s conviction of the incorrectness of the descriptions of the distribution of these two nerves, as given by several authors, is based on repeated dissections of the nerves in the horse, ox, sheep, pig, dog, cat, and rabbit; in the woodchuck and the badger the nerves were dissected but once. In most cases the nerves were traced their entire length, i.e., from their superficial origin from the brain to their respective muscles. All of the dissec- tions were made with the greatest possible care under a binocular dissection microscope. In the pig the nerves were also traced microscopically in a 42 mm. embryo cut into sections of 20 microns. In all of these animals the dorsal branch of the N. oculomotorius was distributed to the Mm. rectus dorsalis and the levator palpebrae; the ventral branch was distributed to the Mm. rectus ventralis, rectus medialis and to the obliquus ven- tralis. In all cases one or more small branches were given off ~ vs % Co 82 SD Sh 21 23 = Mi: . M. = MI = Py wh vi Ae [A wary 8 - - ween ne . Cut surface of supraorbital process. A Ai Wee ji i ik Fig. 1 24. A. sphenopalatina. 25. N. maxillaris, cut and one end . Frontal sinus. . Cut surface of the zygomatic proc- ess of the temporal. Cut surface of the malar. . Palpebrae. . Gl. lacrimalis (somewhat reflected). . M. rectus dorsalis or superior. . M. levator palpebrae dorsalis or superior. . M. rectus lateralis. M. rectus ventralis or inferior. rectus medialis. obliquus dorsalis or superior. obliquus ventralis or inferior. retractor oculi or bulbi. (The greater part of the muscle has been removed; only the two ex- tremities, 14 and 14’ are shown). . Cut edge of sphenoid. . maxillaris interna. . ophthalmieca. . temporalis profunda anterior. . supraorbitalis or frontalis. . Small artery to the mass of adipose tissue in the temporal . A. infraorbitalis. . A. orbitalis or malaris. . A. buecinatoria. SM: > PPP ossa. turned aside. . lacrimalis, cut and turned aside. . supraorbitalis or frontalis. . nasociliaris or palpebronasal. . ethmoidalis. . infratrochlearis. . N. trochlearis. . Sensory root of Ganglion ciliare. and 34. N. oculomotorius, dorsal and ventral branches. 35. Small branch from the N. oculo- motorius to the M. levator palpebrae dorsalis. . N. abducens. 37. N_ orbitalis or zygomaticus (the peripheral portion has been re- moved). . Ganglion ciliaris. . Nn. ciliares. . N. opticus . N. sphenopalatinus. . Ganglion sphenopalitinum showing many small nerves leaving it. 3. N. palatinus anterior or major. . N. palatinus posterior or minor. 5. Cut edge of the periorbita or ocu- lar sheath. AZAAZAAZA INNERVATION OF MUSCLE RETRACTOR OCULI 205 to the ciliary ganglion. In no case were there found the slightest indications of filaments from either the dorsal or ventral branches of the N. oculomotorius to the M. retractor oculi as stated by so Many. In all cases the N. abducens, figure 1, 36, supplied all portions of the M. retractor ocult. Most of the statements regarding the form of the M. retractor oculi and its relation to the optic nerve are somewhat mislead- ing. In some of the domestic animals as the horse, ox, sheep and pig this muscle is not readily divisible into four distinct portions—dorsal, ventral, medial and lateral as it is in the dog, eat and rabbit, but forms a continuous sheet which surrounds the posterior part of the eyeball and a part of the extra cranial portion of the optic nerve. The medial side of the optic nerve for a distance of one and one half centimeters from the apex of the orbit, in the horse, is entirely uncovered by this muscle all the fibers of which are attached to the lateral side of the optic nerve as shown in the figure (14’). LITERATURE CITED Benstey, B. A. 1910 Practical anatomy of the rabbit. P. Blakiston’s Son and Company, Philadelphia. Brabiey, O. C. 1897 Outlines of veterinary anatomy. London. Also W. R. Jenkins, New York. 1912 A guide to the dissection of the dog. London, Longmans, Green and Company. Brinton, Wm. 1847-49 Cyclopaedia of anatomy and physiology, vol. 4, part I, p. 622. CHAUVEAU, A. 1857 Traite d’ Anatomie Comparee Animaux Domestiques. Cornine, H. K. 1900 Ueber die Entwicklung der Kopf und Extremitaten Muskultur bei Reptilien. Morpho. Jahrbuch, Bd. 28, pp. 28-104. ELLENBERGER U. Baum 1891 Anat. des Hundes. Berlin. 1908 Handbuch der Vergleichenden Anat. der Haustiere. Berlin. FiscHER 1890 Bronn Klassen des Thier Reichs VI, III, 16-42. Reptilien, p. 753. Foutz, J. C. E. 1862 Recherches d’ Anat. et de Physiologie Experimentale sur les Voies Lacrymales Jo. de la Physiologie, Tome V, pp. 226-247. Franck, L. 1894 Handbuch der Anat. der Haustiere. Stuttgart. Gurut, E. F. 1873 Vergleichende Anat. der Saugethiere. HorrMann, C. K. 1890 Bronn Klassen des Thier Reichs, VI, III, I, 15, Rep- tilien. 206 G. S. HOPKINS Hopkins, G. 8. 1913 Directions for the dissection and study of the cranial nerves and blood vessels of the horse. Published by the author. Jounson, C. E. 1912-13 :The development of the prootic head somites and eye muscles in Chelydra serpentina. Am. Jour. Anat., vol. 14, pp. 119-186. IKkrause, W. 1884 Die Anat. des Kaninchens. Leipzig. LEISERING. 1899 Atlas der Anat. des Pferdes und der ubrigen Haustiere. Leipzig. Martin, P. 1904 Lehrbuch der Anat. der Haustiere. Stuttgart. Mivart, Sv. G. 1889 Lessons in elementary anatomy. Macmillan and Com- pany, New York. M’Fapynan, J. 1902 The anatomy of the horse. W. R. Jenkins Company, New York. MonTANE AND BourRDELLE. 1913 Anatomie Regionale des Animaux Domes- tiques, vol. 1, Paris. Owen, R. C. 1868 Comp. Anat. and Physiol. of Vertebrates, vol. 3, pp. 258- DAC 259. REIGHARD AND JENNINGS. 1901 Anatomy of the cat. Henry Holt and Com- pany. Reuter, K. 1897 Ueber die Entwickelung der Augenmuskultur beim Schwein. Anat. Heft., Bd. 9, pp. 367-389. SHARE-JoNES, J. T. 1906 The surgical anat. of the horse. London, part I, p. 154. Sisson, 8S. 1914 The anatomy of the domestic animals. Stranceway, T. 1909 Veterinary anatomy, 12th Am. ed. Srruska, J. 1903 Lehrbuch der Anat. der Hausthiere. Wien u. Leipsig. VaraAtpl, L. 1899 Anatomia Veterinaria, vol. 2. Milano. WirepersHEIM, R. 1902 Vergleichende Anat. der Wirbelthiere. 5th ed. Wiper AND Gace. 1886 Anatomical technology as applied to the domestic cat. ZIMMERL. U. 1909 Tratte di Anatomia Veterinaria, vol. 3. Milano. THE ANATOMY WITH ESPECIAL CONSIDERATION OF THE EMBRYOLOGICAL SIGNIFICANCE OF THE STRUCTURES OF A FULL-TERM FETUS AMORPHUS EBEN CAREY Department of Histology and Embryology, Creighton Medical College, Omaha, Nebraska NINETEEN FIGURES INTRODCUTION Although teratological literature is replete with descriptions of the various types of the fetus amorphus the specimen here studied is of sufficient interest to record, for no description, to which the author had access, was found that entirely coincides with this case. The outstanding characteristics of this inter- esting group of monsters are according to Ahlfeld (04) the absence of a heart, normal bilateral symmetry and generally the brain. If the latter is present to any extent at all it is very rudimentary. The subcutaneous tissue is usually hypertrophied, cystic and oedematous. The cord possesses but one artery and one vein. A partial list of the authorities consulted is the following: Ahlfeld, Ballantyne, Beardsley, Bevill, Brodie, Charlton, Clau- dius, Embleton, Hall, Herholdt, Houston, Hicks and Baukart, Hirst and Piersol, Jacobi, J. Jackson, J. S. B. Jackson, Le Cat, Lusk, Mall, Marchand, Meckel, Rauber, Schatz, Schwalbe, Simpson, Tiedmann, Von Winckel, Vrolik, Willey and Windle. The specimen that is the subject of this paper was received by the author, upon taking charge of the department in 1914, from Dr. J. S. Foote, professor of histo-pathology, Creighton Medical College. Dr. Foote came into possession of the monster in 1901 shortly after it was born. It was well preserved in Kaiserling’s fluid. The co-twin was a normal female fetus. The period of gestation lasted the full nine calendar months. The 207 208 EBEN CAREY normal fetus was the first to be expelled at parturition. No other clinical data were recorded; the condition of the fetal mem- branes and maternal deciduae were not tabulated. Ten years ago Dr. Foote heard that the normal co-twin was a healthy, robust girl of five years of age. GENERAL APPEARANCES, WEIGHT AND DIMENSIONS A systematic description of each aspect of the fetus follows in the text. The general appearance of the monster was that. of an irregularly rounded, potato-shaped, skin-covered mass with the external indications of the two lower limb buds; a well marked outgrowth for the right upper limb and a slight pro- tuberance over the region of the left upper limb. The ventral surface presented a thoracic and a hypogastric elevation. Upon the cephalic slope of the latter the umbilical cord was located which contained but one artery and one vein. The parietal, occipital and mid-dorsal regions carried hair. There was no external indication of a neck. No skeleton would be suspected by palpation. Upon percussion a tympanic note was elicited in the left lower thoracic region. There proved to be a large cavity, (1/7, fig. 18.) in this area, outside the skeleton, in the subcutaneous tissue. The facial and genital aspects, the limb buds and right glandular area were interesting from the embry- ological standpoint. The specimen weighed 1130 grams after excellent preservation in Kaiserling’s solution for fifteen years. The greatest length from the tip of the frontal region to the end of the rump was 19 cm. (4 to 16, fig. 2). The crown-rump measurement was 17 cm. (1 to 10, fig. 3). The greatest width through the upper limb buds was 13.5 em. (7, fig. 4). Through the lower limb buds the width was 6.4 em. The greatest thickness 8 cm. (4, fig. 3) was at the mid-ventral point on the line of greatest width. At the location of the external genitalia, the fetus amorphus was 4.5 em. in thickness. Ontheventralsurface there were two eleva- tions with an intervening furrow (6, fig. 3). The apex of the caudal elevation (8, fig. 3) was just below the umbilicus in the hypogastric region. The thickness of the embryo at this point was 6 cm. ANATOMY OF A FULL-TERM FETUS AMORPHUS 209 SYSTEMATIC DESCRIPTION OF THE TOPOGRAPHY) Right ventro-lateral aspect (fig. 1) The cephalic part of the fetus 1s inclined obliquely dorsad, so as to bring more prominently into view and to show the con- tinuity and relationships of the lower limb buds, /2 and 13, and external genitalia; 9, body of the clitoris, 70, glans clitoridis, 11, ostium urogenital, 75, right labia majora, of the ventro- caudal aspect of the fetus, with the rest of the ventral surface Fig. 1 (About one-half natural size.) 1, left globular process; 2, left maxil- lary process; 3, oral pit; 4, groove at ventral junction of mandibular arches; 4, Nn depression of right glandular area; 6, right upper extremity; 7, umbilical cord; 8, the left ventro-latero-dorsal fissure; 9, body of the clitoris; 10, glans clitoridis; 11, ostium uro-genital; 12, left lower limb bud; 13, right lower limb bud; 14, imperforate anus; 15, the right labia majora; ventral aspect. gn 210 EBEN CAREY of the body. The embryological consideration of the structures of the external genitalia will be reserved until plate 2 is described. Directly cephalad from the genitals is seen the remnants of the umbilical cord 7, which contains only one umbilical artery instead of two as are normally found. In the right cephalo-lateral aspect is seen the marked protuberance of the right upper ex- tremity 6. The slight protuberance, directly across the body on the left side, in the region of the left upper extremity is seen to a better advantage in figure 4, no. 7. The location of the imperforate anus 1s marked by 14. The interesting fact to be considered in studying this aspect is the depression 5, of the right glandular area. No similar depression is seen on the left side of the body. This depression is a persistency of the depressed right glandular area, which appears in an embryo of about 25 cm., according to Pinkus (10) and gradually deepens until it is well marked in a fetus of eight months. At this time the nipple is also supposed to be partly formed, but no sign of a nipple is seen in this specimen. The genetic significance of the structures of the facial aspect; 1, left globular process, 2, left maxillary process, 3, oral pit, 4, groove at ventral junction of mandibular arches, will be dis- cussed when plate 1 is considered, figures 6 to 10. Left ventro-lateral aspect (fig. 2) This view of the fetus is presented in order to show the con- tinuity of the rudimentary facial structures with the ventral aspect of the thorax and abdomen. ‘The cephalic part of the fetus is inclined obliquely ventrad, in contra-distinction to the position as shown in figure 1, in which the inclination was ob- liquely dorsad. The most important fact clearly presented is the absence externally of the neck separating head and thorax. Dorsal aspect (fig. 4) The left frontal region 7, is prominently marked. The hair is characteristically arranged over this area in converging whorls. On the other hand the hairs over the crown or vertex 4, are ANATOMY OF A FULL-TERM FETUS AMORPHUS 211 arranged in diverging whorls, the direction of the hairs being toward the right, over the left side of crown as can be made out from the photograph. The literature on the subject of the whorls of the hair, together with a complete bibliography, 1s presented by Pinkus (710). The external indication of the loca- tion of the superior sagittal suture is seen as a suleus, 3. The c Qn = 5 6 T 8 9 Fig. 2 (About one-half the natural size.) 1, right nasal pit;2, right globular process; 3, suleus between right and left globular processes; 4, ventral indica- tion of prosencephalon; 5, left globular process; 6, left nasal pit; 7, palpebral folds of left eye; 8, tongue in oral pit; 9, left maxillary process; 10, ventral junc- tion of mandibular processes; 1/1, depression for right glandular area; 12, right upper extremity; 13, left ventro-latero-dorsal fissure; 14, umbilicus; 15, left lower limb bud; 16, external genitalia, left ventro-lateral aspect. 212 EBEN CAREY right frontal and right crown regions are not so clearly differ- entiated morphologically in the fetus as the left regions. How- ever, the converging and diverging whorls of the frontal and crown areas respectively, are seen, although they do not show so clearly on the right side as upon the left. The normal and abnormal facial aspects (figs. 6, 7, 8, 9, and 10) The abnormal facial structures of the monster are exceedingly interesting, due to the fact that they retain the external facial Fig. 3 (About one-half natural size.) 1, frontal area; 2, palpebral folds of left eye; 3, occipital area; 4, protuberance over left upper extremity; 5, cephalo- thoracic elevation; 6, ventral depression; 7, left ventro-latero-dorsal fissure; ventro caudal elevation; 9, left lower limb bud; 1/0, imperforate anus; left lateral aspect. ANATOMY OF A FULL-TERM FETUS AMORPHUS 213s characteristics, however considerably distorted, of an embryo of the second month. In the light of normal facial develop- ment, as first satisfactorily studied by His (’80), and later by Keibel and Elze (08), Hertwig (06), Mall (’91 and 93), Retzius (04) and others we are enabled to interpret the facts here pre- sented. Although considerably distorted, the two large globular proc- esses 5 and 10, developed from the lateral aspects of the median Fig.4 (About one-half natural size.) 1, left frontal region; 2, palpebral folds of right eye; 3, superior sagittal sulcus; 4, occipital region; 5, deep furrow dorso- mesiad of right shoulder; 6, right upper extremity; 7, protuberance of left upper extremity; 8, dorsal line of the body; 9, ventro-latero-dorsal fissure; 10, right lower limb bud; /1, imperforate anus; dorsal aspect. THE ANATOMICAL RECORD, VOL. 1!, No. 5 214 EBEN CAREY Fig. 5 (About one-half natural size.) 1, apex of right maxillary process; 2, right upper extremity; 3, depression of right glandular area; 4, ventro-cephalic elevation; 5, tip of the right upper extremity; 6, umbilical cord; 7, ventral depres- sion; 8, ventro-caudal elevation; 9, button-shaped right lower limb bud; 10, location of external genitalia; right lateral aspect. Figs. 6-10 The indication numbers of figures 6, 7 and 10 mark out similar structures. 1, crown; 2, frontal area; 3, sulcus between the right and left globu- lar processes; 4, palpebral folds of right eye; 5, right globular processes; 6, naso- lacrimal furrow; 7, nasal pit; 8, palpebral folds for left eye; 9, left nasal pit; 10, left globular processes; 17, left maxillary processes; 12, left mandibular proc- esses; 13, ventral furrow intervening between mandibular arches; 14, right maxillary process; 7’, in figures 6 and 7, lateral nasal processes. Figures 6, 7, 8 and 9. Development of the face of the human embryo by His taken from Heisler: figure 6, embryo of about twenty-nine days. The nasal frontal plate differentiating into processes globulares, towards which the maxillary processes of first visceral arch are extending. Figure 7 of about 34 days: the globular, lateral, frontal, and maxillary processes are in apposition; the primitive open- ing is now better defined. Figure 8 embryo of about the eighth week: immediate boundaries of mouth are more definite and the nasal orifices are partly formed, external ear appearing. Figure 9, embryo at the end of the second month. ANATOMY OF A FULL-TERM FETUS AMORPHUS 215 ai Let AINDaA OW 1 2 216 EBEN CAREY nasal process are seen separated by a well marked fissure 3. The right and left nasal pits 7 and 9 respectively are shown as cicatricial depressions. The lateral nasal processes are not dis- tinct on either side. However, their location is differentiated from the maxillary processes by the persistent naso-optic fissures best marked on the right side by 6. The palpebral folds of the right and left eye, 4 and 8 respectively, are clearly made out. The right is better developed and shows a fissure as does also the left, between upper and lower eyelids. At the inner angle of the right orbit a groove or furrow con- tinuous with the fissure between the two primitive eyelids is well marked, not so clearly seen on the left side, which courses to the lateral aspect of the anterior nares and is no doubt the persistent naso-optic fissure. The fissure is normally present as seen in figures 6 and 7, No. 6, but later becomes obliterated as seen in figure 9. On the left side the naso-optic fissure is not so well marked. The right and left maxillary processes, 14 and 11 respectively, are seen as prominent lateral bulgings of the cheeks. The external indication of the left mandibular arch 12, is apparent, separated by a narrow shallow furrow, 13, from the external indication of the right mandibular arch. This groove 13, leads cephalad to the distorted tongue, in the oral pit, shown darkly colored and protuberant in the photograph. The primitive oral cavity is pentagonal in outline, bounded cephalad by the left enlarged globular process, caudad by the two indistinct external indications of the mandibular arches, and laterad by the external indications of the right and left maxillary processes. The facial aspect thus presents a double hare-lip due to lack of complete fusion of the maxillary and globular proc- esses of their respective sides and the complete split-nose 3, due to lack of union of the right and left globular processes. No bony nor cartilaginous structures were found in the meso- derm immediately underlying the superficial facial structures considered in this topographical description. No ears were present. ANATOMY OF A FULL-TERM FETUS AMORPHUS 217 The normal and abnormal genital aspects (figs. 11, 12, 13, 14 and 15) The structures of the genitalia of the monster are interesting, for to a certain degree they retain the characteristics of an embryo, however, considerably enlarged, at the beginning of the third month. At this time the indifferent phallus, by the direc- tion which it takes, shows a transitory sexual differentiation. According to Herzog (’04), the phallus in the female bends down- wards and in the male it is perpendicular to the long axis of the body. By now referring to figure 15, we see an abnormally enlarged phallus 3, which has practically differentiated into the clitoris. The female phallus at the beginning of the third month may even be larger than that of the male; the downward direction as seen is diagnostic of the female embryo. Surmounting the phallus is the glans 4, at the base of which is an encircling fold, the praeputium. The urogenital sinus 6, is seen patent as a groove; the genital folds which bound this groove have receded inwards due to the overgrowth of the genital tubercles or swell- ing which now form the labia majora, 5. The urogenital open- ing distally, towards the glans clitoridis, on the anal slope of the phallus has closed and has formed the urethral groove. Proximally, towards the anus, the urogenital opening 6, remains patent, which is also diagnostic of the female embryo. INTERNAL ANATOMY Subcutaneous and muscular tissues The fetus was opened by a median incision running to the left of the umbilicus. It was then observed that the skin was firmly adherent to the underlying dense oedematous connective tissue and fat. This tissue was less fibrous as the bony struc- tures were approached. Frozen sections of the tissue contigu- ous to the skeleton were stained with hematoxylin and eosin. Isolated fibers of voluntary muscle were found intermingled with the connective tissue. There were no well defined groups of 3 4 —5 6 T -8 9 Figs. 11-15 (Figs. 11, 12, 18, and 14. 1, phallus; 2, glans clitoridis; 3, ostium urogenital; 4, labia majora; 5, anus; 6, coccygeal eminence; 7, labia minora. Fig. 15 1, ventral surface of fetus amorphus; 2, umbilical cord; 3, body of the 4, glans of clitoridis; 5, left labia majora; 6, urogenital sinus; 7, right lower limb bud; 8, dorsal aspect of right upper limb; 9, imperforate anus. 218 phallus; ANATOMY OF A FULL-TERM FETUS AMORPHUS 219 musculature. In the irregularly arranged muscular fibers were seen various sized vacuoles, which when stained in an alkaline alcoholic solution of scarlet red fat stain, which is Bell’s modifi- cation of the Herheimer method, proved to be fat. Counter staining was made with Delafield’s hematoxylin and the sec- tions were mounted in glycerin. This method proved an excel- lent one for the detection of fat within the muscular fibers. The fat vacuoles were found in the angles of Cohnheim’s areas and in the spaces between the individual sarcostyles. Blood vascular system (fig. 16) The chief features of the blood vascular system of the fetus amorphus are the absence of a heart and, according to Ahlfeld (80 and ’82), the reversal of the flow of blood. In the diagram the arrows indicate the reversed flow of blood according to Ahlfeld’s theory. However this idea is refuted by Breus (’82). The blood enters the body of the fetus amorphus through its single umbilical artery 31, figure 16. As the latter turns to become continuous with the dorsal aorta, there are two branches given off, 35 and 36, the left and right common iliacs, respec- tively. The single umbilical artery is seen to belong to the right side. There is no left umbilical artery present. The right com- mon iliac 36, branches into the internal and external iliac arteries, 37 and 38 respectively. The internal iliac artery ends as shown diagrammatically in figure 16 ina dilatation representing the capillaries of the pelvic viscera. This vessel evidently carries the blood supply to the viscera as no left internal iliac vessel is present. The right external iliac is also shown diagrammati- cally as ending in a dilatation representing the capillary system of the right caudal limb bud. The left common iliac 35, pursues a straight course to the left caudal limb bud where it breaks up into a capillary network. The next vessel given off as we follow the blood current up through the aorta is the right renal artery 30, next the left renal 29, and lastly the hepatic artery 26, which comes off on the ventral surface of the aorta. At the same level, on the BEN CAREY Bf x 1 S ¥ = er 2 S f H : ls 3 Si : HEI 4 BH i re) 8 Wy ; Dorr ~ com idan, 5 ye UD =" Sey MISTS wy re e 2 : ey) 3 Gx 8 S CAA Fy 5 E x E Yo. Fy B® 4 ree FIG 1G ANATOMY OF A FULL-TERM FETUS AMORPHUS 221 left lateral aspect, to the origin of the hepatic artery, the superior mesenteric artery 27, is found. There was no vessel comparable to the inferior mesenteric. The coeliac axis is represented by the single trunk of the hepatic artery. The superior mesen- teric artery 1s a good sized trunk and evidently supplied the regions of the intestine that are normally supplied by the coeliac axis and the inferior mesenteric arteries as well as its own region. At 8, the aorta is seen to bifurcate. The right trunk is very much the larger of the two. The branches given off from these two trunks are comparable on each side. The first branch on the left side is the common carotid artery 3, which bifurcates into the external 7, and internal 2, carotid arteries. Caudad the internal mammary artery is given off, 7, from the sub- clavian, 6. From the dorsal aorta the paired intercostal and lumbar arteries which are not represented in the diagrammatic reconstruction, are given off. The blood is returned from the left and right sides of the head and neck and upper extremities through the left and right superior vena cava 1/5 and 16, respectively. The left external 4 and internal 5, jugulars unite to form a common trunk which in turn with the left subclavian vein 12, unite to form the left superior vena cava 15. At the junction of the two veins men- Fig. 16 1, external carotid artery (left); 2, internal carotid artery (left); 3, common carotid artery (left); 4, left external jugular vein; 5, left internal jugular vein; 6, left subclavian artery; 7, left internal mammary artery; 8, dorsal aorta; 9, vertebral vein; 10, point of fusion of left and right azygos veins; 1/1, trunk of left common jugular vein; 12,-left subclavian vein; 13, left internal mammary vein; 14, common trunk of azygos veins; 15, left superior vena cava; 16, right superior vena cava; 17, common trunk of inferior vena cava; 18, left azygos vein; 19, left inferior vena cava; 20, right inferior vena cava; 21, thoracic aorta; 22, umbilical vein; 23, an anastomotic vein between left and right inferior venae cavae; 24, diagonal branch of umbilical vein draining the intestinal pelvie, and left lower limb buds; 25, the hepatic vein; 26, the hepatic artery; 27, superior mesenteric artery; 28, left renal vein; 29, left renal artery; 30, right renal artery; 31, umbilical artery; 32, superior mesenteric vein; 33, vein draining pelvic region into right inferior vena cava; 34, continuation of femoral vein with the right inferior vena cava; 35, left common iliac artery; 36, right common iliac artery; 37, internal iliac artery; 38, external iliac artery; 39, vein draining pelvic area into diagonal branch of umbilical vein; 40, vein draining left lower limb bud into diagonal branch of umbilical vein; 41, left inferior vena cava. 222 EBEN CAREY tioned above to form the left superior vena cava, the left internal mammary vein 13, joins the left subclavian. The branches forming the trunk of the right superior vena cava are similar to those forming the left superior vena cava but they are not labeled in the diagram. The left vertebral vein is represented by 9; the right is not labeled. The regions drained by the left superior intercostal vein and the azygos system of veins are represented by 18. The right side is not labeled but is similar to the left. The vessels of the two sides after receiving the left and right vertebral veins form a common trunk 14, which flows into the arch of the um- bilical vein. There are no pulmonary veins as well as no pul- monary arteries. The blood is returned from the pelvic viscera through a vessel 33, which empties into the right inferior vena cava and a vessel 39, which empties into a diagonal branch 24, which in turn pours its blood into the umbilical vein shortly before it enters the umbilical ring. The blood is returned from the left caudal limb bud by two veins, one 40, which goes to form with the left pelvic visceral vein 39, the diagonal vein 24, and one 41, which is continuous with the left inferior vena cava. From the right caudal limb bud there is but one vein 34, which joins the vein 33, draining the pelvic viscera. These two latter veins unite to form the right inferior vena cava and no doubt represent the right and left common iliac veins. Upon the left side the left inferior vena cava does not bifurcate into right and left common iliac veins as is found on the right side. The right inferior vena cava receives the right renal and hepatic veins, 25. The left inferior vena cava receives the left renal vein 28. The two inferior venae cavae 19 and 20 unite to form a single trunk which empties into the dorsal aspect of the arch of the umbilical vein. Before these two veins unite there is an anastomotic branch 23, coursing cephalad from left to right. The blood draining the intestines empties through the superior mesenteric vein 32, into the di- agonal vein 24, which in turn pours its blood into the common vein of exit, the umbilical vein, 22. ANATOMY OF A FULL-TERM FETUS AMORPHUS 223 The abnormal reversal of the direction of the blood stream, through a single umbilical artery, with its subsequent accentua- tion of certain arteries and degeneration of others, increases the difficulty of interpreting the significance of the vessels dissected. The arch of the aorta is obliterated and at its location we find a bifureation, with the right integer which supplies mainly the right upper limb, considerably the larger of the two. The left 15, and right /6, superior venae cavae are the persistent left and right anterior cardinal veins. Instead of the two posterior cardinal veins joining their respective anterior cardinal veins, they join each other to form a common trunk 14, which empty into the umbilical venous arch, and is no doubt a per- sistency of the sinus venosus of the embryonic heart. The inferior vena cava is double 79 and 20, up to within one inch of its termination, in the umbilical venous arch where its two components of the right and left sides fuse to form a common trunk, 17. These two posterior venae cavae are undoubtedly the persistent remains of the two subcardinal veins. There is no portal system comparable to that found in the normal adult. The small rudimentary liver possesses but one artery and one vein. The diagonal vein 24, draining the blood from the intestines, pelvic viscera and left limb bud is evidently the remains of the portal system. The blood through this vein empties directly into the umbilical vein. Respiratory system (fig. 17) The lung tissue was completely degenerated. In the thorax there was found a putty-like mass of degenerated tissue through which the oedematous connective tissue was growing. Broken off portions of the cartilaginous bronchi were found throughout the above mass. The small bronchi which contain portions of cartilage and those in the stage of pre-cartilage, proved to be very resistant. The trachea 3, is seen tapering to its bifurcation into the right and left bronchi at 5. There were rudimentary respiratory passages with anterior and posterior nares leading into the dorsal aspect of the deformed mouth. The larynx /, is considerably dilated as seen in figure 17. ee) bo i EBEN CAREY Digestive system (fig. 17) The alimentary tract began at the mouth, and at the caudal part of the pharynx dorsad to the larynx the esophagus 2, figure 17, began. A small dilatation 6, is detected in the region of the stomach. A duodeno-pyloric flexure is made and at the base of the umbilical ring 7, and extending into the umbilical cord, a few coils of small intestine 9, were unraveled. The small cecal evagination 8, was found at the base of the umbilical ring. The large intestine had already made the primary twist across the small intestine. From the cecum the large intestine 13, is traced to its termination in the rectum 25. The descend- ing and part of the transverse colons are marked out at this stage of development. The relationships of the intestinal coils to the umbilical vein 1/0 and umbilical artery 17, and urachus 12, at the base of the umbilical ring, are also shown in figure 17. There was no pancreas present. The common bile duct was absent. The rectal cul-de-sac is connected to the utero-vagi- nal tube by a fissure-like opening 22. Uro-genital system (fig. 17) The urinary apparatus is separated from the genital struc- tures as shown in the figure. Both kidneys 16, are tri-lobed, the left being slightly larger than the right one. A ureter 17, leads from each to the bladder, 2/. From the bladder leading to the uro-genital sinus is the urethra 23. From the tip of the bladder there is a patent tube, the urachus 12, which leads up to and out through the umbilical cord. Fig. 17 1, The larynx; 2, the oesophagus; 3, trachea; 4, broken off bronchi in thorax; 6, bifurcation of trachea into the right and left bronchus; 6, spindle shaped stomach; 7, umbilical ring; 8, cecum; 9, loops of small intestine; 10, um- bilical vein; 11, umbilical artery; 12, urachus; 13, descending colon; 14, left renal vein; 15, left renal artery; 16, tri-lobed metanephros; 17, left ureter; 18, fimbri- ated end of left Fallopian tube; 19, left Fallopian tube; 20, dichotomus division of the uterus into the two Fallopian tubes; 21, bladder; 22, opening between rec- tal cul-de-sac and the utero-vaginal tube; 23, urethra; 24, vaginal portion of ureto-vaginal tube; 25, distal end of rectal cul-de-sac. ANATOMY OF A FULL-TERM FETUS AMORPHUS Mm i CCC GN Ss & \} : n PAO oom} dp ff j SD yyy dj bo or 226 EBEN CAREY The genital system is composed of a utero-vaginal tube 20 to 24, continuous with the two oviducts which branch immedi- ately in a Y-shaped manner. The latter end in a fimbriated dilation 18, the left slightly larger than the right. There were no ovaries. The alimentary tract is connected with the utero- vaginal tube at the location marked 22, which represents the persistent connection between the alimentary tract and the uro- genital systems in the cloaca. Nervous system There was an entire absence of the brain and cranial nerves. The upper part of the cord was absent as far as the seventh cervical nerve. From this point to the end of the lumbar vertebra the cord was present. There was a rudimentary cauda aquina extending from the caudal end of the cord. There were irregular net works of nerves in the regions comparable to the brachial and lumbo-sacral plexuses. Skeletal system From the tip of the frontal bone, the highest point on the skull, to the tip of the fourth sacral vertebra the bony skeleton measured 14.5 cm. (2-12, fig. 19) From the tip of the left shoulder girdle to the tip of the rudimentary right ulna along indication line 9, figure 18, the width was 8.5 em. A compari- son of the intact fetus amorphus can easily be made with its skeleton when it is remembered that before the dissection the monster measured 19 em. greatest length and 13.5 cm. the greatest width. At least 2.5 to 3 em. of dense connective tissue and fat were interposed between the tip of any bony structures and the outer skin which precluded the palpation of the under- lying skeleton. The skeleton was composed of a deformed cranium which was composed of, a fused occipital, sphenoid, ethmoid, temporals, and frontals. The two parietals were respectively distinct Many of the bones of the face and those of the bony vault of the cranium were fused into a distorted mass and could be made FIG 18 Fig. 18 Skiagraph No. 1. 7, right parietal bone; 2, right temporal bone; 3, occipital bone; 4, atlas bone; 5, spine of scapula; 6, glenoid fossa; 7, right clavicle; 8, right humerus; 9, left scapula; 10, proximal end of ulna; 11, left sub- cutaneous cavity; 12, first lumbar vertebra; 13, liver tissue; 14, first sacral verte- bra; 14, center of ossification of right ilium; /6, fourth sacral vertebra; 1c, first cervical vertebra; /d, first dorsal vertebra; 1L, first lumbar vertebra; /s, first sacral vertebra. 227 FIG 19 Fig. 19 Skiagraph No. 2. 1, fused mandible and temporal bones; 2, frontal bone; 3, right parietal bone; 4, anterior fontanelle; 4, left parietal bone; 6, occipi- tal bone; 7, styloid process of left temporal bone; 8, left clavicle; 9, liver tissue; 10, first lumbar vertebra; 11, first sacral vertebra; 12, center of ossification of left ilium. ANATOMY OF A FULL-TERM FETUS AMORPHUS 229 out only with difficulty and then only by location and vague similarities to the normal bones. The nasal, lacrimal and vomers were completely fused and these in turn were fused to the mid- point of the mandible by the plowshare of the vomer. The mandible was completely continuous by bony union with the temporal bones on each side of the cranium. ‘The paired max- illae, zygomatic and palate bones, six in all, were entirely absent. A rudimentary hyoid bone was present. The vertebral column presented complete spina bifida from the atlas to the fourth sacral vertebra 16, figure 18. The laminae were widely separated which deformity is well seen in figure 18 in the dorsal view of the skeleton. In the cervical, most of the dorsal, lumbar and sacral regions the laminae were fused to each other. The atlas was completely fused with the occipital bone at the base of the skull. As can readily be seen by reference to figure 18, the vertebrae presented right lateral scoliosis with the vertex of the curvature at the ninth dorsal vertebra. There is also a slight dorsal kyphosis, sharply angular, from the second to the sixth dorsal vertebrae, somewhat obscured by the opacity in figure 19. The last sacral and coccygeal vertebrae were absent. | Upon the left side of the thorax the first nine ribs were fairly well formed with the remaining three merely attenuated carti- laginous rods. Upon the right side the first seven ribs were fairly well formed and the remaining five represented only by fine rods of cartilage. Upon the ventral aspect the thorax pre- sented a complete cleft sternum. The cartilaginous ribs over the cardiac area presented a distinct bulging. Upon the left side the upper extremity presented a compound clavicle and scapula the remaining bony structures being absent. Upon the right side in addition to the distinct clavicle and scapula (which are not compound as on the left side) there was a well formed humerus 3 em. long and the proximal end of the ulna 0.5 em. long. No radius nor other bones of the upper extremity were present. Both clavicles were completely ossified. The left scapula presented ossification along the acromial proc- ess, where it is continuous with the left clavicle. The spine THE ANATOMICAL RECORD, VOL. 11, No. 5 230 EBEN CAREY and supra-spinous fossa were also completely ossified. ‘The corocoid process was not present. The right scapula was com- pletely cartilaginous except the acromial process and contiguous parts of the spine and corocoid process. The pelvic girdle presented centers of ossification for the illum, ischium and pubis. The sacro-iliac synchondrosis was cartilagi- nous on its iliac and sacral aspects. Both pubic elements of the symphysis were cartilaginous. Besides the pelvic girdle there were no other bones of the lower extremity present. In conclusion, I wish to express my gratitude to Dr. J. 8. Foote for the fetus amorphus recorded in this article; to Prof. J. C. Heisler and the Saunders Publishing Company for their permission to reproduce figures 6, 7, 8, 9, 11, 12, 18 and 14 of this article from Heisler’s Embryology; and to Dr. A. F. Tyler, pro- fessor of Roentgenography, Creighton Medical College, for the skiagraphs herein reproduced. ANATOMY OF A FULL-TERM FETUS AMORPHUS 231 LITERATURE CITED AwLFeELp 1880-82 Die Missibildungen des Menschen. Leipzig, Parts I and II. BALLANTYNE 1894-95 Journal of Anatomy and Physiology, vol. 29, p. 466. Cited by Windle (Report on Recent Teratological Literature.) Jour- nal of Teratologia, no. 1. 1904 Antenatal Pathology, 2 vols. Edinburgh. Bearpstey 1858-59 An acephalous fetus. Boston Medical and Surgical Journal, vol. 59, p. 39. Bevitt 1885 A case of acephalous, with spina bifida. St. Louis Cour. Med. vol. 13, p. 280. Breus 1882 Zur Lehre von dem Acardiacus. Med. Jahrb., Wien, pp. 57-72. Bropie 1819 Account of the dissection of a human fetus, in which the circu- lation of the blood was carried on without the heart. Phil. Tr. Lon- don, 99, p. 161-168. CHARLTON 1910 Cited by Adami. Principles of Pathology. Plate VII, vol. 1 Ciauptus 1859 Die Entwicklung der herzlosen Missgeburten. Kiel. EMBLETEN 1865 Case of a human monstrosity, with sketch; absence of heart and lungs. Edinburgh Medical and Surgical Journal, vol. 56, p. 423-27. Hatt 1843 On the circulation in the acardiac foetus. London and Edinburgh Monthly, Journal of Medical Science, vol. 3, p. 541-7. Herzoc 1904 Beitrage zur Entwicklungsgeschichte und Histologie der Mann- lichen Harnrohre. Arch. f. Mikr. Anat. u. Entw., vol. 63. Herwotpr 1889 Reference Handbook of the Medical Sciences, vol. 7. Cited by Fischer in his article on Teratology. Houston 1836 An account of a human fetus without brain, heart, or lungs; with observations on the nature and cause of the circulation in such monsters. Dublin Journal of Medical Sciences, vol. 10, pp. 204-20. Hertwic 1906 Handbuch, vol. 1, chapter 6. Hicks anp BauKArT 1866-67 Dissection of acephalous monsters without head, heart, lungs or liver. Guy’s Hosp. reports. Hirst AND Pigersot 1891 Human monsters. 4 vols. Philadelphia. His 1880-85 Anatomie menschlicher Embryonen. Leipzig. 1904. Die Entwicklung des menschlichen Gehirns Leipzig, Jacost 1873 Report of Committee on case of acardiacmonsters. American Journal of Obs., vol. 6, p. 631. Jackson, J. 1843 On circulation in acardiac fetuses. London Medical Gazette, vol. 37, p. 467. Jackson, J.S. B. 1837-8 Case of monstrosity in which the brain, heart, lungs, stomach, liver, spleen, pancreas and right kidney were wanting. American Journal of Medical Sciences, Philadelphia, vol. 21, p. 362- 68. Kerpet AND Exze 1908 Normentafeln zur Entwicklungsgeschichte des Men- schen. Jena. 232 EBEN CAREY Le Car 1767 A monstrous human fetus having neither head, heart, lungs, stomach, spleen, pancreas, liver nor kidneys. Translated from the French by M. Underwood. Philosophical Transactions, London, vol. 57, p. 1-20. Lusk 1874 Case of acardia. New York Medical Journal, vol. 19, p. 176-79. Matt 1891 A human embryo twenty-six days old. Jour. Morph., vol. 5. 1893 A human embryo of the second week. Anat. Anzeiger, vol. 8. 1900 Pathology of early human embryos. Johns Hopkins Hospital Reports, vol. 9, p. 57. 1908 A study of the causes underlying the origin of human monsters. Jour. Morph., vol. 19, No. 1. Marcuanpd 1897 Missbildungen. Eulenburg’s Real Encyclopedia, vol. 5, Third edition. Mecxet 1902-1905 Handbuch d. pathol. Anatomie. Vols. 15-19. Pinxus 1910 The development of the integument. Keibel and Mall, Human Embryology, vol. 1. RavBeER 1878 Die Theorien der excessiven Monstra. Arch. f. path. Anat., Berlin, vol. 78,°p. 551-94, and vol. 74, p. 66-125. Rerzius 1904 Biologische Untersuchungen, neue Folge, vol. 11. Scoatz 1901 Archiv f. Gynak. ScHwaLBE 1906-1907 Die Morphologie d. Missbildungen des Menschen und der Thiere. Jena, Pt. I, 1906. Pt. II, 1907. Simpson 1874-77 Description of an acardiac fetus. Transactions of Edin- burgh Obstetrical Society, vol. 4, p. 384-89. TIEDMANN 1828 Observations sur l’etat du cervau et des rerfs dans les mon- sters. J. cumpl. du dict. d. se. med. Paris, vol. 31, p. 142. Von Wincket 1904 Ueber die menschl. Missbildungen. Samml. klin. Vor- trage, Leipzig. Vrouik, W. 1811 Beschrij ving eeniger merkwaardige misgeboorten. Ver- handel v. h. Genootsch t. Bevord. Cited by Fischer, Reference Hand- book of Medical Science. Wititey 1869-70 Birth of an acephalous. California Medial Gazette, San Francisco, vol. 2, p. 20. WINDLE 1890-1910 Report on recent teratological literature. Journal of Anat- omy and Physiology. THE THORACIC DUCT IN THE RABBIT RAY HENRY KISTLER From the Division of Anatomy of the Stanford Medical School SIX FIGURES Notwithstanding the above title I intend to describe not only the thoracic duct, its beginning and termination, but also the large lymphatic vessels in the abdominal cavity which are its real beginning. In my description I will also take into ac- count the main lymph channels from the brim of the pelvis. The injections were made on a series of rabbits, the exact species of which because of cross breeding I was unable to de- termine. They were bought at random from rabbit farms where they had been bred promiscuously. I used 26 adult animals about two years of age, sixteen males and ten females. Immediately after killing by illuminating gas the animals were taken from the jar and placed on an animal board. The hind and front foot pads were immediately injected by means of an ordinary hypodermic syringe with a medium-sized needle. I used India ink for the injection mass and found that by insert- ing the needle just under the skin in the pad of the foot, this fluid would immediately travel on up the leg in the lymphatic vessels. Sometimes the ink would go only as far as the nodes in the popliteal fossa, while in other animals it would go clear up into the abdominal vessels, and even into the thoracic duct. Out of the 26 animals I succeeded in getting the injection fluid to go from the pad of the hind foot clear up the thoracic duct in five cases. In all the rest it stopped at the popliteal nodes. In three of the five cases I found that there was an anastomosis of the vessels around the popliteal node but in the other two cases it looked as though the injection had gone right through the node. The inner part of the node was usually darker than 233 234 RAY HENRY KISTLER the periphery and in one instance just one portion of the node was blackened. Whether this can be explained by a difference in the arrangement of the lymphatic vessels or is due to the effect of the strong pressure created by the syringe I cannot say. However, my classmate, Clattenberg, found that young kittens and guinea pigs, in same way, always show the injection clear up the thoracic duct. It occurred to me that perhaps I did not have enough pressure to force the injection on through. I therefore used a larger syringe, in a few cases, and injected as much as I could into the subcutaneous tissue, but the results were as before for I did not get the injection to go beyond the popliteal nodes in any of these cases. After injecting the pad of all four feet in this manner I tied the animal to the board and made an incision through the skin from the mandible to the xiphoid. I then dissected out the two external jugular veins, which are very large in the rabbit and ligatured them close to the clavicle, and opened the thorax and tied off the two superior venae cavae. In this way I pre- vented the injection mass from either going on into the heart or cranially through the large veins. Next I skinned the hind leg and dissected out the popliteal nodes. There may be one or two of these and they are of considerable size. Upon in- jecting these nodes the ink usually went through into the thoracic duct very easily if the leg was repeatedly flexed and extended. I also always opened the abdomen and injected the large group of mesenteric nodes in order to distend the duct and vessels so that they stood out distinctly. Injections into the pads of the anterior extremity usually stopped in the axillary nodes. I succeeded in injecting these nodes only a few times and not as easily as the popliteal nodes. In almost every case about 14 or 15 ce. of ink was used per rabbit. Of this 4 ec. was injected from the hind and 3 cc. from the front pads, 4 cc. from the popliteal nodes, 1 to 2 cc. from the axillary nodes and 3 ce. from the mesenteric nodes: Since I was not able to dissect the vessels very well in the fresh specimen the whole animals were preserved in a dilute solution of formalin. After this I could THORACIC DUCT IN THE RABBIT 235 finish my dissection easily especially that of the duct at its ter- mination into the great veins of the neck. The injection went through the nodes at the bifurcation of the abdominal aorta to the vessels above. This raises the ques- tion as to whether an injection can go through a node. This Baum ’11 answered in the affirmative. Baum also always found connections of the lymphatics with the peripheral veins but this was never noticed in my injections. In my series of animals I found that the thoracic duct and abdominal vessels were very constant and similar in distribution and appearance for only minor variations were observed. Hence a single scheme can represent the arrangement in all the speci- mens injected with the exception of a few variations which will be spoken of later. : From 3 to 5 lymph nodes were always found at the brim of the pelvis or better at the bifurcation of the large abdominal blood vessels. The more usual number was three nodes. One lay on each common iliac and the third ventral to the bifurcation of the abdominal aorta. However, the position of these nodes varied somewhat for the third or central one was sometimes found in the pelvis just caudal to the bifurcation of the aorta and the iliac nodes also sometimes lay more caudal. The nodes lay in the subperitoneal tissue always ventral to the vessels and could be moved about very easily. They varied in size from 0.5 em. to 1.2 em. in diameter and were connected together by vessels thus forming », kind of a plexus. From this group of nodes a group of 3 to 7 lymph vessels started cranially but in every case small branches of these ves- sels would connect with the adjoining vessels or a more dorsal vessel so as to form a sort of plexus. Nevertheless I could almost always distinguish and trace a certain number of dis- tinct and parallel ‘vessels which ran cranially as far as the renal vein. This group of lymph vessels was always mainly ven- tral to but partly surrounded the great abdominal blood vessels. The number and exact position of these lumbar lymph vessels and their relation to the vein and artery I have tabulated in the accompanying table. The average number of lumbar RAY HENRY KISTLER 236 UBIAB]O -qns puB It] [euse} -nsul -xe jo uoTjOUNL ++ ++ +++ *[BUIO} UT [euse4 -xo Jo uotounr pur deinen [BUuIO} XO NOILVNINHAL AO GOV Id uo1} BU -1uL10} UOUIUIOD 94 0} dn syuns 04 pus SopoU 0} VAO®B SOpIATCT ar I iL I G G g € GI = I l! I G i 1 & IT Vystt 0} Sepou [[BUIS 04 SqIa YZ pus yg 4B Yo sur -03 S[ASSOA 9]}4I] JLOYS _ I I IT G g G OT lr il G I G I! v i 6 doo] Suru10; y4F 98 surol . pue qit 44g 7B SoprArd = I G I I G Pp & 8 uO1} BUTUL -10} IB9U S9pOU [BIOAVG ata I I SG w I ¢ € i . “Ie I I G € G L € 9 UOT} BU -1ul194 a10joq ysnf doo'y = I I j I I i 14 ¢ a {! G it G G g re v S]OSS9A IOY}O [BIOAIS Aq pauL1ojy Surog ory -CUIUIO} 4B UOT}RIVIIG — I IT I G I F € € rs I -T I if Sj 14 § (a =a I I I O = = Sika a eae aos ee O RG NOILVNINUGL BE g & & 2 5 2 ey z es s NOILVNINUGL Rae iy sioad a = _ _ & 5 a ° UVON UO LV NOILVIUVA : OIOVuUOHL 7 S s Ss & M s 5 a Om ON JO UAAWAN ce 2 ° 1oad au Eg = ONINUOI STASSAA HAWAT JO UAANON = & a T aTav 237 THORACIC DUCT IN THE RABBIT + ++ + + ++ + + + ++ ‘Bn “yuy “UIA “UIOU “Ul OFUT | pag ye yoursq qt YI 78 spou oF Jo] 07 YO Jassoa ]]BUIG XB1OY} Ul YSII 0} YO sjassoa |[vuUg SapoU 4 Ja] 0} Suros ouo pig 7B SOplAIq, ‘opou [Teas OF Qu Ig 7% JYSII 0} YO [OSsaa ][BUIg qONp 19.10% OF [BIUBIO qysn{ sapiAIp yond ‘aainssoid youd Aq poqoolur suo] “90 7 BAB BUA JOTIOdNS 4jo] Japun pues odie] ql pag ye doo'T snxojd woJ1j sjonp Z UonVyeIp oss1eT UOTPVUIUIIOY 4B SoyrTuN puv yo18 Japun soprlaAtd BY10OB JO YO Japun yonp jo SUIpPIAIC yonp Jo SUIPIAId ‘sopou ysnory) dn pus v4108 0} [BsIOp yovq usy} Jyoti 04 16 WIJ STaSsoOA OMT, ++ a+ 41 04 STos -s0A Z + I T snxod UlOLy % N bo | G I ine) N ca wD wD SH ud oH oe) N 9% GG VG &G GG 1G 02 61 ST LT 9T ST v1 €1 238 RAY HENRY KISTLER lymph vessels was about four. Some might lie ventral and to the right of the vena cava, while others in the same group lay on a level dorsal to the vein but ventral to the artery. Still others lay dorsal to the artery. These vessels could be easily moved and rolled with the blood vessels and their number and position determined although the same vessel changed its rela- tive position in many cases running more dorsal around the vein or artery or more to right or left. At the level of the left renal vein the group of lymph vessels joined to form two or three vessels which always ran dorsal to the left renal vein and to the left suprarenal gland where they formed the cisterna chyli at the level of the second lumbar vertebra. In this region the mesenteric, diaphragmatic and hepatic lymph trunks join the main lymph vessels. In some cases these join by a common trunk. Generally they flow into a large dilation to the right of the aorta just cranial to the renal veins which is connected to the cisterna chyli by a single trunk or a separate vessel and extend cranially from it and running along the right erus of the diaphragm join the thoracic duct above. In some cases a trunk from the mesenteric nodes also runs caudally passing ventral to the renal veins and joins the lymph vessels before they reach the cisterna chyli which was almost always distinct. It was a well marked dilatation partly surround- ing the aorta, found between the second and third lumbar verte- brae or on the body of the second. It usually lies mainly ventral to the aorta but curves dorsally around the right side.. In some cases it was formed by two dilatations one ventral and to the right and the other dorsal and to the left of the aorta which were connected by a large trunk. However, in two cases out of the series of 26 rabbits instead of a distinct cisterna a dense plexus was present. The dilatation into which the vessels from the abdominal viscera terminated was usually located a little cranial and ventral to the cisterna chyli which formed the beginning of the thoracic duct proper. The latter usually left the cisterna by one large trunk which lay dorsal to the aorta and ran in this position through the aortic hiatus in the dia- THORACIC DUCT IN THE RABBIT 239 phragm up into the thorax always lying to the left and dorsal to the aorta. In some cases a plexus took the place of a single vessel and in others there was a second trunk which started from the ventral part of the cisterna or from the dilatation made by the combination of the visceral lymphatics. This vessel passed to the right side of the aorta along the right crus and at about the 11th intercostal space usually turned dorsally and to the left to join the main trunk. If it did not do this it con- tinued on up on that side of the aorta to a plexus always present at the level of the tenth intercostal space dorsal to the aorta. The thoracic duct runs up dorsal to the aorta to the level of the 11th rib where it turns to the right and as it crosses always breaks up into a plexus of vessels which lie dorsal to the aorta. The occurrence of this plexus dorsal to the aorta at the level of the 11th rib may be explained by the fact revealed by Lewis (’05) in his work on the lymphatic development in the rabbit. Lewis says that the thoracic duct arises from a plexus of lymphatics surrounding the aorta. Hence it may be that in the rabbit only this part of the plexus persisted into adult life. The duct reappears as a single vessel to the right of the aorta at the level of the tenth rib or a little cranially to it. The dorsal plexus which is made up of from 3 to 5 vessels is always present. As the duct extends cranially on the right side of the aorta and dorsal to it, it lies ventral to the azygos vein. It thus extends to the level of the third rib where it comes to lie on the longus colli muscle. At the level of the second rib it begins to cross to the left and passes dorsal to the aortic arch as one vessel. It then runs up in the interval between the innominate and the left subclavian arteries to its termination into the left external jugular vein at the angle of junction of the internal jugular where the pres- ence of nodes is very common. Hence forming the main lymph channels of the trunk we find, as shown in figure 1, a group of vessels running up through the abdomen around the large blood vessels and with the visceral ves- sel forming a cisterna at the level of the second lumbar vertebra. From this on extends one duct which lies to left of aorta to the tenth intercostal space where it crosses forming a plexus dor- 240 RAY HENRY KISTLER a lexus Under Aorta ‘, i Cisterna i i a= pee K PEE S | a prea s LP. ene N. Fig. 1 Thoracic duct. Figs. 2, 3, and 4 Modes of termination of the thoracic duct. ABBREVIATIONS I.J., internal jugular A.V., azygos vein E.J., external jugular I.V., innominate vein T.S., transverse scapular vein N, node Sub., subclavian vein S.R., suprarenal gland A, aorta L.P., lumbar gland T.D., thoracie duct K, kidney G., gland THORACIC DUCT IN THE RABBIT 241 sal to the aorta. From this plexus a single trunk extends crani- ally ventral to the azygos vein and to the right of the aorta to the second rib. At this level it crosses to the left again dorsal to the arch of the aorta and extends on up to its termination into the veins to the left as a single vessel. Variations from the type of duct just described were very few. In 2 out of the 26 cases nos. 13 and 18 I found a plexus in place of a cisterna chyli. The plexus in each case surrounded the aorta and was longer than the average cisterna found in the other cases. In case of one of these the plexus extended on cranially as a narrow plexus on the dorsal side of the aorta up to the plexus at the 10th intercostal space which was described before as being always present. In 9 cases out of the 26 small vessels were seen in the lower thoracic region running laterally at right angles from the duct to the right and dorsalwards, some lying ventral to the azygos, while others were dorsal. The usual place for these was at the plexus found dorsal to the aorta where the duct crosses over, or slightly above in the eighth or ninth intercostal space. There were never more than four and in most of the cases only one or two vessels. They came off at irregular intervals some close to- gether while others were in the intercostal spaces. In 5 out of these 9 cases they were traced to small nodes found in the inter- costal spaces near the sympathetic ganglia. In the other 4 the vessels were small and extended dorsally but it was not possible to determine distinctly where they went and in no case could I trace these to the azygos vein as reported by Witzer (34) in a human subject and by Boddaert (’99) in the rabbit. Indeed in only one case a vessel was given off from the duct to the left at the level of the twelfth rib. It terminated in a small node. Another variation found was the presence of two ducts in two cases. In each of these instances two ducts came off from the retro-aortic plexus. In one case it came off from the plexus at the tenth intercostal space and ran to the left of the aorta to its termination. This vessel passed through a large group of nodes in relation to the left vena cava, from which it received tributaries and continued on up running dorsal to the 242 RAY HENRY KISTLER left subclavian artery and also dorsal to the main thoracic duct and terminated cranially to it at the junction of the external and internal jugular veins. The main duct on the other hand terminated at the innominate vein farther down and received a communicating vessel from the accessory duct as it passed ventral to it. In the other specimen the accessory duct at first turned to the right of the main duct for a length of two centimeters, then curved back to the left and dorsal to the main duct and aorta and ran up on the left side receiving vessels from nodes around the left vena cava and terminating caudal to the main duct in the innominate vein. Another variation was the presence of a vessel extending from the thoracic duct over to a node of variable size which usually lay under the right innominate vein. This occurred in 10 out of the 26 cases and hence was fairly frequent. The vessel usually came off from the duct at about the level of the third rib and ran cranially and to the right on the longus colli muscle to the node which lay at the first rib. In one case two vessels came from or joined this node one on each side of the right vena cava. The presence of small communicating loops was noticed in many cases. These were formed by a small vessel branching off from the thoracic duct and then running cranially a little distance and again joining the duct. This occured in about 9 cases at a region between the retro-aortic plexus and the aortic arch. In some instances the loop was longer than in others. In one instance it was about 4 em. in length but others were shorter even as short as 5 mm. Most of these loops occurred where the duct lay on the longus colli muscle just before pass- ing dorsally to the arch. In some cases small vessels were given off from these loops to surrounding nodes and in one case also ventrally to the aorta. Figure 2 illustrates the termination of the thoracic duct in rabbit no. 12. It has a single termination at the junction of the left internal with the left external jugular. It divides and is joined by vessels from surrounding nodes which are always present and finally empties by a single trunk into the large vein. THORACIC DUCT IN THE RABBIT 243 This is the type of termination found in 16 out of the 26 rabbits. In some instances instead of dividing just before reaching the termination the duct formed a large dilation and in others it passed directly through nodes lying in this region. In every ease the lymph nodes especially those on the left side and around the three great vessels here illustrated, were completely injected. In some of the cases vessels could be seen coming from the nodes and joining the duct just before it terminated. These were most likely efferent vessels which must have been injected back from the duct. In one ease a node fully 2 cm. in length lying dorsal to the left innominate vein was completely filled by the black injection fluid. This indicates that the valves in these vessels were either incompetent or had broken down. In figure 3 I have illustrated the type of termination found in 5 out of the 26 cases. The termination is double. One trunk terminates at the junction of the internal with the exter- nal jugular and the other joins the common jugular vein further caudally. In two of the cases it was hard to determine whether the most cranial duct terminated in the internal jugular itself before the junction was reached or whether it terminated at the junction of the jugulars. In all the five cases it divided after it had emerged from the dorsal side of the aortic arch. Injected nodes were also seen in this region. The termination of the second duct varied a little in its position being close to | the main duct in some cases while in others it was a considerable distance caudally. Figure 4 represents the type of termination found in three out of the 26 rabbits. The termination was single in these cases and was found at the external jugular cranial to its junction with the internal jugular as a rule and usually running dorsal to the internal jugular in its upward course. In two of these cases a large dilation was found just before the duct terminated. From this dilation a lymph vessel always ran up the neck along with the internal jugular vein. Figure 5 represents the mode of termination of one of the dou- ble ducts found in this series. The left duct came off from the plexus dorsal to the-aorta at the tenth intercostal space and ran 244 RAY HENRY KISTLER up on the left side. These ducts empty separately. The one at the junction of the jugulars and the other into the common jugular. After passing through a group of nodes the left duct passes on up dorsal to the subclavian artery and to the main duct to which it sends a communicating vessel and then forms a large dilation which joins the junction of the jugulars. The main duct takes the usual course and empties into the common jugular below. Figure 6 represents the other case of a double duct found ‘in this series. The accessory duct branched off from the main duct at the eighth rib extending to the right and gradually Figs. 5 and 6 Modes of termination of the thoracie duct. curving back to the left, dorsal to the main duct and the aorta. It then passed through a group of nodes to the left and terminated in the innominate vein while the main duct followed the usual course and terminated at the junction of the external jugular with the subclavian vein. These five types here described include all of my series. Me- Clure and Silvester (’09) have illustrated the termination of the duct in four rabbits and their results are very similar to those obtained by me. In two out of their four cases the termination was single and into the junction of the internal and external jugular veins. This was the predominating place of termina- tion in my series. In their rabbits they found two with double ducts which are similar to those cases in which I found two ducts. THORACIC DUCT IN THE RABBIT 245 ‘Upon comparing the termination of the thoracic duct in the rabbit with other animals we find a considerable difference. In monkeys as found by McClure and Silvester the most general termination was a single trunk terminating in the jugulo-sub- elavian junction. According to McClure and Silvester the two jugulars and the subclavian veins in monkeys usually join at a single point. According to Ellenberger and Baum the ter- mination in the dog is usually double. One duct joining the junction of the left internal and external jugulars but the other the external jugular, a condition very similar to the rabbit... The relative size of the position and junctions of the veins is also very much like that found in the rabbit. But according to MeClure and Silvester and of Sisson (’11) and of Chauveau (10) the thoracic duct of the cat usually terminates in the ex- ternal jugular by two trunks. According to Sisson the thoracic duct in the horse takes quite a tortuous course and usually terminates in one trunk which joins the anterior vena cava just behind the angle of junction of the two left jugular veins. The duct in the horse is said to develop from a plexus and hence varies considerably. Often there are two ducts. - In the ox the duct is said to be very variable, hardly ever being single but often double or plexiform. Its termination also varies being single or double but it usually empties into the junction of the jugular and brachial veins. Parsons and Sargent (’09) who have investigated about 40 cases in man found that the duct in 75 per cent of the cases terminated as a single trunk. This trunk joins the internal jugular below the left flap of the valve where the jugular joins the left subclavian vein. In only 7.5 per cent of the cases was the termination at the junction. In 22 per cent of the cases the termination was by two trunks into the last centimeter of the left internal jugular vein. In many of the cases the duct bi- furcated and then reunited sometimes only in the wall of the vein. In 2 eases out of the 40 multiple terminations were met with but never more than four. Clattenberg found the termination in the guinea pig usually single and into the innomi- nate vein just at the junction of it with the internal jugular. THE ANATOMICAL RECORD, VOL. 11; no. 5 246 RAY HENRY KISTLER Nevertheless the duct is usually double up to within a very short distance of its termination. The arrangement of the great vessels is quite different in the guinea pig than in the rabbit. | According to Job (’15) the left duct of the rat empties into the jugulo-subclavian junction but is said to carry lymph from only about half of the body. Upon comparing my results with those found in other animals I find that there is a considerable difference in some respects while in others the differences are slight. In the horse, it is said, the cisterna chyli is about 10 cm. in length, ampullated and lies dorsal to the aorta and to the right opposite the first and second lumbar vertebrae. It is always very definite and is formed by two trunks from the viscera and one or two lum- bar trunks. It seems that the lumbar trunks are not regular vessels but contain nodes of considerable size and are also joined at intervals by lymph vessels. In the rabbit, on the other hand, a group of parallel vessels always run through the abdomen. These are never interrupted by the presence of nodes along their course. They combine and with those from the viscera and the diaphragm form a cisterna chyli which is not always very definite. From the cisterna in the horse the duct runs up through the aortic hiatus and forward on the right of the median plane between the azygos vein and the aorta. It is generally single and extends up to the sixth or seventh thoracic vertebra where it crosses ventral to the oesophagus and then runs to the left of the aorta to its termination. In ease of the rabbit the duct first lies to the left of the aorta and as it passes through the aortic hiatus dorsal to it. It then crosses to the right as described before at the tenth intercostal space and as it does so breaks up into the plexus dorsal to the aorta. This plexus, it seems, is never found in the horse. Crani- ally from this plexus the duct in the rabbit, lies in the same posi- tion as in the horse but crosses ‘to the left, again much higher up and dorsal to the oesophagus. Comparing the duct in the rabbit with that in the ox we find some interesting variations. Instead of the group of lumbar vessels found in the rabbit there is usually only one large duct THORACIC DUCT IN THE RABBIT 247 extending up from the lumbar glands and joining the large gastro- intestinal trunk caudal to the renal veins dorsal to which it passes to form a small cisterna. From the cisterna in the ox there may be one or two ducts which may pass through the aortic hiatus or there may be several anastomosing ducts. There hardly ever is only one duct as is typical in the horse. These ducts extend up and the one on the right crosses over at varying heights usually joining the left and terminating as one. In the ox the duct is more variable than in the rabbit. Comparing the thoracic duct of the human being with that in the rabbit we note few differences. In the abdominal re- gion of man we have two trunks which extend up from the pelvis and lower extremities and the lumbar glands which lie separated by the large abdominal vessels in the midline. These vessels pass dorsal to the renal veins and are joined by a large intestinal trunk which also passes dorsal to these veins and by two descend- ing trunks from the thorax which pass through the ventral part of the aortic hiatus. All of these vessels go to form the cisterna chyli which is about 5 to 6 em. in length and les to the right between the aorta and the lower part of the vena azygos, pos- terior to the right crus of the diaphragm and opposite the first and second lumbar vertebrae. According to Davis (’15) a definite cisterna is only present in 50 per cent of cases in man. From the cisterna the human duct is single and extends cranially through the aortic hiatus. Lying dorsal and to the right of the aorta but ventral and to the left of the vena azygos, the duct terminates singly in 89 per cent of the cases into the left sub- clavian vein, and in 22 per cent of the cases into the junction of the jugular and subclavian. In the rabbit, on the other hand, we find a group of 4-6 anas- tomosing lumbar vessels closely grouped around the large blood vessels with a relatively smaller and indistinct cisterna. From the cisterna in the human being one duct usually extends crani- ally through the aortic hiatus, lying dorsal and to the right of the aorta but ventral and to the left of the vena azygos. The vessel runs cranially in this position to about the fifth thoracic vertebra. Here it crosses over to the left dorsal to the aorta 248 RAY HENRY KISTLER and the oesophagus and from there extends out of the thorax on the left side of the aorta. In the rabbit the duct crosses the vertebral column, once at the tenth intercostal space in the form of a plexus and once above at the second vertebra. This crossing is very similar to that in the man but a plexus is never present in the latter where the duct crosses the vertebral column. Of the many variations which have been found in the human subject: none known to me have the characteristics found typical in the rabbit. Comparing the cisterna of the rabbit with that of the cat we find that in the cat the cisterna is formed by the intestinal trunks and two or three lumbar trunks. It is definite and lies opposite the second lumbar vertebra. From the cisterna the thoracic duct which may be looped extends up as a single duct but always lies to the left and terminates in the left external jugular vein. In Bos taurus according to Baum, the cisterna is not very large. It is formed by two lumbar trunks which join the vis- ceral trunks caudal to the renal vessels. These vessels join and form one large trunk which runs dorsal to the renal vessels to the cisterna opposite the second lumbar vertebra. From the cisterna the duct runs to the right of the aorta to the fifth thoracic vertebra. Here it. crosses dorsal to the aorta and extends up on the left and terminates into the left external jugular vein. In the dog the cisterna is relatively very much larger than in the rabbit. It is long and ovoid and even extends into the thor- acic cavity between the crura of the diaphragm. From the cis- terna the duct is usually single, extends up on the right and then crosses over at about the fourth or fifth thoracic vertebra to terminate in the left external jugular vein. In the guinea pig according to Clattenberg a definite cisterna is not always present. The abdominal vessels are plexiform extending up in the midline. At the level of the renal vessels a number of nodes are usually found in which the plexus is lost. From these nodes extend vessels which form a dilation opposite the first and second lumbar vertebrae dorsal to the blood vessels. THORACIC DUCT IN THE RABBIT 249 Cranially from the cisterna another plexus of vessels may leave the nodes just mentioned and pass directly into the plexus above the cisterna. This plexus is usually wide and extends up into the thoracic cavity to the level of the eighth rib. From the eighth rib on up Clattenberg almost always found two separate ducts one on each side of the aorta. The one on the right side crosses over at the third rib and forms another small plexus with the left. Cranially from here a single duct which terminates at the junction of the innominate and the internal jugular veins on the left is found. This form of duct is much like the embryonic plexus found in most mammals. In conclusion I wish to thank Professor Meyer for the help he gave me in the course of this investigation. LITERATURE CITED. Baum, H. 1911 K6énnen Lymphgefiisse direkt in Venen einmiinden. Anato- mischer Anzeizer, vol. 39. 1911 Konnen Lymphgefisse ohne einen Lymphknoten passiert zu haben in den Ductus Thoracicus einmiinden. Zeitschrift fiir Infec- tions krankheiten der Haustiere, vol. 9. Bopparert, Ricuarp 1899 Etude sur une communication exceptionelle entre le canal thoracique et la veine azygos chez le lapin. Annales de la societe medecine de Gand, Tome 78. | Boune, A. 1907 Zwei Faille von Verletzungen des Ductus Thoracicus. Deutsche Zeitschrift fiir Chirurgie. Leipzig Band 87. Burier, C. 8S. 1903 On an abnormal thoracic duct. Journal of Medical Research, Boston, vol. 10. Cuauveau, A. 1910 The comparative anatomy of the domesticated animals. New York and London. Davipson, A. 1910 Mammalian anatomy with special reference to the cat. Davis, Henry K. 1915 A statistical study of the thoracic duct in man. Am. Jour. Anat., vol. 17. GerHarpt, W. Das Kaninchen zugleich eine Einfiirhung in die Organi- sation der Siugetiere. Leipzig. Jos, THeste T. 1915 The adult anatomy of the lymphatic system in the common rat. Anat. Rec., vol. 9, no. 6. Jossirow, G. M. 1906 Der Anfang des Ductus Thoracicus und dessen Er- weiterung. Arch. fiir Anat. u. Phys. Anat. Abt. Kampmetier, Orro F. 1912 Thoracic duct development in the pig. Am. JOURPAMAGe Vole 1S: Lewis, Freperic T. 1905-06 The development of the lymphatic system in rabbits. Am. Jour. Anat., vol. 5. 250 RAY HENRY KISTLER Mituter, Apam M. 1913-14 The thoracic duct in the chick. Am. Jour. Anat., vol. 15. : Moran, H. 1894 Note sur une anomalie du canal thoracique. Comp. Rend. Soe, de Biol. Paris et Nancy. 105., I. Meyer, A. W. 1915 Spolia anatomica. Addenda I. An unusual thoracic duct. Anat. Rec., vol. 9, no. 7. McCuurg, C. F. W., anp Sintvester, C. F. 1909 A comparative study of the lymphatico-venous communications in adult mammals. Anat. Rec., vol. 3. McCuurg, C. F. W. 1908 The development of the thoracic and right lym- phatie ducts in the domestic cat. Anat. Anz., Jena, vol. 32. Pensa, A. 1908 Osservazioni sulla morfologia della cisterna chili a del Ductus Thoracicus. Review in Schwalbes Jahresberichte, Band 14. Parsons, T. G. anp Sarcent, Percy, W. G. 1909 On the termination of the thoracic duct. Lancet. PaTRUBAN, CARL von 1845 Ueber die Einmiindung eines Lymphaderstammes in die linke Vena Anonyma. Arch. fiir Anat. und Phy. Svirzer, —— 1845 Beobachtung einer Theilung des Ductus Thoracicus. Arch. fiir Anat. und Phys. Stromsten, F. A. 1912 On the development of the prevertebral duct in tur- tles as indicated by a study of injected and uninjected embryos. Anat. Rec., vol. 6. Sisson, S. 1911 Veterinary Anatomy. Philadelphia and London. Wourzer, C. W. 1834 Einmiindung des Ductus Thoracicus in die Vena azygos. Arch. fiir Anat. und Phys. SOME RESULTS AND POSSIBILITIES OF EARLY EMBRYONIC CASTRATION FRANKLIN P. REAGAN Department of Comparative Anatomy, Princeton University SIX FIGURES (FOUR PLATES) The works of Eigenmann (7, 8), Hoffmann (11), Nussbaum (13), and Beard (8) have supported the proposition that the germ-cells in several vertebrates are precociously segregated, and that their locus of origin is not necessarily the ‘germinal epithelium’ or even its immediate region. The classic case of early segregation of the germ-tract of a vertebrate is that de- scribed by Eigenmann, who was able to trace the lineage of the sex cells of Micrometrus aggregatus probably as far back as the fifth cleavage; this is the farthest that any vertebrate germ- cell has ever been traced. Hoffmann discovered the existence of primitive ova in the mesenchyme of a number of bird embryos at a time prior to the establishment of germinal epithelia in those embryos. For a review of the works of Beard (3), Nussbaum (13), Rubaschkin (14), von Berenberg-Gossler, Allen (4, 1,2) and Fuss (9), and others, the reader is referred to the introductory discussion by Swift (18). For a discussion of the segregation of the germ-cells in the invertebrates, Hegner’s ‘Germ-Cell Cycle’ may be profitably consulted. The work of Hoffmann and others seems to be borne out by the highly interesting work of Swift (18). The latter has not only strengthened our belief in the extra-regional origin of the sex-cells in the chick, but he has offered a solution of the ques- tion why the primordial germ-cells of the chick had never been found prior to the twenty-two somite stage. He maintains that the sex-cells originate in a crescent-shaped area of the extra- embryonic blastoderm anterior to the body-axis at the line of 251 252 FRANKLIN P. REAGAN demarcation between the areas pellucida and opaca (see his figure 15); that these.primitive sex cells reach the gonad partly by their own wandering, but principally by way of the blood- stream which transports them either to the gonad where they continue to develop, or to some other region where they soon degenerate. He makes the highly interesting suggestion that the large wandering cells which Dantschakoff (5) found to de- generate in the blood-stream following the twenty-two somite stage are really germ-cells which failed to become incorporated into the gonad. At a very short time subsequent to the publication of the work of Swift, Professor McClure in 1914 suggested to one of his stu- dents that this work of Swift’s could be verified or disproved by the early excision of this crescent-shaped area described by that author, provided the operation be done at a time before the germ-cells have wandered away from this area of prolifera- tion. The work was attempted but no results were obtained. Since then the writer has spent a great deal of time trying to develop 2 technique such that the embryos might be maintained despite the extremely low viability of the experimental material. In this work, some few thousand chick embryos have been sacrificed. During the first year of the work it was possible to rear a few embryos to the seventy-two hour stage; later than this, two embryos survived to the age of five days. These were, however, discarded on the assumption that they were unfit for critical study owing to the fact that they had died 2, short time before they were preserved. This is explained by the fact that such embryos were always allowed to develop as far as they would. It was not always possible to find them in the act of dying. It was found, however, that in normal in- dividuals which were bled to death and then allowed to stand meny hours before preservation that the germ cells were quite: easily distinguishable in these corresponding stages; material which has been dead a few hours is of some value, although it is unfavorable for the distinguishing of such fine structures as mitochondria. I have tried to rear the embryos to the more advanced ages for the reason that my chief interest lies not in RESULTS OF BARLY EMBRYONIC CASTRATION 253 the confirmation or disproval of the work of Swift, but rather in determining the later effects of this extraordinary early castra- tion. It occurred to the writer that if this crescent-shaped area, is the seat of the keimbahn, its removal would afford a means of the earliest ‘castration ever yet performed on a vertebrate; it would give an opportunity for studying in pure culture the interstitial cells of the gonad—particularly their supposed effect on the secondary sexual characters. If, as castration of ani- mals subsequent to hatching or to birth has shown, the secre- tion of the gonad profoundly affects the secondary sexual char- acters, it is reasonable to suppose that it might affect some of those sexual characters which are usually considered as primary —such as the persistence or degeneration of the Wolffian and the Miillerian duct. If the latter be true, early removal of the primordial sex cells might possibly cause the retention of the original ground-plan of the urogenital system, namely the simul- taneous existence of both of Wolffian and Miillerian ducts (or at least one of the latter), if however the development and nor- mal functioning of the interstitial cells were in no way impaired by the absence of sex cells, their secretions would cause the alteration of the ground-plan if this were their normal function. But on the other hand, if the gonad were incapable of produc- ing a secretion in the absence of sex-cells, opportunity would be afforded of studying such response or irregularities as might be observed in the development of the other ductless glands. One castrated embryo was recently reared to an age at which one or the other set of genital ducts should have been eliminated. The ground-plan was found to be persistent when the embryo was dissected, but there is a possibility that an arrest of develop- ment due to operation played a part in the result. An ideal way to confirm the work of Swift would be to trans- plant a ‘crescent’ from an embryo of one pure breed to the mesen- chyme of an embryo of a different breed but of the same sex. If such an embryo could be reared to the adult stage its germ- . cells could be tested by breeding. I have been able to rear an embryo into which a crescent was transplanted, to the age of twenty-one days when it died the day on which it should 254 FRANKLIN P. REAGAN normally have hatched. Dissection of this embryo showed it. to be a male. Artificial hermaphroditism would be produced in fifty per cent of such transplantations as this just described. These are some of the problems raised by Professor McClure’s suggestion, and which are now in progress of investigation. Some results of this work at its present status may be of in- terest. I shall describe at this time the results of some experi- ments by means of which I have convinced myself, at least, that the work of Swift has given us a more correct and more ultimate solution of the origin of the germ-cells than any other yet given in case of the chick; that his work is of the very greatest importance owing to the experimental possibilities which have arisen from it. The description consists of a comparison of a few individuals in which castration was complete or at least very nearly so, with normal individuals at corresponding stages. According to Swift, most of the germ-cells have assembled at the base of the mesentery back of the twenty-second somite when the embryo has reached the stage of thirty-three somites. In this position they remain until the germinal epithelium be- gins to thicken. They then migrate into the gonad. This is seen to be in process in my figure 1. In most of the adjoining sections, primitive ova are being incorporated into this thick- ened epithelium. The sections of this embryo are only five micra in thickness, yet it is impossible to find a section in the gonad-region which fails to exhibit sex cells. Some of these cells are found far ventrally in the mesentery. The figure shows the slight protuberance of the right gonad; the region illus- trated is very similar in location to that of figure 3 and the small rectangle in the keyplate for the latter. In the left up- per corner of figure 1, a few erythrocytes have been inserted; they are drawn to the same scale as the rest of the figure. In every case, erythrocytes were used for comparison with the size of the sex-cells. Figures 2 and 3 are from a section of the left gonad of an embryo slightly older than that from which the preceding fig- ure was made. ‘The gonads of the two embryos were, however, about equally prominent. The germinal crescent of this em- RESULTS OF EARLY EMBRYONIC CASTRATION 255 bryo was excised at a time previous to the establishment of any intersomitic grooves and at which the neural folds of the brain were first indicated. The embryo was killed at the age of ninety-four hours. In this section it will be seen that the germinal epithelium is very little thickened. As in all other sections of this embryo, germ-cells are entirely absent. Only a few cells in the mesenchyme approach in size the erythrocytes shown in the figure; these are not germ-cells. None can be found which exceeds the size of the erythrocytes to any such extent as do the germ-cells which are invariably present in sec- tions of the gonads and mesentery of embryos of this age, and which can be most readily identified, no matter what may be the technique of fixation or staining employed. Figure 4 is a section through the left gonad of a normal chick embryo at the age of one hundred and nine hours. This excel- lent series was prepared about fifteen years ago by Prof. A. M. Miller. The gonad protrudes considerably into the coelom. Among the very compact interstitial cells are numerous lightly staining germ-cells of large size. ‘The mesothelium of the mesen- tery adjacent to the gonad is very greatly thickened, darkly staining, and as in all normal individuals of this age, contains numerous ova which cause local protrusions into the coelom. It will be noted that these germ-cells which have been incor- porated into the mesothelium are slightly larger than those which have reached the gonad. Lying in the mesenchyme of the mesentery are large germ-cells. These can usually be found in any section of a mesentery at this age. Pictures quite simi- lar to this are readily obtained in embryos one hundred and ' twenty-five hours old except that the sex cells found in the gonad are relatively somewhat smaller. It will generally be found that this size-relationship obtains in normal embryos. The germ- cells which remain in the mesenchyme are usually the largest. It may be that those in the gonad undergo more rapid multipl- cation. A few erythrocytes sketched indiscriminately from the dorsal aorta are inserted for comparison of size. Germ-cells can be found in the mesenchyme of the mesentery as late as one hundred and seventy-five hours. This is the oldest material which I have examined for this particular point. 256 FRANKLIN P. REAGAN In these older stages, many instances will be found in which nuclear division in such germ-cells has not been accompanied by cytoplasmic division, so that a large multinuclear cell re- sults, having a diameter about equal to that of the mononuclear germ-cells of the thirty-three somite stage. Such a condition is shown in figure 5. A very interesting condition is found here; it will be noted that a large binucleate cell-body projects from the mesenteric surface and touches the gonad from which there is likewise a slight protrusion and on which there is also an interruption of the mesothelium. These are undoubtedly germ-cell nuclei. If this is a case of chemotaxis it is a very remarkable one. It is entirely possible thet such proliferations from the coelomic wall might sometimes be misinterpreted as giant blood-cells. In the gonad will be seen a germ-cell which is smaller than those lying in the mesenchyme. We may now consider the conditions which obtain in a cas- trated embryo five days old. The operation was performed just prior to the establishment of the first intersomitic groove. If castration was not complete, it was very nearly so. In this embryo I have found about five cells which are considerably larger than the average erythrocyte, and several which are a little larger. JI am convinced however, that these are not germ- cells. If one examines Swift’s (18) figure 6, for instance, he will find at least two mesenchyme cells in the left upper corner of the figure which are larger than the erythrocyte in the small endothelial cavity at the lower right corner. There would be little danger of confusing these with the large germ-cells pres- ent in the figure. I have made no attempt to diagnose germ- cells on the basis of the form of the mitochondria, since there is not perfect agreement on the question whether the germ-cell mitochondria of the chick are characteristic. See Rubeschkin and Tscheschin (14, 15, 16, 17, 19) and Swift (18) p. 496. The conditions in this five-day castrated embryo are illustrated in figure 6. The section passes through the right gonad. The mesothelium of the mesentery consists of a single layer of cells. In all normal individuals at this age this mesothelium near the gonad is found to be greatly thickened, stains darkly and con- tains sex-cells. There are no germ-cells in the mesenchyme of RESULTS OF EARLY EMBRYONIC CASTRATION 257 the mesentery, whereas they are invariably present in the nor- mal individual. The gonad contains only interstitial cells, so far as those who have examined the material have been able to detect. The gonad tissue is greatly vacuolated, while in normal embryos even at younger stages the gonad is quite compact and remains so even through rough histological treat- ment. The interstitial tissue displays solid darkly staining lines of intercellular substance bordered by interstitial cells the ar- rangement of which gives a foliage-like appearance. In some cases the plane of section passes through an interstitial cell which projects into a large vacuole. When the diameter of such a vacuole is about that of a germ-cell the picture is some- what similar to that which a germ-cell in a normal gonad would present if the cytoplasm were dissolved out. Professor E. G. Conklin has examined this material and has pronounced the fixation to be sufficiently perfect that germ-cells should be easily distinguished if they were present. If any are present they are certainly very few. amg In the foregoing account, the term ‘stroma tissue’ might well have been used instead of the term ‘interstitial tissue.’ It seems reasonable to believe that the abnormal conditions recorded and the early removal of the crescent-shaped piece of blastoderm are causally related. In conclusion I wish to thank Professors C. F. W. McClure and E. G. Conklin for the aid which has made this work possible. SUMMARY 1. The extra-regional origin of the germ-cells of the chick may be regarded as highly probable. 2. The early location of the germ-tract on the yolk-sac gives opportunity for the earliest castration yet performed on a ver- tebrate. It makes possible an analysis of the functional activity of the sex-cells and the interstitial cells in the production of internal secretions, the effects of the latter on the sexual charac- ters and on other characters. It makes possible the production of artificial hermaphroditism, provided the germ-cells of one crescent can be made to enter the gonad of an embryo of the opposite sex. The work of Swift is of very great importance. 258 FRANKLIN P. REAGAN LITERATURE CITED (1) Auten, B. M. 1906: The origin of the sex-cells of Chrysemis. Anat. Anz., Bd. 29. (2) 1909 The origin of the sex-cells of Amia and Lepidosteus. Anat. Ree., vol. 3. 2 (3) Bearp, J. 1904 The germ-cells. Part 1. Jour. Anat. and Phys., vol. 38. (4) von BERENBERG-GossLER 1912 Die Urgeschlechtszellen des Hiihner- embryos aus 3 und 4 Bebriitungstage. Arch. Mik. Anat., Bd. 81. (5) DantscHakorr, W. 1908 Entwicklung des Blutes bei den Végeln. Anat. Hefte, Bd. 37, S. 471. (6) Dopps, G. S. 1910 Segregation of the germ-cells of the teleost Lophius. Jour. Morph., vol. 21, p. 563. (7) Etiaenmann, C. H. 1891 On the precocious segregation of the sex-cells in Micrometrus aggregatus, Gibbons. Jour. Morph., no. 5, p. 481. (8) 1897 Sex differentiation in the viviparous teleost Cymatogaster. Arch. f. Entw’mech., Bd. 4, p. 125. (9) Fuss, A. 1912 Uber die Geschlechtszellen des Menschen und der Siugetiere. Arch. fiir Mikros. Anat., Bd. 81, Heft 1. (10) 1911 Uber Extraregionare Geschlechtszellen bei einen Menschlichen Embryo von vier Wochen. Anat. Anz., Bd. 39. (11) Horrmann, C. K. 1893 Etude sur le developpement de l’appareil uro- genital des ois@ux.- Verhand. der Koninklyte Akademie von Weten- schoppen, Amsterdam, Tweedie Sectie, vol. 1. (12) Mreves, F. 1908 Die Chrondriosomen als Traiger erblicher Anlagen. Cytologische Studien am Hiihnerembryo. Arch. fiir mikr. Anat. und Entw., Bd. 72. (13) Nusspaum, M. 1901 Zur Entwicklung der Geschlechts beim MHuhn. Anat. Anz, Bd. 19. (14) Rusascuxin, W. 1907a Uber das erste Auftreten und Migration der Keimzellen bei Végelembryonen. Anat. Hefte., Bd. 39. (15) 1907 b Zur Frage von der Entstehung der Keimzellen bei Siugetier- embryonen. Anat. Anz., Bd. 31. (16) 1910 Chondriosomen und Differensierungsprozesse bei Siugetierem- bryonen. Anat. Hefte, Bd. 41. (17) Semon, R. 1887 Die indifferente Anlage der Keimdriisen beim Hiihn- chen und ihre Differenzierung zum Hoden. Jena Zeitschr. Naturwiss., Bd: 21. (18) Swirr, C. H. 1914 Origin and early history of the germ-cells in the chick. Am. Jour. Anat., vol. 15, no. 4. (19) Tscuascutn, S. 1910 Uber die Chondriosomen der Urgeschlechtszellen bei Végelembryonen. Anat. Anz., vol. 37. (20) Watpnyrer, W. 1870 Hierstock und Ei. Leipzig, Englemann. (21) Woops, F. A. 1902 Origin and migration of the germ-cells in Acanthias. Am. Jour. Anat., vol. 1, p. 307. "BAO OAT} -1id “Og {kroyuesou “ys py ‘umnipoyyide jwuruaies “yzda “fy "uoye} ST g OINDY YoryM Woy suoISoI oy} ATYSnor soyvorpur Arojuosou oy} JO 4Jo] VY} OF 9pdULy -0d OY], ‘OpvU SI gE OINSY YOryM UWIOIJ UOTZDES 9Y} JO YOJoysS JoMOd-MOT Z “MOTJIPUOD SUIZSO19} UT UB So} VAYSN]TE FL JN ‘word 44 you st UoTjoos sIy, “wUMIpoyyIda [euIUIES oY} portoyUs AVY S]]90 UES OY} ‘suOIyO0s SuTUIO[pe ysour UT “O08 X ‘ursos-anjq [Aqyota Aq poMOT[O} UT[AXOYVUIOBY UOIT “UOTYVXY 91J90B-O1TY “pO sunoy Ayouru okIquIO Yoryd [BULIOU B JO pvUOs ZYysIA oY YSNOIG} WOT | Saunold JO NOILVNVIdxXo T ALV Id 260 RESULTS OF EARLY EMBRYONIC CASTRATION PLATE +t FRANKLIN P. REAGAN THE ANATOMICAL RECORD, VOL. 11, NO. 5 PLATE 2 ne EXPLANATION OF FIGURES 3 Section through the left gonad of a ninety-four hour chick embryo which was castrated prior to the formation of any intersomitic grooves. Note the ~ absence of germ-cells and the thinness of the germinal epithelium. The sec- tion is viewed from its posterior surface. Benda’s fixation followed by Meves’ _ iron-haematoxylin stain. XX 800. Hndih., endothelium; £rth., erythrocytes; G. epth., germinal epithelium. Msth,, mesothelium. 262 RESULTS OF EARLY EMBRYONIC CASTRATION PLATE 2 FRANKLIN P, REAGAN IOS SOS WOM”, oS Ta TES% WAS O44 OPT SF 263 PLATE 3 EXPLANATION OF FIGURES 4 Section through the left gonad of a normal chick one hundred and nine hours old, showing germ-cells in the gonad, and in the mesothelium and the mesenchyme of the mesentery. Zenker fixation. Delafield’s haematoxylin and orange-G. Princeton Embryological Collection, series no. 46. 800. 5 Section through the left gonad of a normal chick one hundred and sixty- two hours old, showing primitive ova in the gonad and in the mesentery. Fixa- tion and stain as in figure 4. Princeton Embryological Collection, series no. 41. X 800. Coel., coelom; Gon., gonad; Mst., mesentery; Msth., mesothelium; Pr.O., primitive ova. RESULTS OF EARLY EMBRYONIC CASTRATION PLATE 3 PRANKLIN P, REAGAN 265 - PLATE 4 EXPLANATION OF FIGURE 6 Section through the right gonad of a one-hundred-and-twenty-hour chick which was castrated at a time prior to the establishment of the first inter- somitic groove. The mesentery, the mesothelium and the gonad are devoid of sex cells. Note the peculiar foliage-like appearance of the gonad tissue. A few erythrocytes drawn to scale were inserted into the lower part of the figure. Bouin’s fixation followed by iron-haematoxylin and eosin. The left gonad would have afforded a still better comparison with figure 4. The right one was chosen for the reason that the stroma-tissue was less dense than in the left. Both gonads are almost the same size. The mesothelium near the left gonad is very little thicker than that on the right. The left gonad contains no germ cells. > 800. Coel., coelom; Erth., erythrocytes; Gon., gonad; Mst., mesentery; Msth., mesothelium of mesentery. RESULTS OF EARLY EMBRYONIC CASTRATION PLATE 4 PRANKLIN P, REAGAN 267 THE RELATION OF AGE TO FERTILITY IN THE RAT HELEN DEAN KING The Wistar Institute of Anatomy and Biology THREE FIGURES Data have recently been obtained that show the complete breeding history of a considerable number of female rats. An. analysis of these data with reference to the question of fertility and its relation to age seems desirable, since literature dealing with litter size in rodents (bibliography in “The Rat,’ Donald-. son, 715) gives very little information on this point and fails to record the entire litter production of even one pair of animals. The breeding records of seventy-six females that produced a total of 585 litters are used in this study. The majority of the females (50) were piebald or ‘hooded’ rats; the rest were either ‘extracted’ albinos (15) or ‘extracted’ grays (11). All three strains were derived from the F; generation of a cross between the wild Norway rat (Mus norvegicus) and the domes- ticated albino (Mus norvegicus albinus). Mention is made of the kind of rats used merely as a matter of reference. The con- clusions drawn from the results are doubtless applicable also to other strains of rats. All of the females lived to be at least sixteen months of age, the oldest dying at the age of twenty-three months. Under the conditions existing in the animal colony of The Wistar In- stitute a rat is usually in its prime at the age of seven or eight months, and after reaching twelve months of age it is classed as ‘old.’ Very few individuals live for more than twenty months, although all animals are kept under environmental conditions that are seemingly well suited to their needs. The relatively early death of the animals is due, in part at least, to the fact 269 THE ANATOMICAL RECORD, VOL. 11, No. 5 270 HELEN DEAN KING that seasonal changes in temperature’in the region of Phila- delphia render old animals very susceptible to pneumonia, the disease that invariably. proves fatal to a rat of any age. In a more equable climate, like that of California, rats have been kept in good physical condition until they were four years old (Slonaker, 712). In the rat the menopause usually appears at the age of fifteen to eighteen months (Donaldson, ’15, p. 21). Data covering the litter production during the first sixteen months of life, therefore, may be assumed to show the actual fertility of the great majority of females. The word ‘fertility’ is here used as defined by Pearl and Surface (’09) to designate: “the total actual reproductive capacity of pairs of organisms, male and female, as expressed by their ability when mated together to produce (i.e., bring to birth) individual offspring.” Fertility, according to this definition, is a much more comprehensive term than fecundity with which it is often confused. The latter, as suggested by Pearl and Surface should properly be used to signify only ‘‘the innate potential reproductive capacity of the individual organism as denoted by its ability to form and separate from the mature body germ cells.” Litter data for the three strains of rats are shown in table 1. TABLE 1 Showing litter data for the three series of rats eae) ! Bee m > "ea| ee (oo | Be “8a ee ag 2° |e a me 2B mm pare Bas) @2 lassel am n 3 Bas heb Pe fei Bom] 4.6, : = 334 59m] Bae (Para! BO | a 52m Z z < & a & Zz Prebaldseries: -... tance oe aoe 50 406 | 6.9 | 2798 | 1447 | 1351 | 107.1 Extracted albinas)-+...+-- 54. .04|) Lo 88 | 6.2 | 548 | 279 | 269) 103.7 Pxfracted! Stays ae: wee ek 11 91} 6.7 | 609 | 310} 299 | 103.6 76 585'| 6.7 | 3955 | 2036 | 1919 | 106.1 As table 1 shows, the corresponding records for the three series are very similar. The differences in regard to litter size and to the relative proportion of the sexes that are found are well within the limits of the variation that is always to be ex- RELATION OF AGE TO FERTILITY IN RAT 271 pected when the number of records is comparatively small. For further analysis, therefore, the data for the three strains have been combined. The entire series of records, arranged according to the age of the mothers when the litters were cast, is given in table 2. The ‘mean age of the females,’ as given in the first column of table 2, is the median point of a thirty day period in the life of TABLE 2 Showing the number of litters in the combined series, together with the sex ratios and the coefficients of variation for litter size. Data arranged according to the age of the females when the litters were cast COEFFI- 5 AVERAGE NUMBER OF EM. tos 1 oF LITTERS AS ae ‘BER OF 1S MALES FEMALES rang zo VAmIATIONS . LITTER FEMALE BIZB 90 38 6.9 264 126 138 91.3 34.9 120 49 7.9 389 207 182 1b re 32.3 150 57 7.6 433 215 218 98.6 26.5 180 60 7.8 472 252 220 114.5 31.5 210 61 Use 471 243 228 106.5 25.2 240 46 7.3 337 155 182 85.1 36.8 270 44 7.0 314 163 151 107.9 36.2 300 49 7.4 363 187 176 106.2 35.7 330 41 6.0 246 138 108 127.7 37.8 360 3l 6.1 191 97 94 103.1 36.5 390 35 5-1 179 104 75 138.6 51.2 420 26 4.5 118 64 54 118.5 41.8 450 18 4.3 79 35 44 79.5 51.5 480, 13 3.2 42 23 19 121.0 73.3 510 10 3.4 34 17 17 100.0 36.3 540 6 3.6 22 9 13 69.2 AT, 570 1 1.0 1 1 585 6.7 3,955 2,036 1,919 106.1 38.0 each animal, except in the two cases noted below. For example, the mean age ‘120 days’ includes the records for all litters pro- duced by females that were from 105 to 135 days of age when parturition occurred. The ninety day group is one exception to the above rule; it comprises litter records for a twenty day period only, as the youngest mother in the series was eighty- six days old when her first litter was cast. One female gave birth to a litter of one when she was 594 days old. For the 22 HELEN DEAN KING sake of uniformity this record is put under the mean age ‘570 days’ which is thus extended to include a period of forty-four days. ; The majority of female rats that are in good physical condi- tion cast their first litters when they are about three months old. Thirty-eight of the seventy-six breeding females bore young before they were 105 days old; all of the remaining females, TO t p SRREEE Ease + 14 Cee ae! i | Number of Litters 30 Age in Days 90 120 150 180 210 240 300 360 390 42 460 480 510 540 572 Fig. 1 Graph showing, for the entire series, the relation of the age of the mother to litter production (data in table 2). with four exceptions, threw litters before they reached the age of 135 days. As table 2 shows, the number of litters cast in- creased with the age of the mothers until the females attained the mean age of 210 days. After the age of maximum fertility was passed the number of litters cast decreased rapidly, and only a small proportion of the females bore young after they had reached the age of fifteen months. The graph in figure 1, constructed from the litter data in table 2, shows the relation of the age of the mother to litter production. RELATION OF AGE TO FERTILITY IN RAT 273 The graph in figure 1 starts relatively high and rises rapidly to its maximum which comes at the 210 day period. The decline of the graph is much more gradual than its rise, and not until near the 360 day period does the graph drop to the level at which it starts. From this point the fall is more rapid, and the graph reaches zero after the females have attained the mean age of 570 days. Fecundity in the rat, measured solely by the number of litters cast by the females at different age periods, is thus found to accord remarkably well with the law formulated by Marshall (710): “The fecundity of the average individual woman may be described, therefore, as forming a wave which, starting from sterility, rises somewhat rapidly to its highest _ point, and then gradually falls again to sterility.’ There can be no doubt that animals, in general, tend to follow a simi- lar law, as the litter necords for various species collected by Mar- shall, by Pearl (13) and others have already shown. Judging from the data in table 2 a female rat reaches the height of her reproductive capacity when she is about seven months of age. This age represents also the median point in the animal’s breeding career. That is, one-half of the total num- ber of her offspring are produced by the time she has reached this age and one-half are produced afterwards. When the females have reached the age of eighteen months their reproductive activity is usually at an end, as the data in table 2 indicate. Donaldson (06) has shown that the first year of a rat’s life is approximately equal to thirty years of human life. On this assumption a female rat that is eighteen months of age corresponds physiologically to a woman of forty- five. The menopause evidently takes place in these two forms at about the same period in the life span of the individual, but there is no corresponding likeness as regards the age of puberty or of maximum fertility; both of these processes take place in the rat at a relatively much earlier period. The third column of table 2 shows the average size of the lit- ters cast by the females at different age periods. The litters of very young females contained an average of 6.9 young per litter. This is a smaller average number of young than is found 274 HELEN DEAN KING for any group of litters until the females. have past the zenith of their reproductive activity. Such a result was to be expected, since a number of investigations, for instance those of Minot (91) on guinea pigs and of Hammond (14) on rabbits and pigs, have shown that the number of offspring produced by young animals breeding for the first time is usually below the number that is considered normal for the species, and also that litter size tends to increase for a time with the age of the female. The largest litters in the series were those produced by females with a mean age of 120 days. Litter size remained close to the maximum until the females were eight months old when a slight diminution in the number of offspring was noticed. A further decrease to an average of only six young per litter was found in the litters thrown by females that were one year old. Each succeeding month added to the female’s life seemed to lessen the number of her offspring to a marked extent, and after the females were fifteen months old the mean size of the litters cast was only about three young per litter. Not infrequently the offspring of old females were born dead or soon died from neglect as the mothers seemed unable to suckle them. There is, as yet, no standard for litter size in ‘extracted’ strains of rats with which the present series of records can be compared. Miller (11) and Crampe (’84) give 10.5 as the average number of young in a litter of wild gray rats; but Lantz (10), on examining a large series of animals, found an average of only 8.1 embryos in pregnant gray females. According to Crampe the average litter of albino rats contains 6.3 young; data for over 1000 litters, collected by King and Stotsenburg, give the mean number of young in albino litters as 7.0. Accord- ing to the above observations litters of gray rats contain a greater average number of young than do those of albino rats. The 585 litters used in the present investigation contained an average of only 6.7 young. This seems to indicate that litter size in ‘ex- tracted’ strains of rats is less than that in either of the pure strains from which the animals were derived. It must not be forgotten, however, that the litter size for the pure strains, as given above, was not obtained from the complete breeding rec- RELATION OF AGE TO FERTILITY IN RAT 275 ords of a number of females but from a random collection of litters cast by females of unknown age. Litter size in various strains of rats eannot be properly compared until litter records for the several strains have been collected in a similar manner. The relation between the age of the mother and litter size is shown by the graph in figure 2. The data used in construct- ing this graph are given in table 2 The graph reaches its maximum when the females are prac- tically at the beginning of their reproductive activity (i.e., at | HEE EEE HEH Scceeeeeaas os ee Ssssetassesfaee Seacrest teaeesteee a8 Ns COO Seeeneeeees ‘ pusersssisessereee fg eee ee EH Eee HH EES Scie SSRSGTStSosreraasocs 30 a) 33 60 siz 3990 0 ( Fig. 2 Graph showing, for the entire series, the relation of the age of the mother to the average size of the litter (data in table 2). 120 days of age), and then declines very gradually approximat- ing zero when the females are eighteen months old. Fertility in the rat, measured by the size of the litters cast, is thus found to be correlated with the age of the mother at the time that parturition occurs. There is a possibility that the number of the pregnancy is a factor that influences the size of the litters cast. In order to analyze the data on this basis the records have been arranged according to the position of each litter in a litter series and are given in table 3. When the data are arranged as-in table 3 it is found that the second litter is the largest of the series. This result is in accord with the observations of Crampe (’84) and of King and Stotsen- 276 HELEN DEAN KING TABLE 3 Showing the number, the average size of the litters and the sex ratios when the data are arranged according to the position of each litter in a litter series AVERAGE POSITION OF THE TOTAL NUM- NUMBER OF TOTAL NUM- NUMBER OF kien ts | SE coreg ees | Oe | 1 76 ee 553 290 263 110.2 2 76 Oath 591 292 299 97.6 3. 76 6.9 531 276 255 108.2 4 74 Te 543 270 273 98.9 5 66 7.0 463 235 228 103.0 6 56 6.8 384 197 187 105.3 7 43 6.9 300 157 143 109.7 8 36 5.8 210 118 92 128.2 9 30 5.0 151 89 62 143.5 10 21 4.8 100 57 43 132.5 11 14 4.8 67 31 36 86.1 12 9 3.8 35 15 20 75.0 13 7 So 25 9 16 56.2 14 1 20 2, 0 22 | 585 6.7 3,955 2,036 1,919 106.1 burg (15) on the albino rat. The number of the pregnancy, up to five, does not seem to have a very marked effect on litter size. The first five groups of litters have an average of 7.2 young per litter, which is above the average size of litters of albino rats (7.0 young per litter) and considerably greater than the mean size of all the litters in the present series (6.7 young per litter). A slight decrease in size is noted in the sixth litter group, and in the succeeding litters the number of young dimin- ishes steadily. Only exceptionally vigorous females are able to produce more than ten litters and these later litters rarely contain more than one to three young. As a rule female rats begin breeding when they are three months old, and they will produce a litter each month for sev- eral succeeding months if they are in good physical condition. The second litter is cast, therefore, when the female is about four months old and the fifth litter is born when the mother is seven or eight months old. On referring to table 2 it is found that litters born when the females are four months old have a RELATION OF AGE TO FERTILITY IN RAT Son greater average size than litters cast at any other age period, and that females reach the climax of their reproductive activity at about seven months of age. In both tables there is a rapid decrease in the size of the litters towards the end of the series. As far as the question of litter size is concerned the two tables are in complete agreement. Such a litter series as that in table 3 is necessarily an age series, and it is very probable that it is the age of the female and not the number of the pregnancy that is a determining factor for litter size. The size of a newborn litter of rats depends, primarily, on the number of ova shed at a given period of ovulation that are capable of fertilization. Litter size, however, is not always indicative of the actual fecundity of the female, since the off- spring born represent only that portion of the fertilized ova that were capable of normal development. Not infrequently the examination of a gravid female will show one or several fertilized ova in the uterus that are more or less atrophic and so incapable of developing into normal embryos (Huber, 715). Such ova are usually absorbed in situ, and only very rarely are mon- strosities found among the normal newborn young. Accord- ing to Hammond (14), the lower fertility of young sows as compared with that of adult animals is due to the fact that not so many ova are shed at each period of ovulation. This ex- planation for the change in the fertility of swine is doubtless applicable also to a similar change in the fertility of rats and of other animals. Very probably the lessened fertility of old ani- mals as compared with that of animals in their prime is due to the same cause. Whether abnormal ova are more frequent in old females than in young ones and so help to diminish the fer- tility in later life has not, as yet, been determined. The last column of table 2 gives the coefficients of variation for the size of the litters cast by the females at different age periods. These coefficients show that size variation is consider- ably greater in the litters thrown by very young females than in the litters produced by females at the height of their repro- ductive activity when they are seven months of age. The latter 278 HELEN DEAN KING group of litters has the lowest coefficient (25.2) in the entire series. . As the number of litters cast after the females were a year old was relatively small, the coefficients for later litter groups can have little value. There seems, however, to be a very marked tendency for litters cast by older females to exhibit a greater range of variability in size than is shown by the litters of young females, the maximum variability appearing in the litters produced by females when they were about sixteen months - old. The entire series of litters gives 38.00 as the coefficient of variation for litter size. This coefficient is practically the same as that for litter size in the mouse, which is 37.5 according to the records collected by Weldon (’07), but it is 10 points less than the coefficient for the number of human offspring (Powys, 05). The coefficient of variation for fertility is very high in all mammals, apparently, being at least 25 per cent in the sey- eral cases where it has already been determined (Surface, ’08). Different females—even sisters from the same litter—show marked variations in the number and in the size of the litters they produce. Whether such differences depend upon the in- heritance of various fertility factors, or whether they are due to environment or to individual peculiarities of the females them- selves remains to be determined. Table 4 shows the number of litters produced by the seventy- six females whose breeding records are used in the present study. As shown in table 4, the range of variation in the number of litters produced by different females was from three to four- teen with an average of 7.7 litters per female. One of the two females that cast only three litters did not breed until she was six months old when she gave birth to a litter of seven. A second litter, with nine young, was born when the mother was eight months old, and a final litter, containing seven young, one month later. This female lived to be seventeen months old and she appeared to be in good physical condition until shortly before her death. The other female casting only three litters had a very similar breeding history. Some diseased RELATION OF AGE TO FERTILITY IN RAT 279 TABLE 4 Showing the litter production of 76 female rats NUMBER OF BREEDING NUMBER OF LITTERS FEMALES CAST — — mBeonorrwowdonnrwo ® tv © CONT & Cr hm CoO ~] for) 585 condition of the generative organs was doubtless responsible for the small number of litters produced by these two females, as investigations being carried on in the animal colony of The Wistar Institute by Dr. Stotsenburg show that sterility in a female rat is usually due to the formation of ovarian cysts or to degenerative changes in the uterus. According to Crampe (’84), female albino rats, as a rule, do not produce more than four or five litters: records collected by Miller show that the wild gray rat has relatively more litters than the albino rat. The average of 7.7 litters per female, found in the present series of animals, is undoubtedly too high for the general run of females. Twenty-three of the seventy- six breeding females in this series had a total of five or six litters only, and it seems probable that this is about the average number of litters produced by female rats in general. While six females had thirteen litters each, only one female gave birth to fourteen litters. This latter case isso unusual that it seems worthy of special note. The complete litter data are given in table 5. This female, a piebald, gave birth to her first litter on Febru- ary 7 when she was ninety-five days old. This litter was ex- 280 HELEN DEAN KING TABLE 5 Showing the litter production of a female piebald rat, that was born November 4, 1913, and died June 14, 1915 reat DATE OF BIRTH ea MALES FEMALES i "| “Bebruarsy 17; ol GUA eerste cco ics aes 11 5 6 2 March lle 1G 14Atee eres seo cos es eee 13 5 8 3). )Aprill.3, 191 rei eek noe 8 6 2 A | April 30: 319 UA ie oes os, cio oe ici toes 9 5 4 Be a eMiay 23). WO MAS eer eee Sn i8 csvostace eo haces extent ee 9 6 3 6° (| Jamie 20; LOA eee Benes fic tio ae Set onmtees te 10 2 8 7 \ Sially 14719 DA tees tars sree eee 11 5 6 So Are ust U2 ASIA ee creeeene visu: tons eye eset 6 oy 4 9 + September 101914"... fee 55- 6 CAE oa ee 10 6 4 10° October To Ola eee satiate eae 10 9 1 1 | 8November723 slGl4ee aac sc eee homie ae ae + 3 1 12-4) January 28; 01915 acai ince eae eae 3 2 1 13: March: 26; st Ola ae tener ae une hme ergs 3 3 0 PA | SA rt 28; Oa ye jak ee ketene eee cestees 2 0 2 ceptionally large for the first litter of so young a female as it contained eleven young. The second litter, with thirteen young, was cast the following month. It is rather remarkable that both of these litters should be so much larger than normal, since, as a rule, a very large first litter is followed by a compara- tively small one, unless at least two months intervene between the birth of the litters. The female cast two litters in April, and subsequently she gave birth to a litter each month until she was twelve months old. With one exception each of these litters was larger than the average litter of albino rats. A marked decline in fertility was noted after the female was a year old: the intervals between litters became longer and the size of the litters decreased. The fourteenth litter, which contained only two young, was cast when the female was about seventeen months old, and although the female lived to be nearly twenty- two months old she did not breed again. During this long period of reproductive activity a total of 109 young were born, 59 males and 50 females. The median point in this female’s breeding career was the same as that for the entire group of RELATION OF AGE TO FERTILITY IN RAT 281 females, namely seven months, and she produced an average of 7.8 young in each litter. An examination of the individual records for each of the re- maining females in the series that gave birth to a very large number of litters i.e., from eleven to thirteen, shows that in every instance the first litter cast was large, containing from nine to eleven individuals. In those cases where females pro- duced less than six litters the first litter cast, with one exception, never contained more than seven young. The number of rec- ords is so small that no definite conclusions can be drawn from them, but they seem to indicate that the size of the first litter cast is somewhat of an index of the fertility of that particular female: a large first litter indicating that the female, if she keeps in good physical condition, will produce more litters than the average run of females. Crampe states that the second of a rat’s litters is always the ‘best’ and that this litter is indicative of the size of subsequent litters. This observation has been confirmed only in part by the present series of records: the second litter is the largest of the series, but the size of this litter is not as indicative of the later fertility of the female as is the - size of the first litter cast. Individual rats show as marked differences in the number of young produced at one birth as they do in regard to the total number of litters cast. Litters cast by some females are almost- always relatively large. The female whose litter record is given in table 5, for example, cast but one litter in the first ten that contained less than seven young. Some females never have a litter that contains more than seven young, while others females cast a large and a small litter alternately. The litter frequencies in the three series of rats are shown in table 6, the range in litter size being from one to sixteen. In table 6, as in table 1, there are slight differences in the cor- responding data for the three series of rats that may or may not prove to be significant when larger series of records are analyzed. Litters of eight young were most frequent in the piebalds and in the extracted grays, while six was the most common number of young in the litters of extracted albinos. The data for the . 282 HELEN DEAN KING litter frequencies in the combined series is shown in the form of a frequency graph in figure 3. . The graph in figure 3 has two modes, one at the point of six and the other at the point of eight young per litter. The graph thus appears to be compound, and it is possible that one of the two modal points corresponds to the degree of fertility normal for the wild Norway rat and the other to the degree of fertility that characterizes the albino rat, since these are the two strains from which the animals used for this study were derived. As the material is probably heterozygous as regards the factors for litter size, it does not seem advisable to attempt any analysis of the curve. It is of interest in this connection to note that the graph for litter frequencies in swine, as given by Went- TABLE 6 Showing litter frequencies in the three series SIZE OF LITTER 1 Diallo) Apat ieetoon |e ie |f ecm] et tu ee leel| ek Oates tn rt | esd Soe Tea | Piebal ds ee ieee eee 6 | 20} 32) 35) 31) 56) 40} 71} 48] 28) 17) 12; 8} 1) 1 Extracted albinos......... Beh 7) Oy ae Su TIA al Extracted grays.......... SH Gl TIA Oakey, abt | aN aly, 6 | 28} 49) 48) 53) 88) 64) 94) 71) 41) 19} 13} 9) 1] 1 worth and Aubel (716), has three modal points; one at four, a second at eight, and a third at twelve pigs per litter. The first mode corresponds to the degree of fertility in the wild hog, the third is close to that of the most fecund of the domestic breeds of swine, and the third probably represents a heterozy- gous condition. Evidence regarding the relation of the age of the mother to the sex of her offspring is conflicting. Statistics collected by Bidder (’78) and by Punnett (’03) show that there is a great excess of boys among the children of very young mothers, the relative number of boys decreasing at subsequent births un- til the mother is thirty. Among children of old mothers (i.e.,. over forty) the sex ratio is again very high. In the horse Wil- chens (’86) found a relation between the age of the dam and the _ sex of her offspring very similar to that existing, apparently, RELATION OF AGE TO FERTILITY IN RAT 283 in the human race. On the other hand, Schultze’s (’03) in- vestigations on mice indicate that the age of the mother has seemingly no influence whatever on the sex of her young. According to the observations of King and Stotsenburg the normal sex ratio in the albino rat is about 107.5 males to 100 females. As there are no available data regarding the normal sex ratio in other strains of rats the sex ratio in the albino rat is here taken as the standard with which to compare the sex ratios found in the present series of animals. wesee i Sueeace ° ee 10o-) H Ls | Number of Litters a SRSSSRSSSeeanee ve Scasceccscescease Oot Seeeee Ww 12 13° 14 16 16 Fig. 3 Graph for the frequencies of litter size in the entire series (data in table 6). Table 2 gives the sex ratios for the various litter groups when the data are arranged according to the mean age of the females at the time that the litters were cast. The sex ratios in litters belonging to closely related groups are so unlike that it would appear that there is no relation whatever between the age of the mother and the sex of her offspring. The sex ratio for the entire series of 3955 individuals is 106.1 males to 100 females. This shows that in the strains of rats used for this study the normal proportion of the sexes is about the same as that in the pure albino strain. When the litter data are arranged according to the position of each litter in a litter series (table 3), the sex ratios obtained 284 HELEN DEAN KING for the individuals in successive groups of litters are not quite as diverse as those for related litter groups as shown in table 2. The sex ratio among the individuals belonging to the first lit- ters of the series is higher than the standard, and in subsequent litter groups, up to the fifth, there is seemingly a tendency for the number of male offspring to decrease. A similar change in the sex ratios from the first to the fourth litter was noted by King and Stotsenburg in a series of litters cast by twenty-one albino females. Beginning with the fifth litter the sex ratios rise gradually until a maximum of 143.5 males to 100 females is reached at the ninth litter of the series. For the eleventh and subsequent litters, however, the sex ratios are much lower than the standard. From the sex ratios as given in table 2 it would appear that among the individuals of a litter series the sex ratio might be expected to start relatively high and then fall steadily until about the fifth litter, rise again gradually to a Maximum at about the ninth or tenth litter and subsequently drop to a low level which is maintained until the female reaches the menopause. The records under consideration are a special group selected solely because they cover the complete breeding history of a number of females that lived to an advanced age. Perhaps, therefore, they cannot be used legitimately to give evidence regarding the possible effects of the age of the mother on the sex of her offspring. From the data as given the only conclu- sion that can be drawn is that the age of the mother is not a dominant factor in determining the sex of her young. If, as Riddle (’16) maintains, sex is determined by the ‘level of metab- olism’ in the fertilized egg, there is a possibility that the age of the mother may indirectly influence sex through its effects on the metabolic processes in the egg. Age has a profound influence on every tissue in the body, and its effects on the germ cells is a problem that must be attacked from a chemical standpoint, since it can never be solved by sex statistics how- ever extensive they may be. Vt RELATION OF AGE TO FERTILITY IN RAT 28: ™~ SUMMARY 1. Litter data covering the entire breeding history of seventy- six female rats are given in the present paper. All of the fe- males belonged to ‘extracted’ strains that were derived from the F, generation of a cross between the wild Norway rat and the domesticated albino. 2. The material used comprises the data for 585 litters con- taining 3955 individuals, 2036 males and 1919 females. The average number of young in each litter was 6.7. 3. Fertility in the rat, measured by the total number of Lit- ters cast, increases with the age of the female up to the time that the animal is seven months old. There is a sharp decline in fertility after the female is a year old and, except in rare instances, the menopause has appeared by the time that the female is eighteen months of age. 4, Female rats reach the height of their reproductive activity when they are about seven months of age. This age also repre- sents the median point in the animal’s breeding career. 5. The age of the mother is a factor in determining the size of the litter cast. Litters of very young mothers are relatively small, and later litters are large until the female reaches seven months of age. Litter size diminishes with the reduction in the number of litters cast, and litters of very old females rarely contain more than three young. 6. The second litter is the largest of the series, the third and fourth litters are usually a little larger than the first. 7. The serial number of the pregnancy, up to the fifth, does not seem to alter the size of the litter to any great extent. The sixth litter cast, however, is smaller than the preceding ones, and the number of offspring decreases rapidly as the position of the litter in the litter series advances. It is very probable that it is the age of the mother, not the number of the pregnancy, that influences the size of the litters. 8. Coefficients of variation for litter size show that the lit-_ ters cast by very young females have a greater range of variation in size than have the litters cast by females at the height of THE ANATOMICAL RECORD, VOL. 11, No. 5 286 HELEN DEAN KING their reproductive activity. From this point the range of varia- tion in litter size appears to increase as the female grows older, and to reach its maximum in the litters cast when the females are sixteen months old. 9. For the entire series of litters the coefficient of variation for litter size is 38.00. 10. The total number of litters produced by different females raried from three to sixteen, with an average of 7.7 litters per female. 11. The majority of female rats probably produce from five to six litters only. ; 12. The size of the first litter cast seems to be somewhat of an index of the fertility of the female. If the first litter is very large the female will probably cast more litters than the av- erage run of females, provided she remains in good physical condition. 13. The range in litter size was from one to sixteen. Hight was the most frequent number of young in the litters of the pie- balds and of the extracted grays, while six was the most com- mon number for the litters of the extracted albinos. 14. The sex ratio for the 3955 individuals in the series was 106.1 males to 100 females. This sex ratio is very close to the normal sex ratio for the pure albino strain (107.5 males to 100 females). 15. The sex ratios obtained for the various litter groups (tables 1 and 2) do not indicate that the age of the mother is a dominant factor in determining the sex of her offspring. Old females, however, seem to produce relatively more females than male young. LITERATURE CITED Bipper, F. 1878 Ueber den Einfluss des Alters der Mutter auf das Geschlecht des Kindes. Zeitschr. Geburtshilfe und Gynikologie, Bd. 11. Crampr, H. 1884 Zucht-Versuche mit zahmen Wanderratten. II Resultate der Kreuzung der zahmen Ratten mit wilden. Landwirthschaftliche Jahrbiicher, Bd. 13. Donatpson, H. H. 1906 A comparison of the white rat with man in respect to the growth of the entire body. Boas Anniversary Volume, New York. RELATION OF AGE TO FERTILITY IN RAT 287 Donatpson, H.H. 1915 The Rat. Memoirs of The Wistar Institute of Anatomy and Biology, No. 6, Philadelphia, 1915. Hammonp, John 1914 On some factors controlling fertility in domestic ani- mals. Jour. Agri. Sci., vol. 6. Huner, G. Cart 1915 The development of the albino rat, Mus norvegicus albinus. II. Abnormal ova; end of the first to the end of the ninth day. Jour. Morph., vol. 26. King, H. D. anp Storsensura, J. M. 1915 On the normal sex ratio and the size of the litter in the albino rat (Mus norvegicus albinus). Anat. Rece., vol. 9. Lantz, D. E. 1910 Natural history of the rat. Bull. Public Health and Ma- rine Hospital Service. Govt. Printing Office, Washington, D. C. MarsHau, F. H. A. 1910 The physiology of reproduction. Longmans, Green and Co., London. Miter, N. 1911 Reproduction in the brown rat (Mus norvegicus). Amer. Nat., vol. 45. Minot, C. S. 1891 Senescence and rejuvenation. I. On the weight of guinea pigs. Jour. Phys., vol. 12. Peart, R. 1913 Note regarding the relation of age to fecundity. Science, vol. 37. PgarL, R., anp Surrace, F. M. 1909 Data on the inheritance of fecundity obtained from the records of egg production of the daughters of ‘200- egg’ hens. Bull. Me. Agri. Exper. Station, no. 166. ° Powys, A. O. 1905 Data for the problem of evolution in man. On fertility, duration of life and reproductive selection. Biometrika, vol. 4. Punnett, R. C. 1903 On nutrition and sex-determination in man. Proc. Cambridge Phil. Soc., vol. 12. Rippir, O. 1916 Sex control and known correlations in pigeons. Amer. Naturalist, vol. 50. ScHULTZE, O. 1903 Zur Frage von den Geschlechtsbildenden Ursachen. Arch. mikr. Anat., Bd. 43. SLoNaKER, J. R. 1912 The normal activity of the albino rat from birth to natural death, its rate of growth and the duration of life. Jour. Animal Behavior, vol. 2. Surrace, F. M. 1908 Fecundity in swine. Biometrika, vol. 6. Wetpon, W. F. R. 1907 On heredity in mice. 1. On the inheritance of the sex-ratio and of the size of the litter. Biometrika, vol. 5. Wentworth, E. N. anp AvuBEL, C. E. 1916 Inheritance of fertility in swine. Jour. Agri. Research, vol. 5. Wickens, M. 1886 Untersuchungen ueber das Geschlechtsverhiltniss und die Ursachen der Geschlechtsbildung bei Haustieren. Biol. Centralbl., Bd. 6. ee ms ; To es ee as a Woh Saute lle lp bea ATE- dan Melo. 1 Coit) a ie raked : xi Bie. Reet eee beth 9 ae et Ai Buel ‘ Lia cogh te Hehe) «jets ea am on Ae cel ud: oC MM Sa: hie ga nena a vy (bhi (lk Rae a Payee ie Si fet a a ia pe tauen - i <7 7 * noel : a we ~ ~~ ree ’ a ’ v¢ ry t . f A f / iat . ' ' a A i ‘ h @ Miay i : Sy . = ‘ ~ TECHNIQUE NOTES From the Department of Histology and Embryology, Cornell University, Ithaca, New York I. THE APPLICATION OF BENDA’S NEUROGLIA STAIN H. M. KINGERY This note embodies the results of some experiments with Benda’s stain for neuroglia cells and fibers. The stain is, as is well known, the basic anilin dye, toluidin blue, used after a double mordantage of the sections in ferric alum and sodium sulphalizarinate. A previous mordantage of the tissues in a chrome solution, a ‘chromation,’ is an essential part of the method. Benda himself has employed two methods for producing this ‘chromation.’ The first (Benda, ‘00) was the use of Weigert’s chrome alum bath followed by chromic acid, with tissue fixed in formalin. In the second method (Benda, ’10), tissue fixed in alcohol was placed in 10 per cent nitric acid, then in 2 per cent potassium dichromate, and finally in 1 per cent chromic acid. My results show that this ‘chromation’ may be obtained by using a chrome fixer, or better, by a dichromate bath after fixation; and the best results are produced by the use of a fixer containing a dichromate, followed by a dichromate mordantage. This is essentially the method used in preparing tissues for Weigert’s copper hematoxylin, and it may be said that in general, sections which will take a good Weigert stain for myelin will also give good results with Benda’s neuroglia stain. Tissues fixed in Zenker’s fluid, in Zenker’s followed by Mil- ler’s, in Helly’s (Zenker-formol) followed by Miiller’s, in Miiller’s alone, in formalin followed by copper dichromate, and in the copper dichromate-sublimate-acetic mixture as used by Kingsbury, followed by copper dichromate, all have given good preparations with Benda’s stain. It is worthy of note that the tissue fixed in Zenker’s fluid alone gave the poorest neuroglia stain, and that good Weigert preparations were obtained from all the tissues, except this same one. A number of sections of the spinal cord of an animal ( a skunk hap- pened to be available at the time) were fixed in copper dichromate- sublimate-acetic and mordanted in copper dichromate for periods from two to twenty-five days to determine the length of time required for the best ‘chromation.’ These all gave good results w th Benda’s stain but the best preparations were obtained from tissues mordanted four to six days. Sections of this same material were also stained with Weigert’s copper hematoxylin, and good preparations were obtained 289 290 H. M. KINGERY from the tissue mordanted two days. After a longer stay in the copper dichromate solution, the myelin failed to stain with the copper hema- toxylin, remaining yellow, while the neuroglia cells and fibers stained blue. With Benda’s stain, the myelin after a short mordantage stains a light reddish brown, but after a longer mordantage remains yellow, unaffected by the stain. From this it would seem that a short mordanting in dichromate so- lution is best for staining the myelin and a longer is best for neuroglia. After a prolonged mordantage the axis cylinders are brought out more clearly, apparently as a result of their ‘chromation’ for they did not stain with the copper hematoxylin. This does not quite agree with: the results of Smith, Mair, and Thorpe (’08) who state that the order is myelin, axis cylinders, neuroglia. This method of fixation—Helly’s fluid (Zenker-formol) followed by Miiller’s or copper dichromate-sublimate-acetic followed by cop- per dichromate—has given excellent results with tissues from a number of animals: cat, dog, man, mouse, rat, skunk. The spinal cord was used in each case, and in addition, in one form (dog) the op- tic nerve was taken. These results would show that for the ‘chromation’ four to six days in copper dichromate (2.5 per cent solution) is equivalent to two to four weeks in Miiller’s fluid or plain potassium dichromate solution. There is apparently little choice between them; possibly the tissues mordanted in copper dichromate are a trifle less brittle. A 2 per cent solution of potassium dichromate works as well as Miiller’s fluid. My results with material fixed in this way (Miiller’s after Helly’s or copper dichromate after the copper dichromate-sublimate-acetic mixture) have been better than with tissues treated as Benda recom- mends. The preparations correspond to his description; neuroglia fibers and nuclei of neuroglia cells, deep blue; cytoplasm of neuroglia cells, paler blue or purplish; myelin, reddish brown; axis cylinders darker red; connective tissues light red or pink; nerve cells reddish or purplish; and Nissl bodies darker purple. Benda’s method of staining was used, and is given for reference. Paraffin sections 5 to 8 uw are treated as follows: 1. 4 per cent ferric alum, twenty-four hours. 2. Running water, ten-twenty minutes, followed by several changes of distilled water. 3. Amber-yellow solution of sodium sulphalizarinate, twenty-four hours (saturated solution of sodium sulphalizarinate (Kahlbaum) in 70 per cent alcohol, 1 ce., distilled water, 100 cc.). 4. Several changes of distilled water, which is then absorbed with tissue paper. 5. 0.1 per cent aqueous solution of toluidin blue, heated (on the slide) until it steams, and allowed to cool, fifteen minutes or more. 6. After rinsing in distilled water the sections are treated for a few seconds with acidulated alcohol (70 per cent alcohol, 100 cc., concen- trated hydrochloric acid, 6 drops). The length of time required varies TECHNIQUE NOTES 291 with the different methods of mordanting, and is best determined by trial. 7. After the acid alcohol is absorbed with tissue paper, the sections are rapidly dehydrated with absolute alcohol. 8. The sections are then differentiated with creosote under control of the microscope. This usually takes several minutes. If more than ten seems necessary, it may be well to remove the creosote with abso- lute aleohol and treat with acid alcohol again for a few seconds; then the sections may be dehydrated and differentiated with creosote as before. 9. The creosote is absorbed with tissue paper and after several changes of toluene or xylene, mounted in balsam or damar. To prevent the fading of the stain, it is necessary to remove the creosote pretty thoroughly. REFERENCES Benpa, C. 1900 Erfahrungen tiber Neurogliafirbungen und eine neue Far- bungsmethode. Neurol. Centralbl., Bd. 19. 1910 Neurogliafirbung. Enzyk. d. mikr. Tech., 2te Aufl., Berlin, pp. 308-311. Huser, G.Cart 1903 Studies on neuroglia tissue (No. 2), neuroglia cells, and neuroglia fibers of vertebrates. Contributions to medical research (dedicated to Victor C. Vaughan). Wahr, Ann Arbor, Mich., pp. 578- 620. Kinessury, B. F. 1912 Cytoplasmic fixation. Anat. Rec., vol. 6, pp. 39-52. Smitu, J. L., Marr, W. anp THorpe, J. F. 1908 An investigation of the principles underlying Weigert’s method of staining medullated nerve. Jour. Path., vol. 18, pp. 14-27. II. SOME USES OF MALLORY’S CONNECTIVE TISSUE STAIN H. M. KINGERY Mallory’s anilin blue connective tissue stain is of course well known for the purpose for which it was intended, but it has been found quite useful for other purposes as well. As usually employed for collagen fibers, Zenker material is used. I have found that this stain used with tissue fixed in picro-aceto-formal (Bouin’s fluid) gives a very pretty differentiation for skeletal muscle, distinguishing clearly the isotropic and anisotropic bands. In practice, a thin muscle is moderately stretched and pinned out flat on a cork which is then floated upside down on the fixer. The picric acid is sufficiently removed after washing about a week in alco- hol. Thin paraffin sections (4-6) are stained according to Mallory’s directions (five minutes in the acid fuchsin solution and then twenty minutes in the anilin blue solution; differentiation in 95 per cent alco- hol, dehydration in absolute alcohol, toluene or xylene, balsam or damar). Differentiation proceeds rather slowly and may be watched under the microscope. In a finished preparation the dark band (ani- sotropic) is stained blue and the light band (isotropic) is light red or pink. Hensen’s disc (M) appears light in the middle of the blue band and Krause’s membrane (Z) is deep red in the middle of the pink band. 292 GUSTAVE J. NOBACK This method of fixation and staining when applied to insect mate- ria differentiates very nicely chitinised from non-chitinised cuticula. The chitinised is stained red and the non-chitinised is a clear blue. This method was applied particularly to the intestine of the grass- hopper and besides differentiating the cuticula it brought out the striations of the muscle fibers of the muscular coats with almost dia- erammatic clearness. With this same fixation (picro-aceto-formol) Mallory’s stain also brings out the connective tissue very clearly. And I have found that the stain may be used with good results after a number of fixers— aleohol, Carnoy’s 6-3-1, formalin—if the sections are placed for a - short time in picro-aceto-formol and then washed, before staining. Aqueous and alcoholic solutions of picric acid also give fairly good re- sults as ‘mordants,’ but not so good as picro-aceto-formol. The results obtained by this method compare very favorably with those secured after Zenker fixation. The formulae follow. Picro-aceto-formol Saturated aqueous solution of picrice acid .................4-- 75 parts Formalin... 2 .iho fee tls fe ele ane elas sey eBoy) ato aa emaerets 25 parts Glacial acetie acid... oo S20 2. ocd © odes 2 ciate oes a tee create 4 parts Solution A. Acid fuchsin: etait}. Mon er 28 ee oe ote see oho earns 0.2 gram Distilled waters ccd ccic ne soeicus sire «ees aon a eee Le aneaeen 100.0 ce. Solution B. Griibler’s water soluble anilin blue.....................0.... 0.5 gram Orange Gucis adden or Het a eas ete Me oe ice ans mera ates 2.0 gram 1 per cent aqueous solution of phosphomolybdie acid...... 100.0 ce. III. THE USE OF THE VAN WIJHE METHOD FOR THE STAIN- ING OF THE CARTILAGINOUS SKELETON GUSTAVE J. NOBACK While the Schultze and the Spalteholtz clearing methods for bone are well known and frequently used, it would seem to the writer that the van Wijhe staining and clearing method for cartilage, which nicely complements the Spalteholtz method in the study and demonstration of the development of the skeleton, is not so well known and appreciated. I venture therefore to call attention briefly to its value, having used it during the past three years with satisfactory results. The method is very simple; embryos or other material to which it is to be applied should be preserved in alcohol or (better) formalin. The specimen is next placed in 67 per cent alcohol with 1 per cent of hydrochloric acid added, for several days or a week. It is then trans- ferred to the same solution plus 0.25 per cent of methylene blue in which stain it remains for a week or two weeks, until thoroughly stained. Tol- uidin blue may be used instead of methylene blue if preferred (Lundvall TECHNIQUE NOTES 293 04,12). It is then retransferred to the acid alcohol, which is changed at intervals of one or two days or when markedly colored. In this the specimen remains until the color is nearly entirely removed from all parts save the cartilage which remains deep blue. To remove the acid it should be washed for several days with changes of 82 per cent (85 per cent) alcohol and then dehydrated by passing up through 95 per cent alcohol, absolute alcohol, equal parts of absolute alcohol and benzene, into benzene, changed at least once, in which it may remain, or it may be mounted in xylene damar or Canada balsam. The method is particularly serviceable in demonstrating the develop- ment of (a) the sternum and ribs, (b) the auditory ossicles, Meckel’s cartilage and Reichert’s cartilage, (¢) the chondrocranium, (d) the cartilage in the developing bones of the extremities, ete. As has been indicated at the beginning, the method supplements satisfactorily the Spalteholtz method in which the bone has been stained red in the usual way by means of alizarin. Two embryos, or the two halves of the same embryo carefully cut as nearly as possible in the median plane, may be run through, for bone (according to the Spalte- holtz alizarin method) and for cartilage respectively, the skin, central nervous system and viscera having been removed to clarify the view. One arm or leg may be stained for bone and the other for cartilage, ete. A solid mounting medium offers so many advantages over a liquid one that where possible it was used, damar balsam in xylene solution being preferred to Canada balsam because of its lighter color. By using glass supports for the cover-glass, such as small pieces of glass rod, ete., and using care in adding successive amounts of xylene damar, solid mounts of quite large specimens may be made on glass slides or plates. In this way specimens such as arms of the same embryo stained for bone and cartilage respectively may be mounted side by side or in parallel series to show advancing stages of development. Glycerin- jelly may be used as a solid mounting medium in the Schultze method, or with alizarin stained bone, but it may not be used with specimens stained for cartilage with methylene or toluidin blue. In the use of liquid mounting media, it was found that the benzyl benzoate and oil of wintergreen mixtures used in the Spalteholtz method were not so useful for mounting specimens stained for cartilage by the van Wijhe method, since fading was apt to result. Benzene was found to be the most serviceable mounting medium. Lundvall has used for larger speci- mens benzene four parts and carbon disulphide one part (see also Lundvall, 1912). REFERENCES Lunpwatt, H. 1904 Ueber Demonstration embryonaler Knorpelskelette. Anat. Anz., vol. 25, pp. 219-222. Lunpvatt, H. 1905 Weiteres iiber Demonstration embryonaler Skelette. Anat. Anz., vol. 27, pp. 520-523. Lunpvatt, H. 1912 Ueber Skelettfirbung und Aufhellung. Anat. Anz., vol. 40, pp. 639-646. ‘ Sarptey, P. G. anp C. C. Mackin 1916 The demonstration of centers of osteoblastic activity by use of vital dyes of the benzidene series. Anat. Rec., No. 9, July 20, vol. 10, pp. 597-599. 294 B. F. KINGSBURY SpaLtTenonz, W. 1914 Ueber das Durchsichtigmachen von menschlichen und tierischen Priparaten. Leipzig, Ed.:2, 1914. vAN WisHE, J. W. 1902 A new method for demonstrating cartilaginous mikro- skeletons. Koninkl. Akad. van Wetenschappen te Amsterdam, Proc. meeting, May 31, 1902. IV. A CONVENIENT METHOD OF ORIENTATION IN PARAFFIN IMBEDDING WHEN PAPER TRAYS OR BOXES ARE USED B. F. KINGSBURY The following simple method has been used by the writer occasion-. ally for the past fifteen years or so and since I do not recall ever having seen it mentioned, a brief description may not be out of place. The paper box method of paraffin imbedding which I use quite generally is of course well known and described in most books of technique (cf. Lee, Vade-mecum, p. 77). The procedure in obtaining the orientation is the following. By means of a moderately soft lead pencil direction lines or, if desired, the outline of the specimen or embryo (traced from a x 1 photograph), are marked on the inside of the bottom of the box chosen or usually on the paper before it is folded. The box is then floated on cold water and the melted imbedding paraffin poured in. The specimen is then at once transferred from the melted infiltration paraffin to the box. By the time this has been done a thin translucent layer of solidified paraffin covers the bottom of the box, the orientation lines upon the paper showing through. The specimen is arranged as desired accord- ing to the orientation lines or upon the outline and the paraffin allowed to cool, etc. Subsequently after removing the paper from the cold block it will be found that the pencil mark is on the paraffin block which then may be trimmed and by it easily oriented for sectioning. i ON THE ELECTIVE STAINING OF THE ERYTHROCYTE K. OKAJIMA Kyoto, Japan Today we are in possession of numerous excellent staining methods applicable to microscopic researches of the blood. The anilin dyes have been especially serviceable in the coloration of the red blood cells, and among these Eosin and Orange G deserve special mention. Solutions combining a number of dyes, for instance the triacid mix- ture (Ehrlich-Biondi), eosin methylblue (May-Griinwald), methylazure methylblue eosin (Giemsa), have rendered excellent service in the hands of many investigators. However these dyes are designed more particularly for the staining of film specimens of blood, since in staining sections they color not only the erythrocytes, but also the plasmic substances; in a strict sense they are, therefore, not the elective stains for erythrocytes. The need of an elective stain for the hemoglobin bearing erythrocytes is often felt in researches dealing with the genesis of blood cells and in many other types of the histological investigations. I have recently discovered a method of the elective tinction of the erythrocyte. The finding is based on the fact that the phosphomolybdic acid lac of alizarin stains among several tissue elements only haemo- globin. After mordanting with phosphomolybdic acid the great major- ity of the tissues of the’ animal body lose the property of staining with molybdenum alizarin lac while the erythrocyte or more particu- larly haemoglobin is colored with it. In this regard the method here recorded may be regarded as a new method for the microscopic deter- mination of haemoglobin. The various steps of the method are as follows: The material may be fixed in for- malin, sublimate, potassium bi- chromate, etc. 1. The sections are transferred to distilled water. 2. Mordant in 10 per cent phos- phemolybdiec acid solution for 30 seconds to 2 minutes. 3. Wash in water. 4. Stain in following mixture for 20 minutes to 20 hours. Sodium sulfalizarinate, saturated aqueous solution—100 cc. 10 per cent phos- phomolybdic acid, aqueous solu- tion—30 ee. (10-50 ee.). 5. Wash in water. 6. Alcohol. 7. Xylol, balsam. It is not necessary to prepare the staining solution a short time before using. A solution kept for one-half year, exposed to daylight, gave excellent results. On mixing the phosphomolybdic acid and so- dium sulfalizarinate solutions the yellowish brown color first observed later changes to one of bright orange red. Attention is called to the fact that on staining sections according to this method, the erythrocytes of vertebrates, the nuclei of the erythro- cytes excepted, are durably stained a light to dark orange red, other tissues remaining unstained. The method is thus differential. Some- 295 296 K. OKAJIMA times the nuclei and protoplasmic substances, especially in materials fixed in bichromate of potassium, take the stain a little, but it is easy to distinguish the bright orange red color of erythrocyte from the dirty yellowish brown color of nuclear chromatin or from other protoplasmic substances, for in the latter the color bleaches gradually. The con- nective tissue fibrils and osseous tissue are colored a bluish tinge, in- creased in intensity by longer staining so that excellent double staining, with brilliant contrast in orange red and blue may be obtained. It remains to be considered whether the molybdenum alizarin lac stains haemoglobin or some other structure of the erythrocyte. To determine this question the following experiment was undertaken. On a spot on two slides the chemical pure hemoglobin (Merk, Darmstadt), was spread and near it a section of liver fixed in formalin. Both were allowed to dry. One slide was now mordanted in the phosphomolybdie acid solution, the other not. Both were then stained in the alizarin molybdenum lac. On the slide mordanted we observe that the liver cells were not stained while the haemoglobin was colored a very dark orange red. On the unmordanted slide the liver cells were colored brilliant red and the haemoglobin a deep orange red. This observation may serve to show that the erythrocyte or haemoglobin represent the substance which stains after mordanting of phospho- molybdic acid by molybdenum alizarin lac. From the facts given it would seem that molybdenum possesses the property of effecting animal tissues so that mordanting by it diminishes or entirely deprives them of their staining property, this with the ex- ception of haemoglobin. It is a question whether the molybdenum alters the majority of tissues, haemoglobin excepted, or whether there exists a peculiar affinity between the haemoglobin and this lac. It may be of interest to determine precisely the chemical relations of both. It is recommended that before or after the staining by the solution given the sections be treated with some nuclear dye. Haematoxylin may be used for this purpose, the section being first stained in this dye. On the subsequent use of haematoxylin, staining of connective tissue fibrils is obtained by reason of the formation of an haematoxylin molyb- denum lac as in the Mallory’s stain. In conclusion it may be stated that as with the solutions so with the stained preparations, they are durable. The slides made one year ago and kept in the half dark are as yet unbleached. After the present work was completed and ready for publication, attempts to make alcoholic solutions were undertaken. The alcoholic saturated solution of the sodium sulfalizarinate changes a little its color on the addition of the phosphomolybdie acid. The procedure is the same as described for aqueous solutions. By this modification the length of time required for staining has been considerably shortened and the coloration seems more certain. On the durability of both solutions and preparations, when alcoholic solutions are used, a future communication will give information. The alcoholic stain has been prepared by mixing the two following solutions: Sodium sulfalizarinate saturated alcoholic solution, 100 cc.; 10 per cent phosphomolybdic acid aqueous solution, 1 to 2 ce. NEUTRAL RED AS A CELL STAIN FOR THE CENTRAL NERVOUS SYSTEM J. B. JOHNSTON University of Minnesota The method described below has been used for eight years for the study of the tigroid content of cells as well as the size, form and group- ing of cell bodies. Asa ‘Nissl method’ it gives better results with for- malin material than methylene blue or toluidin blue. The method is simple and the stain is permanent and is suitable for photography. The stain is made up in a 1 per cent aqueous solution and kept for months or years until thoroughly ripened. The ripening process is hastened by exposure to the air or by boiling but I know of no way to obtain a satisfactory staining solution in a few days. ' 19; - PROCEEDINGS OF THE AMERICAN SOCIETY OF ZOOLOGISTS FOURTEENTH ANNUAL MEETING The American Society of Zoologists held its fourteenth Annual Meeting jointly with Section F of the American Association for the Advancement of Science and in affiliation with the American Society of Naturalists, December 27, 28 and 29, 1916, in Scher- merhorn Hall, Columbia University, New York City. BUSINESS SESSION Election of Members At the session for transacting business, held at 11 o’clock on Friday, December 29, President D. H. Tennent in the chair, the persons whose names follow, having been recommended by the Executive Committee to the Society for election to membership, we duly elected. ALLEN, Ezra, A.M., Ph.D. (University of Pennsylvania), Professor of Biology, Philadelphia School of Pedagogy, 12th above Spring Garden, Philadelphia, Pa. CarotTuers, E. Exeanor, A.B., A.M., Ph.D., Zoological Building, Univer- sity of Pennsylvania, Philadelphia, Pa. CHURCHILL, Epwarp Perry, A.B. (Iowa), Ph.D. (Johns Hopkins), Assistant U. 8. Bureau of Fisheries, 317 Marshall Street, Hampton, Va. Cort, Wiiti1am Water, A.B. (Colorado College), M.A., Ph.D., University of Illinois), Assistant Professor of Zoology, University of California, Department of Zoology, University of California, Berkeley, Calif. Dottey, Jr., Witi1aM Ler, A.B., A.M. (Randolph-Macon), Ph.D. (Johns Hop- kins), Professor of Biology, Randolph-Macon College, Ashland, Va. Gre, Witson, B.S. (University of S. C.), Ph.D. (University of California), Pro- fessor of Biology, Emery University, Oxford, Ga. Goopricu, Husert Baker, B.S. (Amherst), M.A., Ph.D. (Columbia), Instructor in Zoology, Wesleyan University, Middletown, Conn. Hai, Maurice Crowrtuer, S.B., M.A., Ph.D., D-V.M., Parasitologist, Research Laboratory, Parke, Davis & Co., Detroit, Mich. Issen, Heman Lawntirz, B.S., M.D., Ph.D. (Wisconsin), Assistant in Experi- mental Breeding, University of Wisconsin, Madison, Wis. 467 468 AMERICAN SOCIETY OF ZOOLOGISTS Jones, ORREN Luoyp, B.S., M.S., Ph.D. (Wisconsin), Associate Professor, Ani- mal Husbandry, Jowa State College, Ames, Iowa. Kixcarp, Trevor, M.S., Professor of Zoology, University of Washington, Seattle, Wash. Merz, Cuartes W., B.A., Ph.D., Station for Experimental Evolution, Carnegie Institution of Washington, Cold Spring Harbor, Long Island, N.Y. Mippieton, Austin Raupu, A.B., Ph.D. (Johns Hopkins), Assistant Professor of Biology, University of Louisville, Louisville, Ky. Mosuer, Epna, B.S. (Cornell), Ph.D. (Illinois), Instructor in Entomology, University of Illinois, Natural History, Building, Urbana, IIl. Mutter, Herman J., A.M., Ph.D., Instructor in Zoology, Rice Institute, Houston, Texas. i Packarp, Cuarues, M.S., Ph.D., Instructor in Zoology, Columbia University, Schermerhorn Building, Columbia University, New York City. Ropertson, Witiiam R. B., A.B. (Kansas), Ph.D. (Harvard), Assistant Pro- fessor of Zoology, University of Kansas, 1420 Ohio Street, Lawrence, Kans. Rocers, Frep Terry, A.B., Ph.D. (Chicago), Assistant Professor of Zoology, Baylor University, Waco, Texas. SuepHerp, W. T., A.M., Ph.D., Professor of Zoology and Dean, Waynesburg College, Waynesburg, Pa. Van Cieave, Hartey Jones, B.S., (Knox College), M.S., Ph.D. (Illinois), Associate in Zoology, 300 Natural History Building, Urbana, Ill. Wenricn, Davin Henry, B.A., M.A., Ph.D., Instructor in Zoology, University of Pennsylvania, Zoological Laboratory, Philadelphia, Pa. Wentworts, Epwarp N., M.S. (lowa), Professor of Animal Husbandry, Kansas State Agricultural College, Manhattan, Kans. Wuitinc, Parneas W., A.B., M.S., Ph.D., Harrison Research Fellow, Univer- sity of Pennsylvania, Zoological Laboratory, Philadelphia, Pa. Election of Officers The Committee on Nominations, consisting of E. A. Andrews, Edwin Linton and C. A. Kofoid, having recommended persons for election to the various offices of the Society, and no other nominations having been made, Maynard Mayo Metcalf was elected President to serve for one year, Charles Zeleny, Vice- President to serve for one year, Caswell Grave, Secretary-Treas- urer to serve three years, and H. V. Wilson, Member at large of the Executive Committee to serve five years. Report of the Secretary-Treasurer The Secretary-Treasurer reported that 40 members withdrew from the Society during the year and that 26 members have not paid dues, in protest against the increase in annual dues, but that - FROCEEDINGS 469 257 members have accepted the plan, adopted at the Columbus meeting, for supporting and increasing the circulation of Journals published by The Wistar Institute. Attention was called to the fact that the list of members of the Society now contains 308 names of which 173 (56 per cent) are of members residing in Eastern territory, 185 (44 per cent) are of members residing in Western territory. Financial Statement The financial statement of the Secretary-Treasurer, for the year 1916, is as follows: RECEIPTS January 1, 1916: : ERECTOR DRAG: 5 0k oS Jee cn ee ante © hore ee TO ne eee 809.68 acre dues for the year 1915...45 <5. 4 auvc w alee ae eee eee 2.00 Pree Oued lOrene year TOME Fg h2 sok Ldn at nee er eee eee 8.00 mack dues for. the year 1915.20 ei wel ots Su eae sete eee eee 36.00 January to December: Buedfor 1OlG ati Sli sO eek Gaels sc han tees o Rae ene te eee 126.50 Pires for 1916 at °S.00\(Poreign) 22% 23.3) Ve eee 8.00 Pace for TOlG att) FOOL Cay k a Sop, ae or Bion oe Bre 1225.10 Dues for 19lGsat) 6:50) (hife Members)! e-.9205 0.224. 0e te ae 19.50 PacHierIOIGrat \iGlOO. ce 2008s bs AO CRS it Sl 6.00 Peden fort Si GrGee 75.00: Seis eee Ne So ee clk eee evan 335.10 October 1, 1916: Engerest, ab per cent Om: Cepasitss 2 si a. Oe = oo nie nine cles doin doe = 32.61 BOE ry thes otros pe eu oA payee ls tk Reem sic atuaees NO ee A ere $2608 .49 EXPENDITURES January to December: Appropriation for Councilium Bibliographicum................... 200 .00 BaP EEO ICME ROR ge arty 2 At 52 ede Rags ea ieint Sync Sa 4.35 Pelexrapu telephane and street CAP... 5 25.2.4- spun + Meenas sale’ = =F 2.60 Two copies ‘‘Science’’ containing Proceedings..................--- .30 BRAIONELY. Shamps; POSt-CArdS. 20.6.2). tes cian cece thnk eae e feels 32.82 ypewritine and: clerical assigtance...2 2.2. 66 ses i deeds poke 31.65 Printing announcements and programs................-+2++e+++++ 51.55 OCR aIGGHes TEGHENCH, LOLEEN B Onr oe a8 40: 1 Stk hep aw ke tc eat 5.00 207 Subscriptions for Journals, Wistar Inst. ...............2.2..2 1591.00 Expenses of Secretary at Conference on Journals. ................. 4.80 Expenses of Secretary at New York meeting...................... 34.84 OR eat eee Chae ohh OM ce Aes As, chee lawl aise geen Sapte Cae $1958.91 December 28, 1916: balance om Hands nese. hh fons Sees ease oe viaho ausiee Dae nigral alee $649.58 470 AMERICAN SOCIETY OF ZOOLOGISTS Report of Auditing Committee The Auditing Committee, consisting of Ulric Dahlgren and William H. Longley, reported that the accounts of the Secre- tary-Treasurer had been examined and found correct. Instruction for Executive Committee By motion made by P. P. Calvert, seconded by C. E. McClung, the Society instructed its Executive Committee to continue to make efforts to increase the number and variety of Journals available to members in return for annual dues. Change in By-Laws By vote of the Society, by-law 4 b, which reads as follows; was stricken from the list of by-laws. 4(b) When the annual meeting is held in conjunction with the American So- ciety of Naturalists, the Society shall adjourn for one session (morning or after- noon) to meet in joint session with the American Society of Naturalists, and it shall be the policy of the Society to conclude its annual meeting with the Natur- alists’ dinner, and the Secretary-Treasurer is instructed to urge upon the Natur- alists the propriety of adjusting their program with this point in view. Resolution Adopted K. G. Conklin proposed the following resolution which was unanimously adopted by the Society: “Whereas, the National Academy of Sciences has, at the request of the President of the United States, organized the National Research Council for the purpose of promoting and organizing research in the interest of National Welfare, and “Whereas, we recognize that human progress is dependent upon the advancement of knowledge, and _ “Whereas, one of the chief purposes of the American Society of Zoologists is the promotion of research in Zoology, “Therefore, be it resolved that the American Society of Zoologists agrees to cooperate with the National Research Council in any ways practicable.”’ PROCEEDINGS 471 Committee on Premedical Education The Committee on Premedical Education, failing again to submit a report, was continued and instructed to report at a future meeting. JOINT SESSION WITH NATURALISTS The business of the Society having been transacted, the session adjourned to meet in joint session with the American Society of Naturalists, at 2 p.m., in the Horace Mann School, the program for which was a symposium on the subject, ‘‘Biology and National Existence.” Sessions for Presentation and Discussion of Papers At sessions held during the forenoons and afternoons of Dec- ember 27 and 28 and during the first part of the forenoon session on December 29, the papers listed on the program were read in full or by title. Forty-four papers were presented in full, twenty- seven were read by title. The papers listed in the Genetics Section, as ordered by the Society at the Columbus Meeting, were scheduled for and read at the session on the afternoon of December 28. TITLES OF PAPERS LIST OF TITLES OF PAPERS ARRANGED IN GROUPS IN THE ORDER oo NI OD ON 20. 21. RECEIVED BY THE SECRETARY COMPARATIVE ANATOMY . Cell inconstancy in Hydatina senta. A. Franklin Shull, University of Michigan. . A gynandromorphous cat. Mary T. Harmon, Kansas State Agricultural College. . On the third layer of protoplasm in amoeba. A. A. Schaeffer, University of Tennessee. . Some homologies in the epipharynx and hypopharynx of the nematocerous diptera. Adelbert L. Leathers, Olivet College (Section F). EMBRYOLOGY . The history of the eye muscles. (Lantern illustrations.) H. V. Neal, Tufts College. . A case of superfetation in the cat. Mary T. Harmon, Kansas State Agri- cultural College. . On the mechanism of serial differentiation in the embryonic vertebrate nervous system. O.C. Glaser, University of Michigan. . Embryology of the yellow mouse. W. B. Kirkham, Yale University. . Investigations of the light organs of arthropods. Ulrich Dahlgren, Prince- ton University. . Further experiments on the laterality of transplanted limbs. Ross G. Har- rison, Yale University. . The effect of removal and regeneration of parts upon metamorphosis in am- phibian larvae. Charles Zeleny, University of Illinois. . Life history of Zeugophora scutellaris. B.H. Grave, Knox College. . The results of extirpation of the hypophysis and thyroid glands of Rana. Bennet M. Allen, University of Kansas. CYTOLOGY . An experimental study of cell division. L. V. Heilbrun, University of Llli- nois College of Medicine. . Early castration of the vertebrate embryo. Franklin P. Reagan, Princeton University. Introduced by C. F. W. McClure. . Microdissection studies. The cell aster; a reversible gelation phenomenon.— Illustrated with drawings. Robert Chambers, Jr. Cornell University Medical College. . Multiple chromosomes of Hesperotettix and Mermiria. C. E. McClung, University of Pennsylvania. . The spermatogenesis of Culex pipiens, P. W. Whiting, University of Penn- sylvania. (Section F.) . The segregation and recombination of homologous chromosomes in two gen- era of Acrididae (Orthoptera). E. Eleanor Carothers, University of Penn- sylvania. (Section F). Synapsis and chromosome organization in the male germ cells of Chortippus and Be eee D. H. Wenrich, University of Pennsylvania. (Sec- tion F). The chromosome complex in Apithes agitator. W. J. Baumgartner, University of Kansas. 473 474 AMERICAN SOCIETY OF ZOOLOGISTS 22. 23. 24. 45. 46 New facts and views concerning the occurrence of a sexual process in the life eycle of a myxosporidia, chloromyxum leydigi. Rhoda Erdmann, Yale University. Spermatogenesis in fhe albino rat. Ezra Allen, University of Pennsylvania. (Section F). Multiple complexes in the alimentary tract of Culex pipiens. Caroline M. Holt, University of Pennsylvania. (Section F). GENETICS . Sex-linked inheritance of spangling in poultry. George Lefevre, University of Missour1. . Two classes of factors for color patterns in Paratettix. Robert K. Nabours, Kansas State Agricultural College. . The relation of yellow coat color to black eyed white spotting of mice, in heredity. C.C. Little, Harvard Medical School. . Mutation in Didinium nasutum. S. O. Mast, Johns Hopkins University. . The occurrence of mutations in skunks of the species, Mephitis putida and M. hudsonica. J. A. Detlefsen, University of Illinois, College of Agricul- ture. . The influence of parental alcoholism on the learning capacity of the off- spring. E.C. MacDowell, Carnegie Institution of Washington. . Linkage in the sex-chromosome of a new species of Drosophila. Chas. W. Metz, Carnegie Institution of Washington. Introduced by C. B. Daven- port. . An examination of the so-called process of contamination of Genes. Thomas Hunt Morgan, Columbia University. . An analysis of the effect of selection on bristle number in a mutant race of Drosophila. Alfred H. Sturtevant, Columbia University. . The elimination of males in alternate generations of sex-controlled lines. Calvin W. Bridges, Columbia University. Introduced by T. H. Morgan. . Coincidence of crossing over and the chromosome theory of linkage. Alex- ander Weinstein, Columbia University. Introduced by T. H. Morgan. . Determinate and indeterminate laying cycles in birds. Illustrated with lantern. L. J. Cole, University of Wisconsin. . A-strain of sex intergrades. Arthur M. Banta, Carnegie Institution of Washington. . Effect on fertility of crossing closely and distantly related stocks of Droso- phila ampelophila. Roscoe R. Hyde, Indiana State Normal School. . Are the polyradiate cestodes mutations? Illustrated with lantern. Frank- lin D. Barker, University of Nebraska. EVOLUTION . A revised working hypothesis of mimicry. W.H. Longley, Goucher College. COMPARATIVE AND GENERAL PHYSIOLOGY . Recent studies of nerve conduction in Cassiopea. Lllustrated with lantern. Alfred G. Mayer, (Carnegie Institution of Washington). . The theory of sex as stated in terms of results of studies on the pigeons. Oscar Riddle, Carnegie Institution of Washington. . The adaptive color changes of tropical fishes. Illustrated with lantern. W. H. Longley, Goucher College. . The histological basis of adaptive shades and colors in the flouder, Para- lichthys albiguttus. Albert Kuntz, St. Louis University School of Medi- cine. Further data on the relation between the gonads and the soma of some apes birds. H. D. Goodale, Massachusetts Agricultural Experiment Station. . The sensory potentialities of the nudibranch Rhinophores. Leslie B. Arey, Northwestern University Medical School. 70. a. ie PROCEEDINGS 475 Paramecium grown in pure cultures of bacteria. George T. Hargitt and Walter W. Fray, Syracuse University. . Recognition among insects. N.E. Meclndoo, Bureau of Entomology. 9. The rate of loemotion of Vanessa antiopa in different luminous intensities and its bearing on the ‘‘Continuous Action theory of Orientation.’? Wm. L. Dolley, Jr., Randolph-Macon College. Introduced by 8. O. Mast. . A super-organ for the expansion of Renilla. G.H. Parker, Harvard Univer- sity. . The photoreceptors of Amphioxus. W. J. Crozier, Bermuda Biological Sta- tion. 2. The olfactory reactions of snails. Manton Copeland, Bowdoin College. . The reactions of the crimson-spotted newt, Diemyctylus viridescens to light. Albert M. Reese, West Virginia University. . Reaction of the whip-tail scorpion to light. Bradley M. Patten, Western Reserve University. . The effect of light and dark upon the eye of prorhynchus applanatus, Kennel. W. A. Kepner, and A. M. Foshee, University of Virginia. . Experimental control of endomixis in Paramecium. R. T. Young, Univer- sity of North Dakota. . Orientation to light in Planaria (n. sp.) and the function of the eyes. W.H. Taliaferro, Johns Hopkins University. Introduced by 8. O. Mast. . Sense of taste in Nereis virens. Alfred O. Gross, Bowdoin College. . The Influence of the Marginal Sense Organs on Functional Activity in Cas- siopea Xamachana, Bigelow. Lewis R. Cary, Princeton University. . The relation between the hydrogen ion concentration of sperm suspensions and their fertilizing power. Edwin J. Cohn, University of Chicago. In- troduced by Frank R. Lillie. . Experimental study of ageing eggs and sperm and of their development. A. J. Goldfarb, College of the City of New York. . The consumption of oxygen during the development of Fundulus_hetero- clitus. George G. Scott, College of the City of New York, and William E. Kellicott, Goucher College. . A study of broodiness in the Rhode Island Red breed of domestic fowl. H. D. Goodale, Massachusetts Agricultural Experiment Station. . The vitalty of cysts of Didinium nasutum. §S.O. Mast, Johns Hopkins Uni- versity. . The reactions of Pelomyxa Carolinensis, Wilson, to food. W.A. Kepner and J. G. Edwards, University of Virginia. . The significance of conjugation and encystment in Didinium nasutum. S. O. Mast, Johns Hopkins University. ECOLOGY . Some distributional problems of Okefinokee Swamp. A. H. Wright, Cornell University. PARASITOLOGY . A means of transmitting the fowl nematode, Heterakis papillosa (Bloch). James E. Ackert, Kansas State Agricultural College. . Further studies on changes in Thelia bimaculata brought about by insect parasites. lllustrated with lantern. S. 1. Kornhauser, Northwestern University. Some experiments on the transmission of swamp fever by insects. Illus- trated with lantern. John W. Scott, University of Wyoming. The domestic cat a host of taenia pisiformis (Bloch). James E. Ackert, Kansas State Agricultural College. DEMONSTRATIONS Preparations showing the structure of a transformed plasma Clot. George A. Baitsell, Yale University. THE ANATOMICAL RECORD, VOL. 11, No. 6 476 AMERICAN SOCIETY OF ZOOLOGISTS bo . Demonstrations of the following types of chromosome groups in Drosophila ampelophila, XX (female), XY (male), XXY (female), XXYY (female). Calvin W. Bridges, Columbia University. 3. The innervation of the vertebrate digestive tube (Methylene blue intravitam staining) F. W. Carpenter, Trinity College. _ 4. The phosphorescence of enteropneusta. W. J. Crozier, Bermuda Biological Station. 5. The relation between the gonads and the secondary sexual characters in birds. H. D. Goodale, Massachusetts Agricultural Experiment Station. 5. Mounted skins showing a new color variety of the Norway rat. Helen D. King and P. W. Whiting, University of Pennsylvania. 7. Photographs of Thelia illustrating the changes brought about by the parasitic hymenopteron Aphelopus. 8S. 1. Kornhauser, Northwestern University. 8. Multiple chromosomes of Hesperotettix and Mermiria. C. E. McClung, University of Pennsylvania. 9. Models and specimens showing transplanted limbs. Ross G. Harrison, Yale University. 10. Miscropic slides illustrating spermatogenesis in Culex pipiens. P. W. Whit- ing, University of Pennsylvania. (Section F). 11. Specimens of Polyradiate cestodes. Franklin D. Barker, University of Ne- braska. 1. Cell inconstancy in Hydatina senta. A. FRANKLIN SHULL, Univer- sity of Michigan. The number of cells in the various organs of this rotifer was reported by Martini to be always the same. In at least two organs, however, it is found that in a small percentage of individuals aberrant numbers occur. ‘This inconstancy is more in keeping with the well established variability and modifiability of the life cycle. 2. A Gynandromorphous cat. Mary T. Harmon, Kansas State Agri- cultural College. A cat having an ovary on the right side and a testis on the left, was discovered in our laboratories last winter. The animal had been skinned and partially dissected before its peculiarity was discovered. The scrotal sac and external genitalia had been removed. The testis which is about the size and shape of a navy bean is entirely on the outside of the body cavity ventrad and to the left of the ventral border of the pubis. It has all the appearance of a normal testis. The spermatic cord extends from the testis through the oblique muscle where it divides into the vas deferens and the spermatic vein and ar- tery. The vas deferens extends anteriorly until it curves over the ureter where it continues caudad dorsal to the neck of the urinary blad- der. It pierces the prostate gland and enters the urethra about half way between the base of the urinary bladder and the exterior. The prostate gland of the left side is larger than the one of the right side although there seems to be a gland on the right side. The ovary is located on the right side of the body a little posterior to the kidney. It is slightly smaller than the testis and is quite angu- lar. Anterior to the ovary and partially surrounding it is the ostium tubae abdominale. The ovarian artery and the ovarian vein extend to the left from the ovary. Extending from the ostium tubae abdomi- PROCEEDINGS 477 nale is the uterine tube which continues caudad almost parallel to the vas deferens. It enters the urethra through the abortive right pros- tate gland. The ovary and the uterine tube are held in place by the broad ligament and the round ligament. 8. On the third layer of protoplasm in ameba. A. A. SCHAEFFER, Uni- versity of Tennessee. In addition to the streaming endoplasm and the more or less station- ary ectoplasm in amebas there is found a third layer separating the ectoplasm from the surrounding water. This third layer is extremely thin, probably too thin to be seen easily, but the existence of it can be readily demonstrated by means of the movement of small particles which occasionally attach themselves to it. The most striking feature of the movement of particles attached to this third layer is that they move forward toward the tip of a pseudopod faster than the tip advances through the water. Particles travel from every part of the surface to- ward the tips of the advancing pseudopods. If there is but one pseudo- pod, all the particles travel toward the tip of this pseudopod. The ef- fect is therefore that all the particles tend to collect at the advancing end of the ameba. Most of the particles drop off however when or soon after they reach the front end of a pseudopod. Particles travel at varying speeds, depending upon where they are located; particles on an actively enlarging pseudopod move faster than those near the pos- terior end of the ameba. In a general way, the nearer they approach the tip of a pseudopod the faster the particles travel. The speed of a moving particle attached to the third layer is not directly affected by nor related to the rate of streaming of the endoplasm immediately under it (but separated from it, of course, by the ectoplasm). The granules in the streaming endoplasm of a pseudopod move considerably faster than the particles attached to the outside third layer. But occasion- ally one may see a particle sticking to the third layer move more rap- idly than the slowly and uncertainly moving endoplasm immediately beneath it. When streaming is reversed in a pseudopod, movement in the third layer is likewise reversed, and frequently particles attached to the outside travel away from the tip of the retracting pseudopod more rapidly than the tip is retracted. These particles then move to- ward the tip of another advancing pseudopod. Occasionally a particle sticks so close that it travels all over an ameba before it finally drops off. This third layer is found in Amoeba proteus, A. discoides, A. dubia, A. vespertilio, and A. metaproteus (an undescribed species). Whatever may be the relation of this third layer to endoplasmic streaming, it is clear that from the transportative aspect it is not an aid but a slight hindrance to locomotion, since the layer moves in the same direction as the ameba. The nature of this layer is not clear. It seems to be in process of continual formation all over the ameba, and in process of continual de- struction at the anterior ends of pseudopods. Surface is not therefore made at the advancing tips of the pseudopods, as might be thought 478 AMERICAN SOCIETY OF ZOOLOGISTS at first sight, but destroyed, i.e., converted into interior (non-surface) matter. Surface is made over the entire ameba, but very slowly at the posterior end and at the tips of retracting pseudopods. From in- spection it appears that the layer is protoplasmic in composition rather than aqueous, though there is insufficient evidence at hand to decide definitely. It is also at this time impossible to state whether there is essential connection between locomotion and the movement of this third layer. The pseudopods of Difflugia pyriformis carry foreign particles on their surfaces, but these particles do not move as rapidly as the tips of the pseudopods advance. Diatoms and Oscillatoria likewise carry particles on their surfaces, but in the latter the same agency that car- ries the particles also moves the Oscillatoria filaments. Whether the transportative agencies of these several organisms have anything in common cannot yet be stated. 4. Some homologies in the epipharynx and hypopharynx of the nema- tocerous diptera. ADELBERT L. LeaTuERs, Olivet College, Section F. The literature bearing on this subject is very incomplete and more or less confused. The ‘lateral arms’ or ‘premandibles’ of the hypo- pharynx have been well figured for only the Chironominae, while in the other families and subfamilies of the group no such structures have hitherto been recognized. These structures will be shown by a series of comparative figures with especial reference to the family Chironomi- dae, but will include one or more other families in less detail. In a similar manner the comparative development of the hypopharynx from a rudimentary structure to a highly developed tritrating organ will be compared. 5. The history of the eye muscles. H. V. Newau, Tufts College. (With lantern illustrations.) The attempt is made in this paper to demonstrate on the basis of embryological evidence the exact homology of the first three permanent myotomes of Amphioxus, Petromyzon, and Squalus and to describe the more important stages in the phylogenesis of the eye muscles. The evidence is presented for the first time to support the assertion of Dohrn (’04) and the writer (’07) that the second as well as the third myotome participates in the formation of the external rectus muscle. In the light of the evidence given the familiar text-book formula for the ontogenesis of the eye muscles should be amended as follows: From the first myotome (pre-mandibular head-cavity) arise the muscles innervated by the oculomotor, viz., the Mm. recti superior, internus, and inferior, and the M. obliquus inferior; From the second myotome (mandibular head-cavity) develop the M. obliquus superior and the ventro-lateral portion of the M. rectus externus; From the third myotome (hyoid head-cavity) arises the dorso- median portion of the M. rectus externus. PROCEEDINGS 479 6. A case of superfetation in the cat. Mary T. HarMan, Kansas State Agricultural College. The gravid uterus of a cat had two enlargements of the right horn and three enlargements of the left horn. The enlargement of the right horn next to the ovary and the two enlargements of the left horn toward the ovary each contained an embryo 90 mm. in length exclusive of the tail. From the external features, they appear to be near to term. The limbs are well formed and normal, having joints and on the ends of the digits are claws. The whole surface of the skin is covered with pits but very little hair is present. The tail is more than one-third the length of the body. The fetal membranes fill the entire enlarge- ment and fit very closely to its walls. The enlargement of the right horn of the uterus next to the vagina contained an embryo only 10 mm. in length which seemed perfectly normal and which had no indications of having been dead long before it was preserved. The umbilical cord occupies about one-sixth of the ventral surface. The limbs both fore and hind, are merely buds. The tail is about one-fifth the length of the remainder of the body. There are no indications of hair. The mouth is in the process of formation. The mandibular processes have met in the median line; but they extend only about one-third as far as the maxillary processes. The lip groove is shallow, in fact, there is merely the beginning of the separation of the lips and cheeks from the jaw. The oral pit is rectangular in shape. There is no indication of eyelids; but the eyes are plainly visible from the outside as small dark spheres. This embryo does not seem to be of more than two weeks development. If the size of the blood vessels of the uterus and the condition of the blood vessels to the embryos may be taken as a criterion, the blood supply to the small embryo is as good as to the large embryos. It seems as reasonable to the writer to think of the less advanced embryo as the result of delayed fertilization as to account for it on the ground of arrested development or of a second coition. 7. On the mechanism of serial differentiation in the embryonic vertebrate nervous system. O.C. GuAsER, University of Michigan. Long before the separation of the neural plate from the ectoderm is complete, the developing neural tube prefigures a serial differentiation that culminates in a succession of vesicles highly constant for the ver- tebrate nervous system and, as one of its basic attributes, calling for explanation. More than forty years ago His attempted to account for the vesicu- lation of the embryonic brain in terms of cranial flexure. Substanti- ated by ingenious experiments with rubber models, this theory could account for the early lateral and ventral differentiations of the prospec- tive interbrain. However, since the onset of vesiculation occurs be- fore that of cranial flexure the view that the former is dependent upon the latter involves an anachronism. 480 AMERICAN SOCIETY OF ZOOLOGISTS According to His cranial flexure and the subsequent rearrangement of the vesicles are the outcome of differential growth which produces a state of compression in the antero-posterior axis. It is obvious that such compression, if demonstrable prior to cranial flexure, would nec- essarily be an important element in helping us to understand how the nervous system has imposed upon it its characteristic form. Compression in the antero-posterior axis cannot be demonstrated by the method used by His. It can, however, be shown to exist by com- paring the length of the embryonic head with that of the nervous sys- tem. The relation between these two measurements can be expressed in the form of a fraction derived by dividing half the perimeter of the nervous system into the head length. This fraction, which I have called the neurocephalic quotient, tells how many units of head-length are available for every unit of length in the nervous system. When the quotient is greater than unity, the nervous system cannot be under compression; when it is less than unity, a state of compression must exist. A priori we should expect high quotients in the earlier stages and low quotients in the later, whereas embryos in which the vesicles are telescoped or abnormally overdifferentiated, should have quotients lower than normal for their respective ages. These expectations, as will be shown in the paper, are fulfilled, and the conclusion seems warranted that a rising state of compression in the longitudinal axis, prior to cranial flexure, is one of the important conditions under which the vesiculation of the embryonic brain takes place. The paper will be fully illustrated by tables, drawings, and a curve. 8. Embryology of the yellow mouse. W. B. KirxHam, Osborn Zoologi- cal Laboratory, Yale University. It has for some years been known to breeders that yellow mice did not breed true to coat color, each litter almost always containing one or more young of a color other than yellow, and it has also been found that the average number of young in a litter is smaller from yellow than from other colors of mice. These two observed facts taken together have given rise to the theory that all the available yellow mice are heterozygous as regards coat color, and that the homozygous zygotes which should theoretically exist are for some reason not viable. To test out this theory has been the purpose of this work. Material from non-suckling yellow mice, representing each of the first nineteen days of pregnancy has been assembled, that for the first three days comprising ovaries and Fallopian tubes, that for stages be- yond the third day the uteri as well as the ovaries and tubes. The entire material from each mouse has been sectioned, mounted, stained, and every section examined, while all the embryonic stages found have been compared with those of like age from non-suckling white mice. The results are as follows: (a) The rate of cleavage and of embryonic development is the same for yellow as for white mice. PROCEEDINGS 481 (b) All of the observed two-cell stages of both yellow and white mice appear normal. (c) No degenerating morulae or blastulae were found in white mice, while one or more were present in every yellow mouse containing em- bryos of that stage in development. (d) The material covering the sixth to the seventeenth days of preg- nancy has yielded degenerating embryos in eight uteri out of twenty- eight in white mice, and in eleven out of thirteen in yellow mice. If we eliminate females who, by having still births or by eating up their new-born young, showed themselves abnormal, the figures become more striking, degenerating embryos in white mice appearing only in one uterus out of the twelve examined, while in yellow mouse uteri eleven out of twelve contained them. (e) After the normal time for implantation (sixth day of pregnancy) all the degenerating embryos found, with one exception, had induced a typical reaction on the part of the uterine mucosa, but had themselves failed to undergo any development beyond the blastula stage. (f) No degenerating embryos have been found in either white or yellow mice pregnant more than sixteen days. This is to be expected, as the evidence all indicates that if degeneration is going to take place it starts before the seventh day of pregnancy, and the embryos are then so small that the phagocytes easily remove them, and the uterine wall in their vicinity returns to its resting condition several days be- fore the normal embryos are born. (g) Mendel’s law requires an average of 25 per cent homozygous yellow offspring from heterozygous yellow parents. In this investiga- tion we have found in normal yellow females, six to seventeen days pregnant, 69 embryos, of which 26 or 37.8 per cent were degenerating, as contrasted with 2 degenerating embryos out of a total of 84, or 2.3 per cent obtained from normal white females pregnant for the same period of time. Conclusion. The evidence produced by this investigation is not as absolutely decisive as might be hoped for, but the much greater per- centage of degenerating embryos in yellow mice than in white would indicate that some at least of these degenerating yellow mouse embryos are the missing homozygous animals. 9. Investigations of the light organs of arthropods. UtricH DAHLGREN, Princeton University. The origin of the imaginal light organs in Photorus pennsylvanica was investigated to find out how it was derived during the pupal stage. Larvae, bearing their two round ventro-lateral organs on the penul- timate segment, were collected in the spring and placed in vivaria con- taining soil and dead leaves from their habitat. In latter May these larvae scooped shallow cavities in the ground, built lattice-shaped cov- ers with a mixture of saliva and clay and became torpid. In a few days the skin was shed and the cream-white pupae lay in the cavities when they could be easily observed after breaking off the clay covers and 482 AMERICAN SOCIETY OF ZOOLOGISTS placing loose leaves over the nest. Eight to eleven days at the pre- vailing temperature saw them change into the adult fly. During this time a series of successive browning and blackening of different parts of the underlying integument of the imago marked their gradual de- velopment and gave a series of marks to establish a correct chronology upon which to establish the successive stages of the metamorphosis of the light organs. At first only the two larval organs showed. They were always ready to light up on stimulation until about half way through the change when they lost the power for about twenty-four hours. At about this time the adult organs began to appear as whit- ened surfaces of the same segment that contained the larval light organ and also that segment immediately anterior to it. At about the sixth or seventh day this new surface began to light i a central spindle- shaped area in each side of the two segments and the lighting power spread out from these focii until shortly before hatching when the entire surfaces would light slowly if the pupae was disturbed in any way. The internal changes accompanying this were briefly as follows; the small round larval organs began at an early date to be retired from their position against the cuticle and when the imaginal cuticle was formed the entire surface was lined by a hypodermis that showed no differen- tiation. Signs of histolysis became evident in the larval organ which had moved far in toward the center of the body. The new imaginal centers of the adult organs now showed a deposit of large, yolk-like granules in these cells. The new ventral hypodermis of the imaginal light segments also showed this accumulation of reserve food mate- rial as scattered pupal yolk globules. Shortly afterward the hypodermis proliferated into several layers which grew rapidly into the light cells. The origin of the reflector layer is somewhat obscure but appears to be a differentiation of the dorsal portion of these epithelial-derived cells. The trachae grow in and form the cylinders and the end cells appear just before hatching. The light organs of the Lampyridae are not developed from the fat organs as many authors have surmised but from the ventral integumental epithelium of the insect. The luminous organs of the ostracod, Cypridina hilgendorfii, was also studied. Miiller has already shown that light glands opened in several places on the upper lip of a closely allied, if not the same, form. Do- flein undertook to study the organ more closely and was much at fault in his description. He described and drew a sac-shaped reservoir op- ening at the upper lip by several apertures. The luciferine was _ se- creted by a large gland forming a part of the fundus of the sac. Doflein undoubtedly drew the brain or supra-esophageal ganglion for this gland as Yatsu pointed out to Dr. E. N. Harvey and as the writer con- cluded from reading the article and examining the drawing. Studies of the organ in some beautifully preserved material from Japan collected for the writer by Dr. E. N. Harvey show the following conditions: The luminous organ consists of a group of from twenty to PROCEEDINGS 483 thirty hypodermal cells invaginated from the edge of the upper lip into an elongate series of unicellular glands that reach up to and almost touch the upper brain ganglia. These cells are arranged bilaterally into two contiguous groups. They may be distinguished as of three differ- ent kinds by their secretion and form. ‘The first two kinds secrete a series of granules that are basic in reaction, taking the acid dyes as eosin. One of these secretes a very large heavy weaker staining granule and the other a very small deeper staining granule. The cyto- plasmic bodies of these cells form a compact rounded mass up near the brain. They show large single nuclei each with a very large central plasmosome. The distal part of each cell reaches down to the median part of the upper lip and open through the chiten as a very short papillae. This distal region forms in each case a long hollow tube filled closely with the granules of the cell. It is possible that two cells sometimes open through a single papillae but it is not probable. The papillae are closely set together in a group. The third kind of cells form a wide shallow common sac on each side of this median group. Their cytoplasmic bodies are united into the upper wall of this sac which serves as a common reservoir for the se- cretion. While shallower than in the case of the first cells, the eyto- plasm of these third cells is of the same texture and holds the same kind of nucleus but its secretion is totally different, being much like mucous and staining the opposite (with the chromatin stains) of that of the first two kinds. There are two of these sacs and each opens through a very long slender papilla that hangs down laterally to the opening of the typical ostracod shell. A peculiar point is that one of the coarse granular cells on each side sends its distal end or duct into this long papilla and opens alongside of the sac duct by a separate but closely approximated opening. 10. Further experiments on the laterality of transplanted limbs. Ross G. Harrison, Osborn Zoological Laboratory, Yale University. Additional experiments with the fore limb bud of Amblystoma em- bryos make it possible to state more simply than before the rules gov- erning the laterality of transplanted limbs (Proc. Am. Assoc. Anat., Anatom. Rec., vol 10, 1916). Limb buds were transplanted either to their natural location after removal of the normal bud (orthotopic transplantation), or to another region of the body, as for instance to the flank between the fore and hind limbs (heterotopic transplantation). They were grafted either on the same (homopleural) or on the opposite (heteropleural) side of the body, and were placed either in the upright (dorso-dorsal) or the inverted (dorso-ventral) position. The following rules underlie the determination of the laterality of the appendages which develop out of the transplanted buds in both the orthotopic and heterotopic transplantations, though in the former 484 AMERICAN SOCIETY OF ZOOLOGISTS the limbs are more likely to be modified through the influence of their more normal surroundings (vascularization, Innervation, etc.). Rule 1. A bud that is not inverted (dorso-dorsal) retains its original laterality whether implanted on the same or on the opposite side of the body. Rule 2. An inverted bud (dorso-ventral) has its laterality reversed whether implanted on the same or on the opposite side. Rule 8. When double or twin limbs arise, as is frequently the case in these experiments, the original of the two limbs, i.e., the one first to begin its development, has its laterality fixed in accordance with the above rules, while the other is the mirror image of the first. In heterotopic transplantations abortive development, or even com- plete absorption of the tissue, often takes place, and reduplication may occur in any of the combinations. In the orthotopic series abortive development is more rare and redu- plications, though frequent, are limited to certain combinations and may be further modified. The outcome in the several groups of ex- periments was as follows: 1. Homopleural dorso-dorsal grafts developed normally though at first very slightly retarded. 2. Homopleural dorso-ventral grafts resulted in: a. A single limb of reversed laterality (structurally and functionally perfect right limb on left side). One case only. b. Reduplicated limbs. More than half of the cases. c. Typical non reversed limbs which began their development by growing in abnormal direction, but ultimately assumed normal posture by rotation. ‘These cases form the only exception to the rules and require further investigation. 3. Heteropleural dorso-dorsal transplantations yielded: a. Single non reversed limbs. Two cases only, neither perfect. b. Reduplicated limbs in which the secondary bud being reversed, has the laterality of its new surroundings. c. Cases similar to the above in their early development but differ- ing later in that the reduplicating bud gained the upper hand and de- veloped into a normal functioning limb of reversed laterality (corre- sponding to its new surroundings), while the original bud became reduced to a spur or appendage upon the other. 4. Heteropleural dorso-ventral transplantations developed into: a. Single limbs of reversed laterality somewhat retarded in their de- velopment (Rule 2). The great majority of cases. b. Duplicate limbs. A single case only. Experiments with superimposed limb buds and with half buds, gave corresponding results, and together with other experiments, show that the mesoderm of the limb bud is an equipotential system, with definite asymmetry (laterality) which is subject to modification in accordance with the fundamental rules stated above. The theoretical questions involved, particularly those relating to adaptation in the individual, are of considerable interest. PROCEEDINGS 485 11. The effect of removal and regeneration of parts wpon metamorphosis in amphibian larvae. CHARLES ZELENY, University of Illinois. The effect of removal and regeneration was studied by comparing the time of metamorphosis in a set of individuals subjected to opera- tion with the time in a control set. Comparisons were made as follows: Experiment 1. Rana. The effect on the early development of the hind legs of five successive removals and regenerations of the tail. Experiment 2. Bufo. The effect of removal of the tail after the beginning of metamorphosis upon the time of completion of the process. Experiment 8. Bufo. The effect of four successive removals and regenerations of the tail upon the time of metamorphosis. Experiment 4. Bufo. The effect of two successive removals and regenerations of the tail upon the time of metamorphosis. Experiment 5. Amblystoma. The effect of three successive remov- als and regenerations of the tail upon the development of the legs. Experiment 6. Amblystoma. The effect of removal and regenera- tion of the tail upon the time of loss of the balancers. Experiments 7? and 8. Amblystoma. The effect of removal of the right balancer upon the time of loss of the left balancer. Experiments 9 and 10. Amblystoma. The comparative develop- ment of the legs in individuals subjected to the four following degrees of injury: (1) one fore-leg, (2) both fore-legs, (3) one-half of the tail, (4) one-half of the tail plus both fore-legs. The data from these experiments give no indication that metamor- phosis is delayed by removal and regeneration of parts of the body. 12. Life history of Zeugophora scutellaris. B.H. Grave. Knox College. During the summer months the larvae work in the leaves beneath the epidermis, eating out the pulp and causing blackening of the parts affected. A large part of the chlorophyll bearing tissue may be de- stroyed in this way by the end of summer, thus rendering the leaf ineffective as a starch-making organ. The larva may therefore ap- propriately be called a leaf miner. Late in the season, at the time the leaves. fall it crawls out and enters the ground. After burrowing to a depth of between 14 and 2% inches below the surface, it excavates a little spherical cavity in which it coils up for the winter sleep. About the last of May of the following spring (May 25—June 15) the larvae transform into pupae. The duration of the pupa is about three weeks or possibly a month in cool weather. The first beetles appear by the middle of June. There is reason to believe that they appeared as early as June 10 in the year 1913, which was a rather early spring for that locality. A number of beetles which were hatched from breeding boxes were kept in captivity and one of them laid eggs ten days after it emerged. It seems likely therefore that under normal conditions the eggs are laid upon the leaves and twigs during the latter part of June and the 486 AMERICAN SOCIETY OF ZOOLOGISTS first part of July and that the larvae enter the leaves soon after, and begin their destructive work. The adult beetles,.as might be expected, feed upon the leaves of the cottonwood. They swallow the softer parts and discard the fiber. 13. Extirpation of the hypophysis and thyroid glands of Rana pipiens. BENNET M. ALLEN, University of Kansas. Last spring a series of experiments was performed in the extirpation of the anlage of the anterior lobe of the hypophysis and of the anlage of the thyroid gland. The former experiment was successfully per- formed upon 430 tadpoles and the latter upon 336. Removal of the anlage of the anterior lobe of the hypophysis pro- duced a marked contraction of the black pigment cells of the integument apparent at the end of eight days. The silvery cells expanded giving the animals a bright uniform creamy silver color. At this time the hypophysis shows no evident histological differentiation. These operated tadpoles showed marked susceptibility to unfavorable con- ditions of the water. In the absence of the hypophysis the legs failed to develop, the hind legs appearing as mere buds, up to the maximum stage reached—tadpole of 30 mm. length. Tadpoles deprived of the hypophysis were carefully studied in stages of 16.5, 21.5, 24 mm. comparisons being made with control tadpoles. The gonads, and thymus glands showed no consistent modification. The thyroid gland in all cases, however, showed a decided diminution in the amount of colloid produced. In tadpoles deprived of the hy- pophysis the colloid material occurred in far more irregular masses measuring from one-half to two-thirds the diameter of colloid masses in the thyroids of the controls. The general dimensions of the thy- roid glands of these animals did not show any appreciable modifica- tions at these stages. The extirpation of the thyroid anlage caused the tadpoles to halt in their differentiation at a stage in which the hind-limb rudiments were but 4 mm. in length. At the end of November they had shown no further signs of differentiation. Two specimens from which the thyroid had been removed were fed thyroid extracts. In one instance with forty-five days thyroid feeding the hind legs grew to 9.5 mm. length and the fore-limbs grew to 5 mm. length as compared with 4 mm. and 2.5 mm. respectively in the thyroidless controls. They showed much greater differentiation of structure than found in those of the other thyroidless tadpoles. These also showed a remarkable shortening of the intestine in one case to 68 mm. as compared with 190 in the thyroidless tadpoles not fed with thyroid extracts. Studies are being made by students of mine upon the effects of thyroid removal upon the other glands of internal secretion and upon the skeletal system. Seven thyroidless tadpoles of gigantic size are still living, but show no signs of further differentiation. PROCEEDINGS 487 14. An experimental study of cell division. L. V. HertBRuNN, Depart- ment of Anatomy, University of Illinois, College of Medicine. In the experimental study of a biological process, there are two gen- eral modes of procedure. One can attempt to artificially produce or initiate the process, or one can attempt to modify or block the process after it has started. In my study of artificial parthenogenesis, I adopted the former of these methods for studying cell-division. I attempted to analyze the effect produced by the various agents which cause the unsegmented egg to divide mitotically. More recently I have adopted the other method of attack, and I have studied the effect of various agents, which, without injuring the egg, prevent cell-division. Such a suppression of normal activity is of course an example of anesthetic action, and these experiments have incidentally furnished me with considerable data concerning the actual effect of various anesthetics upon the cell. In a recent contribution it was shown that all agents which cause the egg of the sea-urchin to segment, produce a gelatinization in the cytoplasm. The details of this gelatinization process, as it occurs normally, have now been studied in the same egg. At frequent short intervals after fertilization, the viscosity of the egg cytoplasm was determined by the centrifuge method. After fertilization the cyto- plasmic viscosity rises gradually until it reaches a maximum after about twenty to twenty-five minutes.! It is precisely at this time that the mitotic spindle first makes its appearance. The appearance of the spindle is followed by a gradual decrease in viscosity, the egg cyto- plasm returns to its original fluid state. These viscosity differences are very marked and are easily measured. Similar series of viscosity changes during mitosis can also be demonstrated for the second cleav- age. These facts in themselves lend support to the view that the spindle is coagulated out of the cytoplasm. No doubt the gelatinization is confined to certain chemical constit- uents of the cytoplasm. On the other hand, it apparently extends throughout the cell and it is attached peripherally to the enclosing membrane, the so-called hyaline layer of the egg. Hence when the gelatinized egg is centrifuged, frequently parts of the egg surface are pulled in, and the shape of the egg commonly undergoes considerable distortion. This attachment to the enclosing membrane is retained by the astral rays of the spindle. Oftentimes when an egg which pos- sesses a spindle is viewed from one pole, its surface contour does not appear perfectly smooth, but shows faint indentations at various points. These points probably represent the points of attachment of the astral rays. The inward pull exerted on the egg surface as a result of gelatinization, no doubt affords the best explanation of the decrease in cell volume which follows fertilization. This is borne out by the fact that in the Cumingia egg, decrease in volume does not immediately follow fertilization. In this egg both gelatinization and shrinkage only occur after a certain time interval has elapsed. 1 Of course this time varies greatly with the temperature. 488 AMERICAN SOCIETY OF ZOOLOGISTS If the normal gelatinization of the cytoplasm is prevented, then the spindle never forms, the egg remains quiescent and does not divide. Even after gelatinization has begun, it may be reversed. Ether, chloroform, acetone, paraldehyde, propyl alcohol, isoamyl alcohol, ethyl butyrate, ethyl acetate, ethyl nitrate, acetonitrile, nitromethane, chloral hydrate, phenyl and ethyl urethanes, all prevent or reverse cytoplasmic gelatinization. In the study of the action of these anes- thetics, the experimental procedure was usually as follows: soon after fertilization, the eggs were placed in a graded series of concentrations of the anesthetics. Then the viscosity of the various sets of eggs was determined. If the series of concentrations was well chosen, it was found that the highest concentrations produced coagulation, the low- est concentrations no effect, and the intermediate concentrations a liquefaction or reversal of gelatinization. As my studies progressed, I was soon able to predict the fate of the eggs. In the lower concen- trations with no marked effect on the egg viscosity, the eggs would go on to divide, in the intermediate concentrations which produced lique- faction, development would be interrupted, but removal from the anesthetic (after a few hours), would be followed by a resumption of development. On the other hand, the coagulation produced by the higher concentrations was generally irreversible, although in a few cases the eggs thus coagulated were able to undergo a few cell-divisions on being returned to normal sea-water. The actual effect of the vari- ous anesthetics mentioned above, was to dissolve the lipoids of the egg, or at least to increase their degree of dispersion. In concentra- tions slightly above that best for anesthesia, the lipoids appeared to be completely dissolved, and no longer separated out when the egg was centrifuged. Such a solution of the lipoids was usually followed, after a short time interval, by coagulation of the cytoplasm. Low temperatures (e.g., —5° to 5°C.) also prevent or reverse gela- tinization. But this effect of cold is not due to the same cause as is the effect of the other anesthetics just mentioned. In fact when eggs are subjected to both cold and ether the effect of the one tends to coun- teract the effect of the other. But not all anesthetics prevent gelatinization. Hypertonic solu- tions of various salts, although they act as anesthetics, produce quite the opposite effect on the cytoplasm. They intensify the normal gelatinization and in this way prevent cell-division. A similar effect is also produced by chloretone. The action of potassium cyanide is especially interesting. In concentrations of potassium cyanide, far above those sufficient to check development, the early stages of mitotic division are not suppressed. The spindle proceeds to form, but with its formation, development stops abruptly.2, Apparently the cyanide renders irreversible the normal gelatinization process and no lique- faction follows. If eggs are placed into cyanide solutions during the * The fact that the egg can begin its development in these solutions of potas- rb cyanide is a strong argument against the oxidation theory of artificial par- thenogenesis. PROCEEDINGS 489 anaphase stage of mitosis, after liquefaction has begun, then cell- division is not prevented. Since the normal process of mitosis involves both gelatinization and liquefaction, it is easy to understand why agents which cause an in- tensification of either the one process or the other can check the divi- sion of the cell. As to the direct cause of the gelatinization which follows fertiliza- tion, there is some evidence that it is due to salts rather than to an acid. Thus when the cytoplasm is diluted by endosmosis, the gelatinization is reversed. Moreover, whereas coagulation of the cytoplasm by hypertonic salt solutions can be reversed by ether, no other type of cytoplasmic coagulation can be so reversed. It is possible that the gelatinization is produced by the abstraction of water from the cyto- plasm by the growing pronuclei. ‘This gelatinization is then to a large extent reversed, when owing to the rupture of the nuclear membrane, the nucleus discharges its water back into the cytoplasm. But these interpretations are more or less hypothetical. They do not stand on the same firm basis of fact as the observations: (1) That all agents which stimulate to cell-division produce gelatinization, and (2) that any agent which prevents or reverses this gelatinization, pre- vents spindle formation and cell-division. These two facts, taken together with the observed time relations of the gelatinization process, furnish strong evidence that the force which underlies spindle forma- tion is a cytoplasmic gelatinization. 15. Early castration of the vertebrate embryo. FRANKLIN P. REAGAN, Princeton University (Introduced by C. F. W. McClure). In 1870 Waldeyer advanced the view that the germ-cells of the verte- brate embryo arise in that portion of the coelomic mesothelium which covers the gonad. Since then a number of observers have described transitional stages between these epithelial cells and primitive ova. Eigenmann (91) is justly to be considered the leader of an opposing school who believe that the vertebrate germ-cells are of extra-regional origin. In the viviparous fish Micrometrus, he was able to trace the germ-cells probably to the fifth cleavage—certainly to a time prior to the closure of the blastopore, before there were distinct entodermal and ectodermal layers. Hoffmann (’93) discovered primordial germ-cells in bird embryos which had not yet formed germinal epithelia. His work has been confirmed by a number of observers. In connection with the work on avian embryos it is important to note that sex-cells could formerly be found only subsequent to the 22- somite stage; earlier than this their whereabouts was a complete mys- tery. In fact, stages transitional to them were here less evident than in pictures to be found later in the gonad. An interesting observation was that of Danchakoff, who found in the blood stream very large wandering cells of entodermal origin; these were believed to disappear at the age of 23 somites. Their disappearance was as mysterious as 490 AMERICAN SOCIETY OF ZOOLOGISTS the first appearance of the germ-cells. It required the insight of Swift (14) to correlate these two facts and establish a morphological continuity of these large cells from the time of their proliferation by the entoderm, to their incorporation into the gonad. Swift found that these cells originate in a crescent-shaped area of entoderm anterior to the body axis of the very early chick embryo; that they enter the mesoderm which later invaded this region; that they enter the blood vessels forming here and are carried by the blood stream many of them to the base of the mesentery from which they migrate into the gonad. In 1914, Prof. C. F. W. McClure suggested that the work of Swift could be proved or disproved by the early excision of this crescent- shaped area. I have been able to rear a number of embryos so treated to a stage in which it is possible to determine that the gonads are quite devoid of sex-cells. In normal individuals five days old, the mesen- teric mesothelium adjacent to the gonad usually contains many large germ-cells which cause it to protrude locally. Also the mesenteric mesenchyme generally contains some of these big cells. The embryos treated as described have no trace of germ-cells in the gonads, and the neighboring mesothelium remains thin and barren of sex-cells. The stroma tissue of such gonads presents a peculiar foliage-like appearance; it is much vacuolated. My own interest in this problem lies in another direction. It oc- curred to me that since castration of individuals subsequent to birth or hatching greatly affects the secondary sexual characters, embryonic castration might produce even more profound effects—that some sexual characters usually considered primary might really be secondary. Such a possibility is heightened by the recent work of Lillie on Free- martin. At present I am led to believe with Lillie that the fate of the Wolffian and Miillerian ducts is dependent on the internal secretion of the gonad. I believe further that if the stroma or interstitial cells are responsible for this secretion, they are unable to produce it in the absence of germ-cells in the early ontogeny; that the nature of this secretion ‘is related only indirectly to the factor or factors for sex by way of emanations from the sex-cells themselves; that the only primary sexual character is the constitution of the germ-cells themselves. These points are not yet completely proved. Attempts are being made to produce hermaphroditism by trans- plantation of these entodermal crescents. The effects thus produced on the ductless glands are also being investigated. So far, the trans- plantation of adult gonad-tissue to young embryos has had no effect on the embryonic genital system, though it has always caused hyper- trophy of the spleen of the host. I wish to thank Professors McClure and Conklin for confirming many of my observations on my material. It was found impracticable here to discuss thoroughly the results of all previous observers. PROCEEDINGS 491 16. Microdissection studies. The cell aster: a reversible gelation phe- nomenon. Roperr ‘CHAMBERS, JR., Cornell University Medical College. (Illustrated with drawing, to be used in projection ap- paratus.) 1. The centrosphere is an optically hyaline fluid area occupying the center of the aster and increasing steadily in size until the aster reaches full development. 2. The increase in amount of the centrospheric fluid is apparently due to the accumulation of fluid flowing into the centro- sphere from all parts of the cytoplasm. 3. The aster rays are the channels along which the centripetal flow occurs. 4. The cytoplasm between the rays is in the gel state giving a certain amount of rigidity to the aster. The gel state is most pronounced centrally and periph- erally passes gradually into the sol state beyond the confines of the aster. When the aster reaches the periphery of the cell the entire cell is comparatively rigid. 5. In the maturation figures of the egg nucleus the peripheral aster forms a continuous coagulum with the surface layer of the egg to which the entire figure is thus firmly attached. The confines of the central aster pass insensibly into the surrounding liquid cytoplasm. 6. A periodic reversal of the sol to the gel state and vice versa has been demonstrated in the cell protoplasm during division. The steps taken may be divided into the following series: a. When the monaster is fully formed the greater part of the cell is a gel. 0b. As the centrospheric fluid collects on the two poles of the nucleus the cytoplasm reverses to a sol state and the monastral radiations fade out. c. The formation of radiations about the centrospheres, one on each pole of the nucleus, produces the diaster and is accompanied by a return to the gel state. d. A return to the sol state later takes place in the equator of the cell. e. The nuclear spindle now divides fol- lowed by a constriction around the middle of the cell which continues until the cell is cut in two. 7. The reversal of the gel to the sol state usually starts in the equator of the cell and spreads to the poles. The reversal of the sol to the gel begin immediately about the centrosphere and spreads in all directions peripherad. 8. There are appreciable - differences in the sol state of the cytoplasm in certain regions and at various times. The interior cytoplasm of the unfertilized and fer- tilized egg before the aster is formed is slightly viscous. The archo- plasm in the centrosphere and in the rays also the hyaline area in the vicinity of the forming polar body are very fluid. 9. What is described as the gel state in living protoplasm cannot be considered as an inert solid coagulum. Even to the eye there is always a constant but very gradual change among the granules imbedded in the cytoplasmic gel. One may conclude that one of the factors concerned in cell-division lies in the peculiar colloidal property of protoplasm, viz., a periodic reversibility in its sol and gel states. THE ANATOMICAL RECORD, VOL. 11, No. 6 492 AMERICAN SOCIETY OF ZOOLOGISTS 17. Multiple chromosomes of Hesperotettix and Mermiria. C. E. Mc- CiunG, University of Pennsylvania. A restudy of the chromosome complexes of Hespewretne and Mer- miria, upon greatly enlarged collections, has made possible the cor- rection of some errors in the earlier account and the discovery of im- portant new facts. Upon the basis of the present undertanding of conditions in the germ cells of the Orthoptera, numerical variations of the chromosomes are found to be a strong support to the individuality hypothesis instead of militating against it. The multiple chromosome of Mermiria bivittata, at first thought to be a decad, because of certain constrictions and even divisions in meta- phase, proves to be a hexad like the one in Hesperotettix, consisting of a tetrad joined to the accessory chromosome. An explanation of its form and behavior became possible upon the discovery of the J-. shaped tetrads in Trimerotropis by Carothers. Full collections make the determination of numbers in the different cell generations certain and consistent with the interpretation of the multiple as a hexad. So far, conditions within the species appear to be constant, but others than bivittata may not have multiple chromosomes, e.g., neomexicana and texana. Within the taxonomic group bivittata, as at present constituted, there are certain sub-groups, first distinguished apart by the form of the multiple chromosomes, that appear to be specifically distinct upon careful study of somatic characters. As in Mermiria, a study of extensive series of specimens of Hespero- tettix shows that the universality of multiples in all species, a condi- tion realized in my earlier collections, does not exist. Moreover the constancy of occurrence within the species sometimes is lacking, as in viridis, and this is associated with a tendency to form multiples be- tween certain of the euchromosomes, producing octads, a name given to such structures in an earlier paper (’05) in advance of their realiza- tion in experience. The presence or absence of multiples in viridis results in variations of numbers from 9 to 12 in the first spermatocyte and a supernumerary in one individual out of 37 studied raised the upper limit to 138. The number 11 may be constituted in three different ways. Despite the apparent lack of definiteness in organization sug- gested by the range in numbers, there is abundant evidence that funda- mental conditions are not thus disturbed, because if the final mitotic units, the chromatids, are considered the same 46 are present in each first spermatocyte except in the one individual with a supernumerary. All the evidence indicates that the constitution of the individual estab- lished on zygosis are maintained, for no variation was found within the individual. The chromosome conditions of the first spermatocytes so far ob- served fall within six different classes; (1) Twelve separate chromo- somes consisting of eleven tetrads plus the accessory chromosome dyad—a total of forty-six chromatids; (2) eleven separate chromo- somes, ten tetrads plus a hexad—again forty-six chromatids; (3) ten separate chromosomes or eight tetrads plus one hexad plus one octad— PROCEEDINGS 493 forty-six chromatids; (4) nine separate chromosomes or six tetrads plus one hexad plus two octads—forty-six chromatids; (5) again ten separate chromosomes but in this case consisting of seven tetrads, plus two octads plus one accessory chromosome dyad—forty-six chromatids; (6) again eleven separate chromosomes but consisting of nine tetrads, one octad and the accessory chromosome dyad, forty-six chromatids. In the individual with a supernumerary there are eleven separate chromosomes with the other condition similar to class 5 above. Criteria for resolving the varying numbers of free metaphase chro- mosomes jnto identical series of units of lower order are furnished by comparisons of form, size and behavior and by the structural condi- tions of the elements. The conditions in class 1 are typical for large numbers of the short horned grasshoppers, the earliest modification of which appeared in the first members of the genus Hesperotettix which I studied, representing class 2. Here the easily recognizable accessory chromosome is joined to a first spermatocyte tetrad, produc- ing a hexad which acts as a unit in this mitosis. Because of this mitotic relation the hexad is properly called a chromosome, but it is just as definitely different from the other members of the complex, for the morphologically distinct accessory chromosome exhibits all its struc- tural peculiarities quite as clearly as if it were a free unit, while the associated tetrad passes through its usual history. The principle of chromosome union is thus definitely and unequivocally established. Only in very recent material have the conditions in classes 3, 4, 5 and 6 been encountered. These involve an extension of the principle of chromosome association to combinations between two tetrads in the first spermatocyte and a persistent union of the non-homologous ele- ments involved throughout the cells of the individual. These combina- tions result from the endwise union of contiguous sized members of the complex. Thus in class 3 the largest two tetrads are so united, while in class 4 the next two in size also form into another octad. In every case the joined tetrads clearly exceed in sizé the next in order, just as they do in the free condition, and as the gradation in the com- plex requires. In synopsis and during the first spermatocyte prophase their behavior is not altered by the association. Union may involve one or both ends of the tetrads producing rings or V’s. In the latter case first spermatocyte anaphase groups differ accordingly. Such conditions are accounted for upon the assumption of persistent chromo- some individuality and chance union of the classes of gametes actually seen to form. Similar combinations between the accessory chromo- some and a tetrad in the long horned grasshoppers were reported by me (’02) and more recently between euchromosomes (’15) by Robert- son and by Woolsey. The conditions are therefore not abnormal but represent the action of definite forces of chromatin integration. No evidence is at hand to: show how such associations arise or to indicate their later dissolution. The facts demonstrate that numbers are re- duced by definite and gradual steps of which the chromosomes are the measure. The normal number is not exceeded by the addition of other 494 AMERICAN SOCIETY OF ZOOLOGISTS normally constituted chromosomes. Supernumerary chromosomes when present, show their aberrant nature by extra-nuclear position, irregular behavior and great variability. Persistent association of non-homologous chromosomes with elimination of intermediate stages would produce a permanent reduction in the number of free chromo- somes. It is possible to account for the occurrence of the lesser num- ber in Stenobothrus in this way, but the criteria for this determination, urged by Robertson, are not completely valid since V-shaped chromo- somes with achromatic bridges occur in complexes where no reduction in number exists. 18. The spermatogenesis of Culex pipiens L. P. W. Wuitine, Univer-. sity of Pennsylvania, (Section F). In the spermatogonia of Culex pipiens there are three pairs of V- shaped chromosomes, the members of which are usually approximated. Before division the pairs always lie parallel. One of the pairs is smaller than the other two. In the first spermatocytes three characteristic tetrads appear, any one of which may form either a cross or a ring. The four elements of the tetrads are distinguishable in late prophase and in metaphase. The dyads separate into monads in the anaphase. Nucleolar elements are found in spermatogonia, in first and second spermatocytes and in spermatids. In spermatogonia they are asso- ciated with one of the large pairs; in first spermatocytes, with a large tetrad; and in second spermatocytes, with a large dyad. 19. The segregation and recombination of homologous chromosomes in two genera of Acrididae (Orthoptera). E. ELEANOR CarorueErs, Univer- sity. of Pennsylvania. The subjects of this report are the chromosome conditions in the male germ cells of certain species of two closely related genera of short- horned grasshoppers. A microscopical study of these cells has shed light on three points: (1) the manner of segregation of morphologically distinct homologues, (2) the zygotic composition of the species in regard to these dissimilar homologues and (3) a possible cytological basis for separating confused species of the two genera. 1. The chromosomes are constant in number, size and shape in each animal. In size there is the usual double series, one homologue of each pair being derived from each parent. Contrary, however, to any- thing heretofore reported these homologous chromosomes may differ in shape—one being a straight rod, the other V-shaped. In one species seven of the twelve first spermatocyte chromosomes may be composed of such dissimilar homologues.. This peculiarity affords an oppor- tunity to trace the segregation in the gametes of certain chromosomes derived from both parents. It was found that this segregation occurred according to the law of chance. 2. A study of the chromosome complexes of ninety-five individuals, both male and female, showed the zygotic composition of the species to PROCEEDINGS 495 be such as would result from the random union of these gametes. These facts furnish an extensive physical mechanism for the operation of Mendel’s laws of heredity. 3. One of these genera, Circotettix, has eleven chromosomes in the haploid series of all species studied instead of the usual twelve as found in the other genus, Trimerotropis; on this basis the debated species, Cirecotettix suffusus which has twelve chromosomes, should be changed to Trimerotropis suffusa. 20. Synapsis and chromosome organization in the male germ cells of Chor- tippus and Trimerotropis. D. H. WrENRICcH. In a recent paper! the writer showed that pairing of chromosomes in the first spermatocytes of Phrynotettix magnus is by parasynapsis. All the chromosomes of Phrynotettix are of the rod-shaped type. A study has been made of the first spermatocyte chromosomes of Chor- tippus (Stenobothrus) curtigennis which has three pairs of C-shaped and five pairs of rod-shaped chromosomes, and of Trimerotropis siffusa, which Dr. E. Eleanor Carothers has found to possess not only pairs of rod-shaped and pairs of V-shaped chromosomes but also pairs consist- ing of one rod-shaped and one V-shaped chromosome. In all cases the mode of synapsis is found to be the same, viz., a side-to-side union. This mode of synapsis makes it impossible to determine whether the first or the second maturation mitosis is the segregating division unless there is a recognizable difference between the conjugants of a pair. The V-shaped chromosomes of both Chortippus and Trimerotropis may have the point of spindle-fiber attachment indicated by a constric- tion, or by a small non-chromatiec region, or by both. Since Trimero- tropis has a larger total number of chromosomes. and (usually) a large number of V-shaped chromosomes than Chortippus, the existence of this ‘‘weak place” cannot safely be taken as an indication that these chromosomes are compound. In Chortippus the point of fiber attachment of the V-shaped chromo- somes may be further marked by the presence of a small, appendant, plasmosome-like body, which, when present, is constant in this position on the chromosome. Other such bodies are found on two of the rod- shaped chromosomes of Trimerotropis. Their positions are constant for each chromosome. The constancy of position which these bodies exhibit with reference to the chromosome to which they are attached is additional evidence of the constancy of organization possessed by the chromosomes. 21. The chromosome complex in Apithes agitator. W.J. BAUMGARTNER, University of Kansas. Apithes agitator is a small brown cricket often called the shrub cricket. It lives on various shrubs, especially the coral berry. 1 Wenrich, D. H. 1916. The spermatogenesis of Phrynotettix magnus with special reference to synapsis and the individuality of the chromosomes. Bull. Mus. Comp. Zool., Harvard College, Vol. 60, pp. 57-133, 10 plates. 496 AMERICAN SOCIETY OF ZOOLOGISTS The chromosome complex is very instructive as the number is small and the individual chromosomes are quite distinct in size and shape and behavior. In the spermatogonia the number is thirteen, two small straight rods, and eleven U-shaped rods. The largest of these always lies on the periphery of the group and its end may be swollen or split or bent at a sharp angle. This has no mate and is the accessory. The other ten easily group themselves into five pairs. The largest pair show a tendency to lie in or toward the center of the equatorial plate. They are frequently somewhat straightened, sometimes showing a double- wave-like curve. The other eight chromosomes with the accessory usually lie in a circle forming the periphery of the equatorial plate. ° All have the free ends extending outward in the typical way. There is an evident tendency for the two of a pair to lie near together. In size these pairs grade down from nearly as large as the largest to about half of its size. In the spermatocyte the accessory appears as a sausage-shaped rod, and behaves as described in my earlier papers and confirmed by other observers. The small pair are now a longer rod. It frequently shows a constriction and divides precociously. The two dyads may have separated even before the larger tetrads have been drawn into the equatorial plate of the spindle. The other ten chromosomes now form five very definite rings. All of these enter the spindle parallel with the direction of fibers and not in the plane of the plate as most of the rings do in the grasshoppers. The fiber attachment is terminal in the small chromosomes, and median or nearly so in all the others. In dividing one end sometimes separates before the other so that the tetrad may appear like a printed capital C for a short time. Sometimes the sides of the rings (ends of dyads) are swollen. When such a tetrad is seen from the side it may ap- pear as a cross with a very short cross arm. But in either the cross or C shapes the fundamental shape is a ring. The seven elements in Apithes are one accessory sausage shape, one small rod and five rings lying in the plane of the spindles fiber. I be- lieve these shapes are constant, i.e., are assumed in every cycle of te- trad formation. I think this fact is a strong evidence of chromosome individuality. The five large rings may be multiple chromosomes. If each is counted as a double multiple then the original number would be 5 times 4 plus 2 rods plus 1 accessory equals 23. This number is found in some crickets and most grasshoppers, and seems to be kind of a basic num- ber for these families. Several other species of crickets would have this number if these large rings were counted as double multiple chro- mosomes. But in two species studied this number is not obtained if the rings are so counted. The interpretation of the large rings as multiples can be stated only as a probability. PROCEEDINGS 497 22. New facts and views concerning the occurrence of a sexual process in the life cycle of a myxosporidian Chloromyxum leydigi. Ruopva ERDMANN, Osborn Laboratory, Yale University. In a myxosporidian life cycle the sexual process is generally be- lieved to be before spore formation. After a shorter or longer asexual life in which the formation of a vegetative body with either two or many nuclei is effected spore formation begins. Since 1910 Auerbach and Erdmann (’11) have verified the suggestion of Doflein that the sexual process might not occur at the above mentioned place in the life eyele but as soon as the young animal leaves the spore. Auer- bach and Erdmann found young animals (amoeboidkeim) which had left the spore and possess either two or one nuclei. They are the first step in the new life eycle. Erdmann (’11) could further produce these young forms in a culture which had been made on gall plates. In my recent work, finished in 1913, which does not appear until 1916 in consequence of the war, I figure those young animals experimentally freed from the spore after fixation and staining. Besides myself two other authors, Georgevitch (714) and Davis (15) assure that at the beginning of spore formation no sexual process could be found. It shows that the recent investigators have finally left the old view that the sexual process of myxosporidia occur immedi- ately before spore formation. To support this view I can point out that all processes which were thought to be sexual, i.e., the formation of residual (reduction) nuclei and the heteropole division at the first beginning of spore formation, are only connected with the develop- ment of the spore membrane. The glykogen which I could point out to be present in the vegetative myxosporidian body, is used up dur- ing spore formation. The small cells and their nuclei being the prod- duct of the above mentioned heteropole division form the membrane of the spore. Also smaller or bigger chromatic lumps (residual nu- clei) are extruded by the nuclei of the sporoblast-cells and are used in forming the sporogenous membrane, and the polar bodies. They cannot be thought to be reduction nuclei. Having pointed out what I believe to be the real significance of these processes, it is more in accordance with the facts presented that the sexual process in the life cycle of myxosporidia is to be found in the beginning of the life cycle after the young animal left the spore. 23. Spermatogenesis in the albino rat. Ezra ALLEN, University of Pennsylvania (Section F). The haploid, or reduced number of the chromosomes is fundamen- ally nineteen. The spermatogonial number is consistently 37. There is one accessory. This divides in the second spermatocyte division. One chromatoid body in the cytoplasm and one nuclear plasmosome are present. There is no evidence of double reduction. The chromo- somes are of different sizes. In the spermatogonia the forms assumed are rods; in the first spermatocytes, rods, crosses, rings, and single and double loops; in the second spermatocytes, they are all rods. The 498 AMERICAN SOCIETY OF ZOOLOGISTS organization and behavior resemble the orthopteran. The first sper- matozoa are ripe when the rat has reached the age of forty days. The most satisfactory method of fixing and preparing the material for study is described in a paper giving the results of his experiments on technique published by the author in the Anatomical Record, vol. 10, p. 565. 24. Multiple complexes in the alimentary tract of Culex pipiens. Caro- LINE M. Hour, University of Pennsylvania (Section F.) 1. During metamorphosis in Culex pipiens, the number of chromo- somes in the cells of the larval intestine is considerably increased. 2. Before disintegration of the cells begins, the chromosomes of each larval gut cell pass through a number of longitudinal divisions result- ing in three or four multiplications. 3. The number of chromosomes in the multiple complexes is always a multiple of three—oftenest 6, 12, 24, 48; but frequently 9, 18, 36, and even 72 may appear. 4. The triplex divisions of the chromosomes apparently arise through premature splitting of one member of each pair of daughter chromo- somes from the original complex of three bivalents or by a precocious division of one of each of the homologous elements of bivalent chromo- somes. 5. The size relation between nucleus and cytoplasm is extremely variable. 6. It appears that in the resting stage of those cells of normal size which contain multiple complexes, there must be an accelerated growth of each chromatin thread which splits ‘nto normal sized chromosomes in prophase, or else the cytoplasm of such cells must fail to divide and to grow while the chromosomes continue to do both. The former seems to be the probable explanation. 7. There is evidence of a parasynaptic union of sister chromosomes in the resting stage, followed by reseparation through longitudinal splitting in the prophase. 8. These sister chromosomes, the multiples of each member of the original complex, tend to remain together throughout the mitotic changes. 9. The individuality of the chromosomes is maintained until the cell disintegrates. 10. The disintegrated cells appear to be digested by the cells of the newly formed lining of the adult alimentary tract. All these facts suggest that increased metabolism of the older epithe- lial cells may be a means of supplying needed food material to the developing cells of the adult gut during the pupal changes. That this great increase in the amount of chromatin in cells which have attained their growth, functioned for a time, and are about to be ab- sorbed, is not accidental, or simply a process of degeneration seems reasonably clear from the uniformity and universality of this increase in the intestine of Culex. Every cell of the larval gut epithelium apparently passes through the whole series of changes above described PROCEEDINGS 499 before it reaches the stage of disintegration. If this were simply a process of degeneration, it would be hard to account for the tremendous growth in the chromatin material and for the retention of the individual- ity of the chromosomes to the end. One would expect the processes observed in the disintegration of the cells, to come directly without these elaborate preparatory phenomena. It would seem that we have here not the hit-or-miss phenomena of degenerating cells, but a definite adaptation to provide for the support of the organism during metamorphosis. 25. Sex-linked inheritance of spangling in poultry. Grorak LEFEVRE, University of Missouri. Spangling is the term applied by poultry fanciers to the occurrence of a well-defined spot or ‘‘spangle’’ of distinctive color at the tip of the feather. In the breed of Silver Spangled Hamburgs, for example, the feathers are white and each is tipped with a black spangle which is generally proportionate to the shape and size of the feather. The color-pattern is the same in both sexes. A series of experiments has been carried out for the purpose of deter- mining the mode of inheritance of spangling, as it was thought that so definite and simple a color-pattern would be favorable for genetic analysis. It is the experience of breeders, however, that the spangled patter is not reproduced closely. The initial crosses were made reciprocally between Silver Spangled Hamburgs and Brown Leghorns, and the material used for the analysis has been obtained from twelve different matings. The conclusion has been reached that spangling is determined in inheritance by a distinct factor which behaves in a typically sex-linked fashion, the cocks being homozygous and the hens heterozygous, for it in Silver Spangled Hamburgs. When spangling is introduced through the male, both sexes in the F, generation show spangles, while the reciprocal cross gives only spangled males, the females being non- spangled and incapable of transmitting the pattern. It has been further shown that the expression of spangling may be greatly modified, or even entirely obscured, by the action of other factors, especially factors for black pigmentation, which, however, segregate independently of the factor for spangling. The disturbing factors may affect the entire body or only some re- stricted region, as, for example, the feathers of the tail. Black pig- ment, moreover, may be present in the feathers in other parts than at the tip, and in varying degrees may obscure the definiteness of the spangles. In fact, in certain individuals the condition is reached in which black is developed to such an extent as to completely cover the body, even in cases in which the bird carries the spangling factor, as may be proven by appropriate breeding tests. In the light of the above facts, 1t would seem probable that multi- ple factors for black, introduced by the Brown Leghorns, are present 500 AMERICAN SOCIETY OF ZOOLOGISTS and that these factors may have a cumulative effect, with the result that pigmentation is developed to varying degrees of extension. The independence of the spangling factor is shown by the fact that, after segregation and recombination of the several factors concerned, some individuals are extracted in which all disturbing factors are ab- sent and the spangled pattern is exhibited in its original purity. A ° number of such birds have been obtained from different matings, and these now breed as true to spangling as do the Silver Spangled Hamburgs themselves. 26. Two classes of factors for color patterns in Paratettix. Roser K. Nagours, Kansas Agricultural College. Fourteen factors for patterns, each allelomorphic to the other, have been used in Paratettix breeding experiments. In this class two fac- tors for any one suffice to make the whole pattern, and two for differ- ent ones produce a hybrid pattern, intermediate in fact, though the one may be more apparent (epistatic) and the other less apparent (hy- postatic). These are well established as multiple allelomorphs. Hach of these factors is invariably allelomorphic to a multiple allelomorph, never to an absence. Another class of factors, existing without exception in connection with and in addition to the multiple allelomorphs, has been discovered. The one factor most studied produces a well marked melanism in addi- tion to any of the other patterns or their hybrids. It is also possible to distinguish in the patterns between the presence of a single and double dose of the factor. This factor is allelomorphic only to its ab- sence and never to anything. : The multiple allelomorphs among themselves produce the typical 1 :2:1 (in some cases apparently 1:3) ratios. When the non allelo- morphic factor is present the typical 9:3 :3:3:1 (actually 1:1:1:1: 2:2:2:2:4) ratios are secured. The clear definition of the pat- terns and the ratios indicate the presence of only one pair of allelo- morphic (allelomorphic each to the other) factors, and one unpaired (allelomorphic only to its absence) factor in the production of the 9:3:3:3:1 ratios. This conception applies perfectly to similar phe- nomena in other forms. Considering one example in peas: it appears that the factors for roundness and wrinkledness are each allelomorphic to the other, while the factor for yellow is unpaired and allelomorphic only to its absence and never to anything. It is completely mislead- ing to assume that the factor for yellow forms an allelomorphic pair with green. Another case is that of combs of fowls: pea and single appear to form an allelomorphic pair, and there is another factor, behaving as the unpaired one for the melanism in Paratettix and yel- lowness in peas, which, when present, modifies single to make rose (the one rose being single heterozygous for this factor, and the other kind of rose being single homozygous for it), and which modifies pea and the hybrid of pea and single to make the four kinds of walnut. [t is misleading to consider rose a character; it is a modified single. PROCEEDINGS 501 Pea with heterozygous and homogyzous doses, respectively, of the modifying factor makes two kinds of walnut, while the hybrid (pea and single) with heterozygous and homozygous doses, respectively, of the modifier makes the other two kinds of walnut. The data and fuller discussion are in press. 27. The relation of yellow coat color to black-eyed white spotting of mice, in heredity. C. C. Lirrin, Harvard Medical School. It has been known for some time that mice homozygous for the factor producing yellow coat color have never been observed. Castle and Little (10) showed that the ratio of yellow to non-yellow young when two yellow mice are crossed together is approximately 2:1. This is explicable on the ground that the homozygous yellow individuals are formed but fail to develop. ' A similar condition has been found to exist in the case of the factor producing black-eyed white spotting in mice. When two black-eyed whites are crossed together they produce approximately two black- eyed whites to one ordinary piebald mouse. Black-eyed whites are always heterozygous and carry the ordinary type of spotting as an hypostatic character. A series of experiments have been carried on to determine whether or not the lethal action of the yellow factor and the black-eyed white factors is identical. It has been shown that they are entirely distinct in nature. This is proved by the breeding tests of the classes of young produced, by the size of litters, and by the ratio of yellow to non-yel- low animals in Fl. In the course of the experiments it became evi- dent that in both “black-eyed white” and piebald animals which are “vellows” the amount of dorsal pigmentation is from 5 to 30 per cent greater than ‘“‘non-yellows” of the same two color types. As yet, there is no evidence as to whether this is due to interaction of the yel- low and black-eyed white factors or whether it is due to some distinct genetic factor linked with yellow color. 28. Mutation in Didintum nasutum. S. O. Mast, Johns Hopkins University. : In a series of experiments on the effect of conjugation and encyst- ment in Didinium extending from April, 1910, to May, 1914, there suddenly appeared a marked difference in the rate of fission in the prog- eny of a single individual. This difference appeared in the latter part of July, 1912, in a line which had at that time produced 721 genera- tions without conjugation and 197 generations without encystment. The difference was still evident, apparently without diminution, when the experiment was closed after having continued 315 days. There was great variation in the rate of fission from day to day depending largely upon changes in temperature, but the difference in the rate of fission in two groups of lines remained fairly constant throughout. During the 315 days over which the experiment extended the more rapid lines produced a total average of 838 generations and the less 502 AMERICAN SOCIETY OF ZOOLOGISTS rapid lines a total average of 634 generations. The death-rate in the two groups was nearly the same, as: was also the tendency to encyst and to conjugate. 29. The occurrence of mutations in skunks of the species, Mephitis putida and M. hudsonica. J. A. DETLEFSEN. Coll. of Agr., Univ. of IIl. Eleven mutant skunks of the species Mephitis putida and three mutants of the species M. hudsonica have been found. In the former species we have, or had, in our possession four living individuals and received hair samples of two other mutants. In the latter species we have one living mutant and hair-samples of two other mutants. The mutants are as follows: Mephitis putida Female: White hair on body;-few brown hairs on face; eyes black. Successfully bred to normal male, producing three normal offspring. Female: White hair; eyes pink except a narrow ring of pigment on the outer margin of the iris. Successfully bred to normal male, pro- ducing seven normal offspring. Two female albinos with pink eyes (in our possession). Male albino, an albino of unknown sex and two solid blacks of un- known sex have been reported to us. Male and two female brown skunks (hair samples received.) Mephitis hudsonica Albino male: White hair and pink eyes (in our possession). Male and female brown skunks (hair samples received). 30. The influence of parental alcoholism on the learning capacity of the offspring. E. C. MacDoweEtt, Carnegie Institution of Wash- ington. Rats from aleoholized parents have been compared with their double first cousins from normal parents. No structural differences between the rats of normal and alcoholic parentage have been found. The number of rats in a litter averages slightly lower in the matings of alco- holics. Three methods have been employed in rating the capacity for learning: 1. Puzzle box. To enter, the rat must release the door by going behind the box and breaking an electric current; results based primarily on the time of operation. 2. Yerkes multiple-choice apparatus. The problem is to choose the correct door, from a variable series of opened doors, according to its relationship to the other opened doors; results based on the numbers of correct first choices, and the numbers of wrong choices. 3. Watson circular maze. Five choices of going to the right or left are offered; the correct path is followed on choosing right and left turns alternately, results based on time, and camera lucida tracings of the distances run in every trial. . PROCEEDINGS 503 Three groups of litters have been studied. These include 64 rats from normal parents, 54 from alcoholic parents. All the rats in each group are double first cousins. Group 1. ‘Tested with the puzzle box, the rats of alcoholic parentage, are faster; tested with the multiple-choice apparatus, two litters in this group show the normals to be the better choosers. Group 2. Tested with the puzzle box the rats of alcoholic extrac- tion are faster. Group 8. Tested with the puzzle box, the normal rats are faster than those of alcoholic parentage; tested with the multiple-choice apparatus the rats from normal parents are again more successful; two litters in this group tested with the maze, show that the rats from alcoholic parents are faster and cover less ground in learning. 81. Linkage in the sex-chromosome of a new species of Drosophila. (In- troduced by C. B. Davenport.) CHas. W. Merz, Carnegie Institu- tion of Washington. In an undescribed species of Drosophila several sex-linked mutants (as well as several non-sex-linked ones) have been obtained, and have been studied for linkage. The factors for these sex-linked characters fall into a linear series when arranged according to their linkage rela- tions, in much the same manner as factors have been shown to do in Drosophila ampelophila. By means of this series it is possible to make a comparison between corresponding linkage groups, and _ per- haps even between corresponding individual factors, in two related species. 82. An examination of the so-called process of contamination of genes. Tuomas Hunt Moraan, Columbia University. A sex-linked mutant factor in Drosophila called Notch produces two effects, a notch in the wing (dominant) and a lethal effect (reces- sive) so that no notch males ever appear. In the heterozygous fe- males the notch varies between a well-marked serration at the end of the wings to an occasional fly with wings having the normal margin. Through several generations females were selected that had the least amount of notching and then through several generations more fe- males were selected that had notch in only one wing. Such females had both the normal factor and the notch factor, but the notch char- acter was so slightly developed somatically that it showed only on one wing. To those who believe that a somatic character can be used as a measure of the ‘potency’ of the factor affecting the character, this one-sided development would appear to fulfill the conditions nearest to ‘genic weakness.’ Finally, the character was carried fur- ther in stock that had one other recognizable factor close to Notch on each side, so that the character could be followed by its linkage even after it had been selected into invisibility, i.e., normal winged flies that carried the factor could be selected and the stock bred from them. Suitable tests will be pointed out by means of which one can find out 504 AMERICAN SOCIETY OF ZOOLOGISTS whether selection has accomplished its result by piling up modifying factors, or by the isolation or allelomorphic mutations, or perchance by causing ‘allelomorphie fluctuations’ occasioned by the ‘contamina- tion’ of the genes. 33. An analysis of the effect of selection on bristle number in a mutant race of Drosophila. A. H. Sturtevant, Columbia University. A mutant race of Drosophila, known as ‘Dichaete,’ was found to be variable in the number of bristles present on the thorax. A selec- tion experiment has been carried out on this character, involving over 25,000 flies and extending through fourteen generations. Both plus and minus races have been obtained. These races have been tested, by means of the linkage method, to see if the differences between them were due to modifying factors or to changes in the Dichaete gene itself. 34. The elimination of males in alternate generations of sex-controlled lines. Catvin W. Brincss, Columbia University. (Introduced by T. H. Morgan.) There have been demonstrated in Drosophila sex-linked genes, which kill all males receiving such genes. Recently a sex-linked lethal (lethal 10) has been found which allows an occasional male with the lethal gene to come through as a pale-colored dwarf. These rare dwarfs are fertile and transmit to all their daughters the lethal gene and consequently the power to produce only half as many sons as daughters. By mating a lethal 10 dwarf to a female carrying another lethal (lethal 12) whose locus in the X chromosome is exceedingly close to the locus of lethal 10, females are obtained that are incapable of producing any sons except the rare dwarfs, although producing 200 or 300 daughters. 35. Coincidence of crossing over and the chromosome theory of linkage. ALEXANDER WEINSTEIN, Columbia University. (Introduced by T. H. Morgan.) It has been found in Drosophila that a crossing over in one region of a chromosome tends to prevent crossing over in a neighboring re- gion. This has been termed interference. The likelihood that one crossing over will interfere with another decreases with increase of distance between them. The present evidence indicates that if this distance becomes sufficiently great, interference disappears entirely; and as the distance increases still further, interference reappears. These results have a definite bearing on the twisting of the chromosomes. They are in accord with the chromosome theory of linkage, and any other theory must be able to explain them. 36. Determinate and indeterminate laying cycles in birds. L. J. COLE, University of Wisconsin. There appear to be two distinct types of laying cycles in birds, one in which the number of eggs which will be laid in the clutch is definitely PROCEEDINGS 505 determined when laying begins and the other in which the number of eggs that will be laid depends upon stimuli received after laying has begun. In other words the stimulus for cessation of laying and in- seption of brooding has already been received and the reaction pre- determined in the first case, while in the second the stimulus is re- ceived later and is followed by cessation of liberation of ova from the ovary, though laying continues for a time afterward until the ova al- ready discharged have received albumen and shells and have been expelled. The most important stimulus for the onset of broodiness, and the consequent cessation of laying, in the second class of cases is probably a physiological reaction of the female to a number of eggs in the nest. As a consequence, if the eggs are removed as laid the stimulus does not occur and laying continues beyond the regular clutch to an indefinite number. Among domesticated birds the pigeon may be taken as an example of the determinate type and the common fowl ofthe indeterminate. Among wild birds experiments have been carried on with the English sparrow and the house wren, which also appear to represent the two types respectively. 37. A strain of sex intergrades. ARTHUR M. Banta, Carnegie. Institu- tion of Washington. From an individual brought into the laboratory in August, 1912, several separate strains of Simocephalus vetulus have been propa- gated for more than 150 generations. Only parthenogenetic repro- duction can have occurred for—in the first place individuals are isolated when released from the mother’s brood pouch—long before the sexual products are matured; in the second place for 130 generations no males or fertilizable eggs appeared in this stock; and in the third place the individual culture bottles are not retained long enough for a fertilized egg to develop if fertilized eggs were produced. Hence there are three reasons, any one of which is sufficient in itself, for stating positively that sexual reproduction cannot have occurred in this strain for 130 generations. There can, then, be no question of a recent hybridiza- tion within this stock. In addition to the character of the gonads (primary sex characters) which is readily determined by examination of the living animal with the microscope, eight definite morphological secondary sex characters are recognized. In October, 1915, in one of the six strains of this line there suddenly appeared sex forms in a remarkable array and this array of peculiar sex forms has persisted for more than 25 generations. None of the other five strains coming from the original mother more than 150 generations, and more than four years ago, has to date produced males. The strain producing the sex forms ig not inferior to its sister strains in vigor or productivity. The sex array in the sex intergrade strain consists of normal females, female intergrades having one to eight male secondary sex characters, 506 AMERICAN SOCIETY OF ZOOLOGISTS hermaphroditic intergrades with various combinations of male and female primary and secondary sex characters, male intergrades with one or more female secondary sex characters, and normal males. Roughly the various sex forms fall into the classes indicated but really no precise and definite classification is feasible. Almost every possible combination of primary and secondary male and female sec- ondary sex characters occurs. There is nearly every gradation be- tween a normal female and a normal male. A single individual, even a single gonad, may produce eggs and sperm at the same time or sperm at one time and eggs at another. There is a distinct, though not very precise, relation between the secondary and the primary sex characters. Usually an individual with most of its secondary sex characters male will have testes, and conversely an individual with most of its characters female will have ovaries. However many female intergradés have five or six male characters. Some have all their secondary sex characters those of a male. Male intergrades usually produce few sperm or have incompletely developed reproductive systems. Female intergrades with all the secondary characters male are sterile. Those with as many as six male characters as usually sterile, while those with four or five male secondary characters are frequently sterile or show a much reduced productivity. Sterile individuals begin to develop eggs but they either disintegrate in the ovary or die in the brood pouch. Females with few male char- acters are usually normal in vigor and productivity. The proportion of the various sex forms produced by the different mothers varies greatly but in general those mothers which are themselves intergrades produce a larger percentage of males and intergrades than normal females in this strain. Sex here is obviously a purely relative thing. There exists a graded series from normal females, to female intergrades with one to several of their secondary characters those of a male, to hermaphroditic inter- grades with various combinations of primary and secondary male and female characters, to male intergrades with, in some cases several, in other cases a single female secondary sex character, to normal males. Maleness and femaleness are not definite and fixed mutually exclusive’ states but are quantitative and purely relative things. 38. Effect on fertility of crossing closely and distantly related stocks of Drosophila ampelophila. Roscor R. Hype, Indiana State Normal School. The fertility of Drosophila ampelophila is probably very high in nature as shown by the fact that about twenty wild stocks taken from widely separated regions have given a fertility of from 80 to 100 per cent when tested in the laboratory. These stocks when inbred have shown without exception a decline in fertility. The per cent of eggs that give rise to mature flies has dropped in some stocks to as low as 25. When these stocks are crossed a marked rise in fertility above that of the parent stocks used for control occurs both in the cross and © its reciprocal. PROCEEDINGS 507 This result stands out in marked contrast to that obtained when a stock is divided into strains and separately inbred. In this case che strains may loose different degrees of fertility. When the stocks are recombined there is no rise in fertility beyond the parent stock with the highest fertility. The control strain with the highest fertility brings the fertility of the lower strain up to its level but not beyond. 39. Are the polyradiate cestodes mutations? FRANKLIN D. BARKER, University of Nebraska. It is not surprising to find terata or “freaks” commonly among such erratic and degenerate animals as the cestodes or tapeworms. Of these abnormal forms the most rare and in many ways the most unique are the polyradiate or ‘‘double” cestodes having the appearance of two worms variously fused giving rise to two, three or more ‘‘wings”’ or sides. These are known as dihedral, trihedral and polyhedral cestodes. Such terata have been found among the cestodes of man, horse, dog and cat. One of the most interesting aspects of these abnormalities, as with all terata, is their origin. A number of theories have been advanced by various parasitologists to account for their origination such as, the partial fusion of two normal adult cestodes (Bremser); the primative malformation of the scolex with subsequent partial fusion of two worms (Vaillant); the fusion of two normal embryos which give rise to a dou- ble adult (Leuckart); several helminthologists have described these anomalies as distinct varieties of the normal species while others have even gone so far as to consider them as new and distinct species (Kucken- meister +). ' A similar anomaly has been reported among six species of cysticerci or larval cestodes and an abnormal number, more than six hooks, is frequently found in the onchosphere or first larval stage of practically all species of cestodes. Foster (’15) has recently reported the results of experimentally feeding to a rabbit, two gravid segments of a triradiate Taenia pisi- formis “shipped in a solution of formaline of unknown strength, and kept in a 2 per cent solution of formalin for one week.” Seven cysti- cerci were found fully grown and entirely normal. We published in Science, 1910, the finding of specimens of trihedral Taenia serrata = (T. pisiformis) and Taenia serialis. Unfortunately the specimens had been killed and fixed before we discovered their trihedral character. On July 29 of this year we had the good fortune to find a perfect mature trihedral Taenia serrata in the intestine of a collie dog picked up on the streets of Lincoln, Nebraska. The last two gravid prog- lottids of the living worm were teased in physiological salt solution and 3 cc. of this solution containing the freed eggs was fed to each of three half-grown rabbits born and raised in our laboratory pens. Three rabbits from the same litter were kept as checks. August 2 one in- paige rabbit died from unknown cause. No trace of cysticerci was ound. THE ANATOMICAL RECORD, VOL. 11, No. 6 508 AMERICAN SOCIETY OF ZOOLOGISTS August 13, one of the uninfected rabbits died from unknown cause. Ne cysticerci were found. October 16 one of the infected rabbits was killed and 31 cysticerci were found attached to the omentum, liver and posterior end of the colon. Twenty-six of the cysticerci had well developed scolices and 5 were immature. Sixteen cysticerci were re- moved from their protective cysts and placed in 200 ce. of digestive fluid made up of distilled water, 0.2 per cent pancreatin, 0.6 per cent NaCl, and 5 per cent saturated solution sodium carbonate and kept at blood temperature, 37°C. in an incubator for ninety minutes. The 11 mature cysticerci completely evaginated their scolices in from thirty to ninety minutes. All scolices were perfectly normal with respect to number of suckers and hooks. October 17 16 cysts were fed to a 6 months’ old dog. November 18, dog was killed and 3 hook- worms (Uncinaria trigonocephala) and 1 Taenia serrata were found firmly attached to the wall of the lower end of the small intestine. The cestode was perfectly normal though immature, measuring in a relaxed condition 5.5 em. . The last two proglottids were asymmetrical indicating their original nature. ‘The small number of worms found indicates a lack of vitality of thé cysticerci, while the absence of other parasites commonly found even in puppies points to a natural immunity or an unfavorable condition of the intestine. November 27 the third infected rabbit was killed. Six cysts were found, 4 attached to the omentum and 2 to the colon. Five cysticerci were mature, and all were perfectly normal. November 28 the two remaining uninfected rabbits were killed but no cysts were found. The results of these feeding experiments prove conclusively that the eggs of polyradiate cestodes develop into normal worms and do not give rise to polyradiate forms and therefore are not mutants, distinct varieties or species, but are terata or abnormalities which probably arise, as we have previously suggested, from the occasional partial and incomplete separation of early blastomeres of embryos of normal cestodes. 40. A revised working-hypothesis of mimicry. W.H. LonNaLEy, Goucher College. There is grave reason to doubt the existence of animals whose con- spicuousness under normal conditions has been exaggerated by natural selection. It seems desirable, therefore, to attempt to discover what explanation of mimetic resemblance is possible upon the assumption that the colors of insects are correlated with their habits, tend to re- produce characteristic tones of their surroundings, and to obliterate their possessors under the conditions in which they live. It is to be noted that this supposition is perfectly consistent with the many verifiable facts that have been discovered by special students of mimicry. Upon the basis it provides, it is to be anticipated that mimic and model should commonly be found living under the same conditions; that certain groups of insects should show the same local color varieties; that diversity of coloration should appear in one group of butterflies or moths and ally them in outward appearance with dif- PROCBEDINGS 509 ferent genera; and that insects with every variety of larval experience, as adults should possess the same type of coloration, and superficial resemblance which they attain in the most diverse fashion. The same may apparently be stated of all other admissible evidence, which has been assumed to prove the validity of the Batesian and Millerian hypotheses. Upon the other hand recognizable deficiencies in’ current explana- tions of mimicry, and criticisms levelled against them, seem adequately met by the revised hypothesis. There is no longer any difficulty in comprehending how even the initial stages in mimetic resemblance could minister to the advantage of their possessor. One’s credulity is no longer overtaxed by the demand to believe that when the patterns of scores of species of one locality present a single combination of colors, they reveal the effect of natural selection directed toward the produc- tion of resemblance. One is able to escape the inquisitor who wishes to know how creatures, which were capable of being deceived by the first vague resemblance between two species, have been able byselec- tion to push the agreement between the two to the point of apparent identity, and even the occurrence of the ‘mimic’ beyond the range of its ‘model’ is capable of rational explanation. It is therefore suggested, as a working-hypothesis, that mimicry has been initiated and advanced by indiscriminate feeders. These’ have exerted bionomic pressure, and forced their accustomed prey to assume color combinations which most effectually conceal it in its normal environment. In the evolution of types of coloration appro- priate to the surroundings and habits of their possessors members of one genus have occasionally followed different courses, and fortuitous resemblances to.various unrelated genera have occurred, capable of deceiving enemies, which exercise discrimination in their choice of food. At this point selection directed to the production of deceptive” resemblance has been superimposed upon processes culminating in the development of types of obliterative coloration without changing the general trend of their evolution. 41. Recent studies of nerve conduction in Cassiopea. ALFRED G. MAYER. Researches conducted at The Tortugas Laboratory of the Carnegie Institution of Washington upon Cassiopea indicative that nerve con- duction in this medusa is due to a chemical reaction involving the cations of sodium, calcium, and potassium; magnesium being relatively non essential. The sodium. and calcium cations together appear to combine with some undetermined proteid element to form an ion-proteid. The prob- ably high temperature coefficient of ionization of this ion-proteid may account in some measure for the high temperature coefficient of the rate of nerve conduction, which is 2.5 as great as that of the elec- trical conductivity of the sea water surrounding the nerve. The rate of nerve conduction is probably accelerated by an enzyme as stated by E. N. Harvey, 1911. 510 AMERICAN SOCIETY OF ZOOLOGISTS R. 8. Lillie, 1916, American Journal Physiol., vol. 41, p. 1383, appears to be mistaken in assuming that the rate of nerve conduction is a func- tion of the electrical, conductivity of the solution surrounding the nerve, for the decline in rate of nerve conduction is practically identi- eal whether we dilute sea water with 0.415 molecular MgCl or with distilled water. In other words whether we maintain a constant electrical conductivity or reduce it in a ratio nearly commensurate with the dilution. Thus Lillie’s “local action” theory appears not to be supported. My former idea that adsorption of Na’, Ca”, and K’ played an im- portant réle in nerve conduction is erroneous. I was misled by the effects due to a slight acidity of the distilled water, and I did not reduce all observations to a constant temperature, which is essential, due to the high temperature coefficient of the reaction. 42. The theory of sex as stated in terms of results of studies on the pigeons. Oscar RippiE, Carnegie Institution. Studies which have demonstrated the reality of the contrel or re- versal of sex in pigeons (Whitman, and later Riddle) have at the same time indicated the nature of the initial difference between germs of prospectively different sex-value. This difference rests upon differ- ‘ent levels of metabolism; and when the metabolic level of a given germ is shifted from the level characteristic of the germ of one sex, suffi- ciently toward the level of the other sex, it develops into an organism of the sex which corresponds to the acquired, or later, level. The initial difference characteristic of the two kinds of (sex) germs, tends to persist and characterize the adults of the two sexes. Sex is based on a quantitative difference; intermediates of the nor- mal extremes have been experimentally produced, and the normal extremes have themselves been experimentally accentuated. There seems to be no known body of facts in contradiction of this view; though the facts obtained from the pigeons are in direct contra- diction of some of the more or less current theories. 43. The adaptive color changes of tropical fishes. W. H. LONGLEY, Goucher College. (Illustrated with lantern.) The colors of tropical reef-fishes are correlated with their habits. Their color changes may occur almost instantaneously, and in many species even those of unconfined individuals are subject to direct con- trol by the observer. In general, they enable the animals that dis- play them to repeat upon their own bodies the characteristic tones of the various environments in which they move. Photographs secured with a submarine camera and diving-hood record some of the changes in coloration which Epinephelus striatus and Lachnolaimus maximus commonly exhibit, and indicate their effect in reducing the creatures’ conspicuousness. Others show how particular elements in a complex pattern such as that of Abudefduf saxatilis blot out their possessor’s contour, when the animal is viewed . against an appropriate background. PROCEEDINGS Srl A great body of evidence indicates that more than minimal conspicu- ousness may not be ascribed rationally to bright colored fishes as a class, and strikes at the foundation of some of the most widely dis- seminated hypotheses of animal coloration. Obviously, however, it suggests that the characters in question are useful, and that their development has been largely controlled by natural selection. 44. The histological basis of adaptive shades and colors in the flounder, Paralichthys albiguttus. ALBERT Kuntz, St. Louis University School of Medicine. Changes in shade and color in fishes are due primarily to changes in the distribution of the pigment granules in the chromatophores in the superficial layers of the skin and changes in the relationships of the guanophores (cells containing guanin crystals) with these chro- matic organs. The skin of Paralichthys albiguttus contains chromatophores of two distinct types, viz., melanophores and xanthophores. The for- mer contain melanin granules which vary in color from dark brown to black, the latter contain xanthine granules which vary in color from yellow to orange. Under experimental conditions pigment granules can be observed advancing toward the periphery and in turn retreating toward the center of the chromatophores along more or less definite radial lines. Ameboid movements of the chromatophores can not be observed in adult specimens. Neither could evidence be obtained from prepara- tions of the skin which indicates that the chromatophores contract and expand in an ameboid manner. Changes in the distribution of the pigment in the chromatophores are accomplished by movements of the pigment granules within the cells and do not involve essential changes in the form of these organs. A comparative study of living material and preparations of the skin of specimens of Paralichthys albiguttus adapted to backgrounds of various shades indicates that shade depends primarily upon the degree of distribution of the melanin pigment in the melanophores and the spacial relationships of the guanophores with these bodies in the superficial layers of the skin. The xanthophores probably play no important part in the determination of shade. The most obvious response to a change in the color of the back- ground is a change in the distribution of the xanthine granules in the xanthophores. Shades of yellow and orange depend primarily upon the degree of distribution of the xanthine granules in the xanthophores containing yellow and orange pigment respectively. In general the particular quality of the color assumed by the fish depends upon a complex group of factors which do not lend themselves readily to a detailed analysis. Some of the colors assumed may be duplicate by mixing pigments of the colors represented in the pigments contained in the chromatophores. These colors depend primarily upon the degree of distribution of the pigment granules in the melanophores 512 AMERICAN SOCIETY OF ZOOLOGISTS and xanthophores respectively. Colors which can not be duplicated in this manner, doubtless, depend upon the relative degree of distri- bution of the pigment granules in the melanophores and xanthophores plus the optical effects due to the diffraction of light by the guanin erystals in the guanophores. The optical effects produced by the guanin crystals are probably modified by the particular spacial rela- tionships of the guanophores with the chromatic organs. Obviously, certain colors are simulated by the fish more perfectly than others. Among the colors used in the present investigation yellow and green were simulated more perfectly than dark red and dark blue. None of the specimens placed on a dark red background showed any color which approximated the color of the background: more closely than the orange pigment in the xanthophores. In view of these facts the conclusion that all colors can be reproduced in the skin of the flounder is unwarranted. Adaptation to a yellow background involves a moderate degree of concentration of the pigment in the melanophores and a marked de- gree of distribution of the pigment in the xanthophores in the superficial layers of the skin. Adaptation to a green background involves a marked degree of distribution of the pigment in the melanophores and a moderate de- gree of distribution of the pigment in the xanthophores in the super- ficial layers of the skin. The resultant yellowish green color probably depends upon the ratio of the distribution of the xanthine to the dis- tribution of the melanin pigment plus the optical effects due to the diffraction of light by the guanin crystals in the guanophores. Adaptation to a dark red background involves almost maximum distribution of the pigment in the melanophores and the orange colored xanthophores and a marked degree of concentration of the pigment in the yellow xanthophores in the superficial layers of the skin. The resultant reddish brown color is due largely to the wide distribution of orange pigment, the effect of which is probably modified by the blending of orange and black and the optical effects produced by the guanophores. Adaptation to a dark blue background involves almost maximum distribution of the pigment in the melanophores and almost maxi- mum concentration of the pigment in the xanthophores in the super- ficial layers of the skin. Many of the guanophores also become ar- ranged with reference to the melanophores and closely associated with them. Doubtless, the resultant greenish blue color depends largely upon the optical effects produced by the guanophores which are closely associated with the melanophores. The dark shade is due to the wide distribution of the melanin pigment. 45. Further data on the relation between the gonads and the soma of some domestic birds. H. D. Goopatn, Massachusetts Agricultural Ex- periment Station. The results of published data on the ablation of the testes and ovary of domestic birds together with unpublished data on the transplanta- PROCEEDINGS 513 tion of the ovary into castrated males has made it clear that different parts of the soma react in different ways to the secretion of the gonads. Each character appears to be more or less independent of every other character just as they are more or less independent in heredity. ‘The various characters fall into several groups. We may recognize, first, characters (including some of the secondary sexual characters) that are independent of either ovary or testis. Such characters are, size in the female, voice and some phases of behavior, and mandible color in ducts. Second, characters affected by the testis, such as comb and wattles, fat deposition, size in the male, and some instincts and sum- mer plumage in ducts. Third, characters that are affected by the ovary, such as plumage form and color and some phases of behavior. No sharp line can be drawn between the better known secondary sexual characters and those commonly considered ordinary somatic characters, for the reaction of a character to the secretion of either gonad varies not only according to the character itself but also ac- cording to the original sex of the individual. Thus, the size of the primary coverts in relation to the primaries is approximately the same in each sex but after removal of the testes they become dispropor- tionately large, though not after removal of the ovary. The spurs always develop in the female after removal of the ovary, but they also develop in the capon and in feminized cockerels, i.e., in the pres- ence of the ovary in the soma of the male. Females from which the ovary has been removed are neutral in sexual behavior but one of the most astonishing things about castrated males with implanted ovaries is that they exhibit only the sexual behavior of the male. The comb and wattles do not develop in the male after castration, i.e., are infantile; in the castrated male with engrafted ovary they are fully feminized; in the ovariotomized female they may be either fe- male-like or male-like. The capon exhibits two characters that are female-like, viz., the amount of fat deposited and the brooding instinct. On the other hand, two masculine characters are intensified by the removal of the testes, viz., body size and plumage length. The ovari- otomized female is approximately the size, however, as her normal sisters, while the castrated male with engrafted ovary has the size of the male. The plumage shape seems to be completely controlled by the ovary, since wherever that is present the shape of the feather is like those found on intact females. The color is less completely controlled by the ovary for while females from which the ovary has been removed develop the colors of the male, only a portion of those males with engrafted ovaries have developed the female’s color, though they do not develop all the colors of the male, particularly the bril- liant colors. Inducks there is a greenish pigment of the mandible that disappears from the mandible of the castrated female, but since the mandible color of the male with engrafted ovaries is unaltered, it appears that castration induces a previously non-existant difference between the sexes. Finally, there are characters that behave one way in some individuals and in another way in others. This is particularly notice- able in the plumage of the ovariotomized ducks, which in particular 514 AMERICAN SOCIETY OF ZOOLOGISTS regions may vary from a purely masculine condition to a purely fe- male condition or to a condition sui generis. If the entire series of altered individuals is examined, it is apparent that it may be looked upon as a series of sex intergrades. ‘That is, characters that are normally found in one sex may be experimentally transferred to the opposite sex while individuals composed of mixtures of such characters may be obtained. 46. The sensory potentialities of the nudibranch ‘rhinophore.’! LESLIE B. Arey, Northwestern University Medical School. Nudibranch mollusca possess a pair of short, robust dorsal tenta- cles which are commonly perfoliate or rmged and which may or may not be retractile. These important looking tentacles have long been designated ‘rhinophores,’ and it is tacitly assumed that they are in- deed specialized olfactory organs. The presence in certain species of long, more or less dorsally placed tentacles, in addition to the oral tentacles and rhinophores, heightens the suspicion that the latter may perhaps serve some particular sensory function. The sole experimental evidence upon which the assignment to the thinophores of an olfactory activity rests seems to be found in the observations of Graber in 1877 (Biol. Centralbl., Bd. 8, No. 24, pp. 743-754). Graber brought oil of rose near the head of Chromodoris elegans and observed the withdrawal of the rhinophores to be quicker and more vigorous than that of the oral tentacles. He emphatically states, however, that the post-branchial region is the most sensitive part of the body. It would thus appear that the convenient term ‘rhinophore’ is of dubious propriety. For this reason Bermudian nudibranchs were subjected to experimentation designed to test their sensory potentialities. Unless otherwise stated the following account applies to Chromodoris zebra Heilprin. Tactile stimulation. When a rhinophore is touched lightly with a glass rod it is jerked back precipitately within its protecting collar. The sensitivity of the rhinophore to gentle stimulation is astonishing and the explosive type of response is, within wide limits, independent of the strength of the stimulus. Fatigue comes on but slowly, responses of somewhat diminished intensity being readily obtained after fifty successive stimulations at ten-second intervals. The oral tentacles, gill plumes and the general body-surface all re- spond to tactile stimulation. It is unsatisfactory to list the several regions of the body in the order of their sensitivity, for the types of re- sponses are not all comparable. It appears, however, that the so-called rhinophore is the most sensitive part of the body and considerably more so than the oral tentacles. Thermal stimulation. The head region and especially the oral tentacles react distinctly to water at 40°-50°C. applied with a pipet. 1 Contributions from the Bermuda Biological Station for Research, No. 52. PROCEEDINGS 51d The rhinophores, on the contrary, give faint and rather doubtful responses except to temperatures as high as 50°C, Chemical stimulation. Equal volumes of various chemical solu- tions were applied from a constant distance with a pipet. Solutions of 1 M of maltose, or sucrose, or M/2 of lactose were without effect upon all parts of the body, although 3 M glycerine did evoke general responses. Several alkaloids had very weak effects or none at all. Alcohols and organic acids in concentrations of M/10 called forth strong general responses. The chlorides of the alkali metals Na, K, NH, and Li likewise stimulated the body in general, the rhinophores and oral tentacles, however, showing the greatest sensitivity. Solu- tions of substances which produce in man the taste sensations recog- nized as acid, bitter, salty and alkaline were applied in various con- centrations. The gills fail usually before other parts, although all responses gradually weaken with increasing dilution. From the foregoing tests it becomes evident that the rhinophore is not only extremely sensitive to chemical stimulation of diverse sorts, but that this sensitivity is only second to, if indeed it does not equal, that of the oral tentacles, which from their position might be suspected a priori of a specialized gustatory or common chemical function. Olfactory stimulation. Saturated solutions in sea water of numerous essential oils and decoctions of decaying marine invertebrates were prepared and applied by the pipet method. The rhinophores react strongly to these solutions, but other parts of the body appear to be, so far as one can judge from the dissimilarity of the responses, equally sensitive. When a drop of oil is held for some time midway between the rhinophores no response ensues. If the rhinophore, or body, be touched gently with a drop of pure oil, the reaction is weaker than to a saturated solution. Here the number of sense organs stimulated is undoubtedly a complicating factor, yet there is suggested further that the response is one to an olfactory, rather than to an irritative or ‘smarting’ stimulus. An essentially similar behavior to odorous substances was found in Chromadoris roseapicta, Elysia crispa and Fiona marina. Besides the rhinophores, Facelina goslingi possesses long, more or less dorsal tentacles, and short oral ones. The longer pair reacts more vigor- ously to solutions of the oils than do the rhinophores. Summary. The entire body of Chromodoris zebra is sensitive to mechanical, common chemical, gustatory and olfactory stimuli. The head region is somewhat responsive to the application of increased temperature. Several other nudibranchs exhibit general olfactory sensitivity. Of the various parts of the body, the rhinophore is most sensitive to touch; is second, if not equal to the oral tentacle, in its response to chemical stimulation; and shares its sensitivity to odorous substances with the oral tentacle. In at least one species, Facelina, the long posterior tentacles react more vigorously to solutions of es- sential oils than do the rhinophores. Only to thermal stimulation is the rhinophore (of Chromodoris) clearly inferior in sensitivity. Hence 516 AMERICAN SOCIETY OF ZOOLOGISTS the so-called rhinophore, like the insect antenna, is a compound sense organ, for which the misleading term ‘rhinophore’ is highly inapt. 47. Paramecium grown in pure cultures of bacteria. GrorcEe O. Har- Gitt and WALTER W. Fray, Syracuse University. Identical hay-infusions, inoculated with bacteria of the air, hay, or tap-water, produced flora quite similar for air and hay inoculation, | somewhat different for water inoculation.. The bacteria present in the infusions after a growth of three weeks were somewhat different than at first; at the end of three months entirely different types of. bacteria had’developed and some of the original forms had disappeared. In general it was found that the bacteria present in the fresh cultures. were more favorable as food, and those of a three months culture were generally unfavorable as food for Paramecium. 36 From fresh and old cultures, from normal and abnormal (fermenting and putrefying) cultures, over thirty different kinds of bacteria were isolated in pure cultures; of these eleven were identified and their morphological, cultural, and bio-chemical characteristics were thor- oughly studied. Sister Paramecia were grown in pure cultures of these eleven kinds of bacteria and their favorableness or unfavorable- ness as food was determined by the rate of division of the protozoa. No single kind of bacteria was as satisfactory a food as a mixture of different kinds of bacteria. Some bacteria were so unsatisfactory as to cause the death of Paramecium more quickly than if the protozoa: were grown in sterile water. Bacillus subtilis was the best single form. for food; in some cases Paramecium feeding on them divided more rapidly, in other cases less rapidly than on a mixed diet. , By using bacteriological methods it was demonstrated that Para- mecium could be rendered absolutely sterile by washing the animals. through five or six changes of sterile fluid. All feeding tests were carried on with sterilized Paramecia of the same strain. All pipettes, slides, moist chambers, and the like were sterilized in a hot air sterilizer at. a temperature of 150°C., or more. The culture medium used was made at one time, placed in test tubes and sterilized before the experi- ments were started; the fluids were therefore identical throughout the investigation. All external conditions were controlled, or at least were the same for all cultures, so that the only difference between the experimental cultures was in the food supplied to Paremecium. The feeding experiments were conducted as follows: A sterile de- pression slide was filled with hay infusion inwhich were growing bac- teria of a single sort (a pure culture of bacteria). A single Paramecium was introduced and the slide placed in a sterile petri-dish, the atmos- phere of which was kept moist by a small amount of sterile water. Hach: day some of the originally prepared, standard, hay-infusion, freshly inoculated with the desired bacteria, was placed in a slide in a sterile moist chamber and one Paramecium from the slide culture of the pre- vious day transferred to it with a sterile pipette. The slide culture for a feeding experiment was usually carried for a period of ten days or two weeks. PROCEEDINGS 517 If proper precautions were taken the culture fluid of the slide at the end of the period of experimentation was generally found to contain only the single kind of bacteria originally introduced. This was deter- mined in each case by making an agar plate of the culture fluid at the end of the experiment. Occasionally strange bacteria gained entrance and contaminated the culture fluid, but the contamination was so slight that the diet of Paramecium was probably not modified to any extent. In the most extreme case a count of the bacteria at the end of the experiment (two weeks) in the slide culture showed the contami- nating bacteria present in the ratio of 1 to 350 of the form originally introduced. The results of the feeding experiments are believed, therefore, to be accurate indications of the effect of different kinds of bacteria as food for Paramecium. 48. Recognition among insects. N. FE. McInpoo, Bureau of Entomol- ogy, Washington, D. C. It has always been a matter of conjecture as to how the various lower animals recognize one another and by what means the sexes of any species distinguish each other. The senses of sight and touch are undoubtedly used considerably for this purpose, but it is probably true that the olfactory sense is the most important factor. Jaeger (Zeitsch. f. wiss. Zool., Bd. 27, 1876, p. 322) goes so far as to assert that most animals emit odors peculiar not only to the individual, variety, race, and species, but also to the genus, family, order and class, and that these odors are the chief means by which one animal recognizes other animals. The experimental results embodied in the present paper deal only with the odors emitted by honey bees. Some of the results are not conclusive, although the following are now fairly well established by von Buttel-Reepen and the present writer. It is certain that a queen gives off an odor, and it seems reasonable that the odors from any two queens would be slightly different. All the offspring of the same queen seems to inherit a particular odor from her. This odor, called the family odor, perhaps plays little or no use in the lives of bees, for it is certainly masked by the other odors. Drones seem to emit an odor peculiar to their sex, but little can be said about it. It seems certain that each worker emits an individual odor which is different from that of any other worker. It is also prob- able that the wax generators and nurse bees emit odors slightly differ- ent from those of the field bees. Of all the odors produced by bees, the hive odor is probably the most important. It seems to be the fundamental factor or principle upon which the social life of a colony of bees depends, and perhaps upon which the social habit was acquired; without it a colony of bees could not exist. The hive odor is composed chiefly of the individual odors from all the workers in a hive, and is supplemented by the odors from the queen, drones, combs, frames and walls of the hive, ete. From 518 AMERICAN SOCIETY OF ZOOLOGISTS this definition it is easily understood why no two colonies have the same hive odor. The hive odor of a queenless colony is perhaps con- siderably different from that of a colony which has a queen. The ab- sence of a queen odor in the hive odor probably explains why the workers in a queenless colony are irritable and never work normally. All the bees—workers, queen and drones—in a colony carry the hive odor of that colony on their bodies among the hairs. This odor serves as a sign or mark by which all the occupants of a hive “know” one an- other. Since the queen and drones are “aristocrats,” they seem to disregard the sign that has been thrust upon them, but whenever a queen enters the wrong hive, she soon “realizes” that she wears the wrong badge. Worker bees returning to the hives from the fields pass the guards unmolested, because they carry the proper sign, although the hive odor that they carry is fainter than when they left the hive, and it is also partially masked by the odors from the nectar and pollen carried by these bees. Bees kept in the open air for three days lose all the hive odor car- ried on their bodies, but each bee still emits its individual odor. When a colony is divided the hive odor in each half soon changes so that by the end of the third day the original colony possesses a hive odor so different from that of the other half of the colony, that when the work- ers are removed from the two new colonies and are placed together in observation cases, they fight one another as though they had been separated all their lives. While a foreign hive odor calls forth the fighting spirit in workers, the queen odor always seems pleasant to workers regardless of whether the queen belongs to their hive or to another hive. Even though the queen odor forms a part of the hive odor, it is probable that this odor to the workers stands out quite prominently from the hive odor. That workers do not miss their queen for some time after she has left the hive, indicates that her odor thoroughly permeates the hive odor and that whenever this odor grows faint the workers ‘know’ that she is not among them. There has been much speculation concerning the ruling spirit or power in a colony of bees. The writer is inclined to believe that a nor- mal hive odor serves such a purpose. The hive odor is a means of preserving the social life of the bees from without, the queen odor which is a part of it insures continuation of the social bfe within. As already stated the workers ‘‘know”’ their hive-mates by the hive odor they carry. This odor insures harmony anda united defense when an enemy attacks the colony. The queen odor constantly informs the workers that their queen is present, and even though she does not rule, her presence means everything to the bees in perpetuating the colony. Thus by obeying the stimuli of the hive odor and queen odor, and being ended by instinct, a colony of bees perhaps could not want a better ruler. PROCEEDINGS 519 49. The rate of locomotion of vanessa antiopa in different luminous in- tensities and its bearing on the ‘continuous action theory” of orventation. Wm. L. Dotuiry, Jr., Randolph-Macon College. If orientation in light is dependent upon the stimulation of both re- tinas by equal amounts of light, energy as is held by Loeb and his “continuous action theory,” butterflies should move more rapidly in bright light than in weak. To test this the rate of movement of 10 specimens of Vanessa antiopa in each of two lights, one about 2,000 times stronger than the other, was ascertained. They did not move faster in the bright light than in the weak, but, on the contrary, 70 per cent of the insects actually moved more rapidly in the weak light than they did in the strong. These results support those presented previously which indicated that the orientation of Vanessa in light cannot be accounted for on the basis of Loeb’s theory. Moreover, some positive evidence has been obtained in favor of the theory that orientation is dependent upon the time rate of change of intensity, since the results of some experiments seem to indicate that Vanessa moves faster in intermittent light than in continuous light. 50. A super-organ for the expansion of Renilla. G. H. Parker, Har- vard University. The Pennatulid Renilla, in its adult state, consists of a flat, kidney- shaped portion, representing the expanded rachis, and a peduncle of striking proportions. The dorsal surface of the rachis is said to carry three kinds of zooids: large autozooids capable of considerable expan- sion and generally scattered over the surface; small siphonozooids also generally scattered; and a single axial zooid situated on the axis of the colony and not far from its middle. The peduncle has long been known to be divided longitudinally into two chambers by a delicate membrane. If a specimen of Renilla is roughly handled, all its zooids contract and its volume is much diminished by the loss of seawater. Reéxpan- sion is accomplished by the peristaltic action of the walls of the peduncle whereby the colony becomes refilled with seawater. The seawater enters through the mouth of the axial zooid; it then passes down one chamber in the peduncle to the neighborhood of the distal end of that part where it crosses through apertures in the membrane to the opposite chamber. From this chamber it makes its way by appropriate channels to the various autozooids which are thus expanded. The pressure under which this water flows is due to the muscular contractions of the peduncle. The peduncle in Renilla, and probably in many other Pen- natulids, is therefore an organ of expansion for the whole colony. If the functional parts of protozoans are to be called organoids, and the functional parts of metazoan individuals organs, the functional parts of metazoan colonies, such as the peduncle of Renilla, may be called super-organs. 520 AMERICAN SOCIETY OF ZOOLOGISTS 51. The photoreceptors of amphioxus.1 W. J. Crozimr, Bermuda Bio- logical Station. An ineandescent filament appropriately mounted was substituted for the ocular on one limb of a binocular microscope; by means of this device it was possible to secure, with properly adjusted diaphragms, an intense beam of light, microscopic in diameter, which was focussed within or upon various portions of Branchiostoma carribaeum. The exact location of the light-spot, and the extent of light scattering by the tissues, were observed through the other tube of the binocular. Practically every portion of the body of a number of lancelets was examined in this way in a dark-room; precautions were taken to avoid mechanical stimulation, to which amphioxus is very sensitive in the dark. No responses were obtained except when the light was focussed upon or within the ventral half of the nerve cord. It was possible to prove, notably by experiments with individuals in which portions of the integument were thoroughly anaesthetised, that this stimulation did not concern photoreceptors in the skin. The integument of amphioxus therefore contains no normal photo- receptors. As indicated by Parker’s less precise tests, the optic cups within the nerve-tube are probably the light-sensitive organs in this animal. This conclusion is substantiated by the details of the il- lumination trials, and particularly by the demonstration of photo- mechanical changes in the pigment cups of the ‘‘Sehzellen.” Some evidence was secured which points to the photosensitivity of the “dor- sal Sehzellen” of Joseph. The region of the anterior pigment spot is insensitive to light. 52. une olfactory reactions of snails. Manton CopELaAND, Bowdoin College. It is well known that certain snails have the habit of collecting about decaying organisms or living bivalve mollusks, in the latter case often causing considerable damage to oyster beds. The reactions to food of two species belonging to the genera Alectrion and Busycon (Sycoty- pus) were carefully studied with the view of determining the general nature of the respoase, the manner in which food is located and the sense organ concerned. Alectrion which showed variation in its rheotropic reactions moved more often against the current when a dead fish was placed at the head of the stream, and extended its proboscis in search of food when stimulated with fish juice squirted from a pipette. Busycon exhibited similar reactions to an extract of oyster. Since the response was ob- tained from dilute juices emanating from distant food, it may be re- garded as truly olfactory. The tentacles of gasteropods have been described as olfactory or- gans, but both species studied showed the same typeof reaction after their removal. The snails had the habit of burying themselves in the sand, leaving only the tips of their siphons exposed. Under such conditions they often came out of the sand when food juices were taken: ‘Contribution from the Biological Station for Research, No. 53. PROCEEDINGS 521 into the siphons. It soon became evident, therefore, that the olfactory organ was situated either within the siphon or mantle chamber. Since Alectrion still reaction to juices after the greater portion of the siphon had been removed, it seemed probable that the osphradium was the olfactory receptor. This conclusion was substantiated by scraping away the osphradia from several specimens of Busycon, which then failed to show olfactory response, although they took food when it was tasted and in other ways exhibited normal behavior. The way in which Busycon locates distant food through its olfactory sense was definitely determined. When the snail is moving the siphon is continually swinging from one side to the other. If the stimulus is applied when the siphon is at the end of its movement to the left the foot turns in the same direction, whereas it turns to the right if stimu- lation occurs at the termination of the dextral swing. There is no difficulty, therefore, in leading a snail in any direction over the floor of the aquarium or up its side by applying oyster juice to the tip of the siphon, provided the organ is first pointed in the direction which it is desired the animal shall follow. When two cheesecloth bags, one containing a piece of oyster, were fastened in front of and lateral to the siphon tip, one on the right the other on the left, the snail turned in the direction of the baited bag, in a single instance completing two and a half circles. The foregoing experiments show that the snail instead of possessing the paired olfactory organs characteristic of most animals has a median one situtated near the base of the siphon. Accordingly, orientation to dilute chemical stimuli involves two distinct muscular activities, first a right and left swinging of the siphon preceding stimulation, and secondly a movement of the foot in the direction indicated by the si- phon at the time it conducts the stimulating materials to the sense organ, the osphradium. By this prodecure the snail continues to move toward and finally arrives at the source of the stimulus, its food. 58. The reactions of the crimson-spotted newt, diemyctylus viridescens, to light. A. M. Rexrsn, University of West Virginia. 1. Phototropic reactions of Diemyctylus are markedly negative; in 30 observations 251 were found in the dark to 95 in the light half of the aquarium. 2. At temperatures near freezing water the animals become so slug- gish as to be more or less indifferent to light; if the temperature be raised to about 36°C. they become abnormally active, and are again indiffer- ent to light; at 40°C. they are seriously affected or even killed. 3. These animals respond in the same way, though less markedly, when half of the aquarium is illumined from below. 4. Diemyctylus is positively phototactic towards even very weak daylight, such as is seen on a cloudy day 20 feet away from an ordi- nary window. 5. With a 25W 115V Tungsten light 6 inches from the end of the aquarium 298 animals faced the light to 90 that faced away from it; 522 AMERICAN SOCIETY OF ZOOLOGISTS and 244 were noted in the near half of the aquarium to 163 in the far half. 6. With an electric: are projection lantern 15 inches from the end of the aquarium 116 animals faced the light to 41 facing away; and 105 animals were in the near half to 60 in the distant half. 7. At low temperatures the phototactic response to white light is inhibited or even reversed; i.e., in 12 observations with the Tungsten light and a maximum water temperature of 11°C. 48 animals faced the light to 72 that faced away from it; and 48 were counted in the near half to 94 in the far half of the aquarium. 8. With an intense white light at each end of the aquarium the ani- mals tend towards the less intense; if neither light be of great intensity, perhaps not reaching a certain optimum, the animals tend towards the more intense. ; 9. Reaction to pure red light is the same (though, perhaps, more marked) as to the white; i.e., 225 animals faced the light, in one ex- periment, to 46 that faced away from the light; and 221 were in the near half of the aquarium to 49 in the distant half. 10. Reactions to blue light are like those to red, but not so marked. 11. The attraction of green light is more marked than the blue but less marked than the red. 12. A small spot of white light from a micro-electric torch produced no effect when thrown on various parts of the body. 13. The animals responded promptly to a beam of sunlight, thrown on various parts of the body, either from above or below, by a small murror; though if the mirror threw a beam of 5 mm. or less there was little or no response. 14. Animals experimented upon in their native pond, under as natural conditions as could be provided, gave essentially the same responses as described above to sunlight and to an acetylene light at night. 54. Reaction of the whip-tail scorpion to light. Braptey M. Parren, Laboratory of Histology and Embryology, School of Medicine, Western Reserve University. The responses of whip-tail scorpions to light were.studied with a view to establishing quantitatively, certain characteristic reactions. No attempt was made to treat exhaustively all phases of their behavior under the influence of light. The object was rather to obtain such reaction measurements as would best serve as a basis of comparison for subsequent work directed toward determining the relative effective- ness of the various parts of their complex photoreceptive mechanism. teactions to photic stimuli of known intensities were recorded in terms of the induced angular deflections from an initia! direction of locomotion. The results obtained may be summarized as follows: 1. The threshold for the kinetic effect of light was at about 0.16 candle meters. The response was clearly negative to all intensities which induced locomotion. Up to an intensity of 1 candle meter the | amplitude of the reaction increased rapidly. In the intensities above 1 candle meter the increase in deflection was much more gradual. PROCEEDINGS 525 2. When started heading away from the source, in a horizontal beam of light of 120 candle meters, animals continued to move along the path of the rays. In 40 trails the average was within 0.6 of a degree of the central ray of the beam. 3. When subjected to a light of 120 candle meters acting on them from the side, the scorpions turned and moved away from the light. The average deflection was 65.8 degrees. 4. When subjected to balanced, opposed lights each delivering an illumination of 120 candle meters, the average trail was within 3.7 de- grees of the norm to the line connecting the sources of light. 5. When started directly toward a light giving an illumination of 120 candle meters, the scorpions turned and moved away from the source. The average deflection was 140.4 degrees. 6. Unilateral elimination of any part of the photorecptive mechan- ism caused an unbalancing of subsequent reactions. The extent of the unbalance was proportional to the extent of the interference with the receptors. With regard to the method of orientation these results point to the conclusion that the negative reactions of the whip-tail scorpion to light, depend on a tendency on the part of the animal to attaim and maintain bilateral balance of stimulation. Moreover, there are, in this form, no indications that rapid changes of light intensity are necessary to the attainment of orientation. There would seem to be no doubt that light of constant intensity acts as a stimulus. It is apparent, also, that the stimulating effect of light of relatively constant intensity is a prime factor in bringing about and maintaining orientation. 55. The effect of light and dark upon the eye of Prorhynchus applanatus kennel. W. A. Kepner and A. M. Fosuer, University of Virginia. 1. Stimulation by light results in a contraction of the ‘accessory cell or pigment-cell. In sustained darkness the cytoplasmic lamellae of the pigment-cell open up or move apart, resulting in the expansion of the cell. 2. The three cytoplasmic regions of the retinula or visual cell show more or less marked changes in response to light and darkness. The nucleus-bearing part of the visual cell is somewhat widened in the dark. The refractive, middle segment-analagous to an ellipsoid of a vertebrate retinula—disappears in continuous illumination and _ is most Representing the Central Branch. W. A. Locy, This Committee made no report at the joint meeting held at Cleveland in 1912, but during the summer of 1913 a meeting of the Committee, called by the president of the Society, H. B. Ward, was held at Woods Hole, at which a con- stitution for the Society was outlined. At this meeting the absent members, H. V. Wilson, M. M. Metcalf and W. A. Locy, were represented by G. H. Parker, George Lefevre and Jacob Reighard. The draft of the constitution formulated at this meeting was later submitted by the chairman of the Committee, G. A. Drew, to all members of the original committee, and the constitution finally agreed upon is practically the same as that adopted by the Society at the joint meeting held at Philadelphia in 1913 (see page 71). LIST OF FORMER OFFICERS AMERICAN MorRPHOLOGICAL SOCIETY President Vice-President Secretary-Treasurer PB Ese WUEESOM 8 eiehsie fecal ois ep J. P. MeMurrich 1891—C. O. Whitman E. L. Mark J. P. MeMurrich 1892—C. O. Whitman H. F. Osborn J. P. McMurrich 552 President 1893—C. O. Whitman 1894—C. O. Whitman 1895—E. B. Wilson 1896—E. L. Mark 1897—C. S. Minot 1898—H. F. Osborn 1899—E. G. Conklin 1900—T. H. Morgan 1901—J. S. Kingsley 1902—H. C. Bumpus AMERICAN SOCIETY Vice-President E. B. Wilson W. B. Scott W. B. Scott H. F. Osborn S. I. Smith T. H. Morgan W. M. Wheeler H. C. Bumpus E. A. Andrews G. H. Parker OF ZOOLOGISTS Secretary-Treasurer J. P. MeMurrich G. H. Parker G. H. Parker G. H. Parker G. H. Parker G. H. Parker Bashford Dean J. M. Kingsley T. H. Montgomery M. M. Metcalf Additional Members of the Executive Committee 1891—E. B. Wilson H. F. Osborn 1892—E.. L. Mark H. Morgan T. H. Morgan C. B. Davenport E. A. Andrews . H. Herrick 1895—T. H. Morgan S. Watase 1896—E. G. Conklin William Patten 1897—J. S. Kingsley Bashford Dean 1898—C. B. Davenport F. R. Lillie 1899—J. P. MceMurrich G. H. Parker 1900—F. R. Lillie Jacob Reighard 1901—C. F. W. McClure C. W. Hargitt 1902—H. S. Jennings R. G. Harrison AMERICAN SOCIETY OF ZOOLOGISTS EASTERN BRANCH G. H. Parker E. A. Andrews W. E. Castle W. E. Castle C. B. Davenport W. M. Wheeler H. 8. Jennings T. H. Montgomery H. V. Wilson A. G. Mayer Raymond Pearl Jacob Reighard W. E. Castle William Patten William Patten F. H. Herrick H. §. Jennings H. V. Wilson President 1903 1904 1905 1906 1907 1908 1909 1910 1911 1912 1913 1903 1904 1905 1906 1907 1908 1909 CENTRAL BRANCH Jacob Reighard C. H. Eigenmann F. R. Lillie C. C. Nutting S. A. Forbes E. A. Birge E. A. Birge C. E. McClung George Lefevre H. B. Ward H. B. Ward H. F. Nachtrieb S. J. Holmes William A. Locy George Lefevre H. B. Ward M. F. Guyer M. F. Guyer LIST EASTERN BRANCH H. H. Wilder H. E. Crampton G. A. Drew Alex. Petrunkevitch . A. Drew . A. Drew . S. Pratt . S. Pratt . J. Herrick L. Woodruff L. Woodruff . W. Rand Raymond Pearl J. H. Gerould Caswell Grave RrPrommoaod OF Vice-President 1910 1911 1912 1913 Secretary-Treasurer 1903 1904 1905 1906 1907 1908 1909 1910 1911 1912 1913 FORMER OFFICERS 553 CENTRAL BRANCH H. F. Nachtrieb R. H. Walcott C. M. Child C. M. Child Frank Smith F. R. Lillie C. E. McClung T. G. Lee T. G. Lee T. G. Lee Charles Zeleny H. V. Neal H. V. Neal W. C. Curtis W. C. Curtis Executive Committeemen EASTERN BRANCH F. R. Lillie T. H. Montgomery . C. Bumpus . S. Jennings A. Andrews R. Coe A. Drew . M. Metcalf H. Tennent G. Harrison . E. Jordan . E. McClung Ont y ZO 4m bby CENTRAL BRANCH George Lefevre T. G. Lee Herbert Osborn C. H. Eigenmann J. G. Needham S. J. Holmes W. A. Locy C. M. Child R. H. Walcott W. C. Curtis Oscar Riddle H. B. Ward Chauncey Juday H. W. Norris C. E. McCluug H. F. Nachtrieb AMERICAN Society oF ZoGLoGists (AMALGAMATED) President 1914. C. E. McClung 1915. W. A. Locy 1916. D. H. Tennent Vice-President M. F. Guyer W. E. Ritter Charles Zeleny Executive Commitieemen H. E. Jordan H. F. Nachtrieb H. V. Wilson 554 AMERICAN SOCIETY OF ZOOLOGISTS LIST OF PLACES OF MEETING AMERICAN MoRPHOLOGICAL SOCIETY 1890—Boston 1894—Baltimore 1899—New Haven 1891—Philadelphia 1895—Philadelphia 1900—Baltimore 1892—Princeton 1896— Boston 1901—Chicago 1893—New Haven 1897—Ithaca 1902—W ashington 1898—New York CENTRAL NATURALISTS 1899—Chicago 1900—Chicago Socirry or AMERICAN ZOGLOGISTS 1901—Chicago 1902—Washington AMERICAN SOCIETY OF ZOOLOGISTS EASTERN BRANCH JOINT MEETINGS CENTRAL BRANCH 1903—Philadelphia 1905—Ann Arbor 1903—St. Louis 1904— Philadelphia 1908—Baltimore 1905—(Mch.) Chicago 1906—New York 1911—Princeton 1907—(Mch.) Madison 1907—New Haven 1912—Cleveland * 1907—Chicago 1909—Boston 1913—Philadelphia 1910—(Apr.) Iowa City 1910—Ithaca 1910—Minneapolis 1912—(Apr.) Urbana AMERICAN Society or ZOOLOGISTS (AMALGAMATED) 1914. Philadelphia 1915. Columbus 1916. New York AMERICAN SOCIETY OF ZOOLOGISTS OFFICERS AND LIST OF MEMBERS OF THE SOCIETY Officers OR ARLCLATE Lc rl cial: > 5's 5.0 SRT aie ak dcop eaccnee es Maynarp Mayo Mrrtcaur RCE OSRGTE Toes «soc g ons chan draco land sek teenies CHARLES ZELENY MPU UrAN Me CMMMLEMEVOT .. . . « « «init.d spe Oa ie kx ke he nee acai te CASWELL GRAVE Executive Committee GEORGE LEFEVRE L. J. Cons A. F. SHULL R. P. BrazEtow H. V. WILtson HONORARY MEMBER James Viscount Bryce, Hindleap, Forest Row, Sussex, England. MEMBERS Aspott, JAMES FrANcIs, A.B., A.M. (Leland Stanford), Ph.D. (Chicago), Pro- fessor of Zodlogy, Washington University, St. Louis, Mo. ACKERT, JAMES Epwarp, A.B., A.M., Ph.D. (University of Illinois), Assistant Professor of Zoélogy, Kansas State Agricultural College, Manhattan, Kan. ALLEE, WARDER CLypDk, S.B. (Earlham College), S.M. (Chicago), Ph.D. (Chi- cago), Professor of Biology, Lake Forest College, Lake Forest, Ill. ALLEN, BENNET MILLs, Ph.B. (De Pauw), Ph.D. (Chicago), Professor of Zodlogy, University of Kansas, Lawrence, Kan. Auten, Ezra, A.M., Ph.D. (University of Pennsylvania), Professor of Biology, Philadelphia School of Pedagogy, 12th above Spring Garden, Philadelphia, Pa. ANDREWS, EtHan ALLEN, Ph.B. (Yale), Ph.D. (Johns Hopkins), Life Member. Professor of Zodlogy, Johns Hopkins University, Baltimore, Md. Arey, LESLIE BRAINERD, Ph.D. (Harvard), Instructor in Anatomy, North- western University Medical School, 2431 Dearborn Street, Chicago, Ill. BAITSELL, GEORGE ALFRED, B.S. (Central College, Iowa), M.A., Ph.D. (Yale), Instructor in Biology in Yale University, Osborn Zoélogical Laboratory, Yale Station, New Haven, Conn. Banta, ArTHUR Maneun, A.B., A.M. (Indiana), Ph. D. (Harvard), Resident Investigator, Station for Experimental Evolution, Carnegie Institution, Cold Spring Harbor, Long Island, N. Y. BARDEEN, CHARLES RussEuL, A.B. (Harvard), M.D. (Johns Hopkins), Profes- sor of Anatomy and Dean of the College of Medicine, University of Wis- consin, Madison, Wis. 555 THE ANATOMICAL RECORD, VOL. 11, No. 6 556 AMERICAN SOCIETY OF ZOOLOGISTS Barker, FRANKLIN D., A.B., A.M. (Ottawa University, Kansas), Ph D. (Ne- braska), Professor of Medical Zoédlogy and Parasitology, University of N:braska, Station A, Lincoln, Neb. Barrows, Wittram Martin, B.S. (Michigan Agricultural College), 8.B., S.M. (in Biology) (Harvard), 371 Harvard Street, Cambridge, Mass. BarTELMEz, GrEorGE W., Ph.D. (Chicago), Assistant Professor of Anatomy, University of Chicago, Chicago, Ill. Basset, GarpNer CuEeney, Ph.D. (Johns Hopkins), University of Pittsburgh, Pittsburgh, Pa. BAUMGARTNER, Wixi1am Jacos, A.B., A.M. (Kansas), Associate Professor of Zoblogy, University of Kansas, 1209 Ohio Street, Lawrence, Kan. BeckwitH, Cora Jipson, B.S. (Michigan), M.A., Ph.D. (Columbia), Associate Professor of Zodlogy, Vassar College, Poughkeepsie, N. Y. Bicetow, Maurice Aupueus, B.S. (Ohio Wesleyan), M.S. (Northwestern), Ph.D. (Harvard), Professor of Biology, Teachers’ College, 525 West 120th Street, New York City. BiceLow, Rosert Payne, 8.B. (Harvard), Ph.D. (Johns Hopkins), Associate Professor of Zodlogy and Parasitology, Massachusetts Institute of Technology, Cambridge, Mass. Bryrorp, Raymonp, B.S. (Earlham), 8.M. (Chicago), Ph.D. (Johns Hopkins), Professor of Zodlogy at Earlham College, Earlham College, Richmond, Ind., Earlham P. O. Biackman, Mavutssy Wiuiett, A.B., A.M. (Kansas), Ph.D. (Harvard), Pro- fessor of Forest Entomology and Head of Department of Forest Zodlogy, New York State College of Forestry, at Syracuse University, 216 Stratford Street, Syracuse, N. Y. 7 Bortna, Atice Mippieton, A.B., A.M., Ph.D. (Bryn Mawr), Associate Profes- sor of Zoédlogy, University of Maine, Orono, Maine. Bruner, Henry Lang, A.B. (Abingdon), Ph.D. (Freiburg), Professor of Biology, Butler College, 324 South Ritter Avenue, Indianapolis, Ind. Bupineton, Ropert ALLYN, B.A., M.A. (Williams), Professor of Zodlogy, Ober- lin College, Oberlin, Ohio. Bumpus, Hermon Carey, Ph.D., LL.D. (Clark), Ph.B., D.Sc. (Brown), D.Sc. (Tufts) President of Tufts College, Tufts College, Mass. Burrows, Montrose T., A.B. (Kansas), M.D. (Johns Hopkins), Acting Resi- dent Pathologist, Johns Hopkins Hospital, Baltimore, Md. Byrnes, Estuer F., Ph.D., 193 Jefferson Avenue, Brooklyn, N. Y. Cauxtns, Gary N., B.S. (Mass. Inst. Tech.), Ph.D., (Columbia) Professor of Protozoblogy, Columbia University, New York City. Catvert, Pattie Powrett, Ph.D. (Pennsylvania), Professor of Zoélogy, Univer- sity of Pennsylvania, Zoélogical Laboratory, Philadelphia, Pa. Carotuers, E. Eveanor, A.B., A.M., Ph.D., Zodlogical Building, University of Pennsylvania, Philadelphia, Pa. CarPENTER, Freperic Watton, B.S. (New York Univ.), A.M., Ph.D., (Har- vard), Professor of Biology, Trinity College, Hartford, Conn. Cary, Lewis R., B.S., M.S. (Maine), M.A., Ph.D. (Princeton), Asst. Prof. of Biology, Princeton University, Princeton, N. J. MEMBERS 557 Casteet, Dana Brackenrincr, A.B. (Allegheny), A.M. (Ohio Wesleyan), Ph.D. (University of Pennsylvania), Associate Professor of Zoédlogy, Uni- versily of Texas, Austin, Texas. Castie, Wiuttam E., A.B. (Denison), A.M., Ph.D. (Harvard), Professor of Zodlogy in Harvard University, Payson Road, Belmont, Mass. CHAMBERLIN, Raupn V., B.S. (Utah), Ph.D. (Cornell), Curator of Arachnida, Myriopoda and handiiin: ‘Sue. Comp. Zoélogy, Harvard University, Museum Comp. Zodlogy, Cambridge, Mass. CuHamBeErs, Ropert, Jr., A.M. (Queen’s Univ., Can.), Ph.D. (Munich), Instruc- tor in Anatomy, Cornell Medical College, 28th Street and First Avenue, New York City. Cuester, WAYLAND Moraan, A.B., A.M., (Colgate Univ.), Professor of Biology, Colgate University, Hamilton, N. Y. CuiLp, CHARLES MANNING, Ph.B., M.S. (Wesleyan), Ph.D. (Leipzig), Associate Professor of Zodlogy, Hull Zodlogical Laboratory, University of Chicago, Chicago, Ill. CuHuURCHILL, Epwarp Perry, A.B. (Iowa), Ph.D. (Johns Hopkins), Assistant U.S. Bureau of Fisheries, 317 Marshall Street, Hampton, Va. Crapp, Cornetia Marta, Ph.B. (Syracuse), Ph.D. (Chicago), Professor of Zoél- ogy, Mount Holyoke College, South Hadley, Mass. Cxiark, Howarp Watton, A.B., A.M. (Indiana), Scientific Assistant United States Bureau of Fisheries, United States Biological Station, Fairport, Iowa. Cor, Westrey R., Ph.D. (Yale), Professor of Biology, Yale University, New Haven, Conn. Cocuitt, Grorce E., A.B., Ph.D. (Brown), Professor of Anatomy, University of Kansas, R. F. D. 9, Lawrence, Kan. Coir, Leon J., A.B. (Michigan), Ph.D. (Harvard), Professor of Experimental Breeding, College of Agriculture, University of Wisconsin, Madison, Wis. Cotton, Haroutp Seuuers, B.S., M.A., Ph.D. (Pennsylvania), Assistant in Zodlogy, University of Pennsylvania), Philadelphia, Pa. Conepon, Epaar Davipson, A.B., A.M. (Syracuse), Ph.D. (Harvard), Leland Stanford Jr. University, 330 Coleridge Avenue, Palo Alto, Cal. ConkKLIN, Epwin Grant, B.S., A.B., A.M., Se.D. (Hon.) (Ohio Wesleyan), Ph.D. (Johns Hopkins), Sc.D. (Hon.) (Pennsylvania), Professor of Biology, Princeton University, Princeton, N. J. Coox, Marcaret Harris, B.S., Ph.D. (Pennsylvania), 122 West Linn Street, Bellefonte, Pa. CopeLanpD, Manton, S.B., S.M., Ph.D. (Harvard), Professor of Biology, Bow- doin College, Pierniick: Sai: Cort, Witt1amM Watrter, A.B. (Colorado College), M.A., Ph.D. (University of Illinois), Assistant Professor of Zoédlogy, University of California, Depart- ment of Zoélogy, University of California, Berkeley, Calif. _ Cowtgs, R. P., B.A. (Stanford), Ph.D. (Johns Hopkins), Professor of Zodélogy, University of the Philippines, Manila, Philippine Islands. Crampton, Guy Cuester, B.A., (Princeton) M.A. (Cornell), Ph.D. (Univ. of Berlin), Associate Professor of Entomology, Massachusetts Agricultural College, Care of Department of Entomology, Amherst, Mass. 558 AMERICAN SOCIETY OF ZOOLOGISTS Crampton, Henry Epwarp, A.B., Ph.D. (Columbia), Professor of Zodlogy, Barnard College, Columbia University; Curator of Invertebrate Zodlogy, American Museum of Natural History, New York City. Crozier, W1tu1aAM Joun, B.S. (College of the City of New York), A.M., Ph.D. (Harvard). Resident Naturalist, Bermuda Biological Station for Research; Sheldon Fellow, Harvard University. Agar’s Island, Bermuda. Curtis, Maynie Rost, A.B., A.M. Ph.D. (Michigan), Assistant Biologist, Maine Agricultural Experiment Station, Orono, Maine. Curtis, WINTERTON Conway, A.B., A.M. (Williams), Ph.D. (Johns Hopkins), Professor of Zodlogy, University of Missouri, 208 Hicks Avenue, Columbia, Mo. DauucGREN, Unric, A.B., M.S. (Princeton), Professor of Biology, Princeton University, 204 Guyot Hall, Princeton, N. J. DanIEL, J(OHN) F(RANKLIN), S.B. (University of Chicago), Ph.D. (Johns Hop- kins), Assistant Professor of Zodlogy, University of California, 1421 Haw- thorn Terrace, Berkeley, Cal. DaveNnporRT, CHARLES BENEDICT, Ph.D. (Harvard), Director of Department of Experimental Evolution, Carnegie Institution of Washington, Cold Spring Harbor, Long island, N. Y. DaveNPorT, GERTRUDE Crotty, B.S. (University of Kansas), Cold Spring Harbor, Long Island, N. Y. Davis, Herspert Spencer, Ph.B. (Wesleyan), Ph.D. (Harvard), Professor of Zodlogy, University of Florida, Gainesville, Fla. Day, Epwarp Carro.u, A.B. (Hamilton), A.M., Ph.D. (Harvard), Dalton Hall, Bryn Mawr College, Bryn Mawr, Pa. Dean, Basurorp, A.B. (College of City of New York), A.M., Ph.D. (Colum- bia), Life Member, Professor of Vertebrate Zodlogy, Columbia University; Curator of Fishes and Reptiles, Amerzcan Museum Natural History, Riverdale- on-Hudson, New York. DETLEFSEN, Joun A., A.B. (Dartmouth), A.M., Sc.D. (Harvard), Assistant Professor of Genetics, University of Illinois, College of Agriculture, 916 West Nevada Avenue, Urbana, Ill. : Dopps, GipEon S., B.A., M.A. (Colorado), Ph.D. (Pennsylvania), Assistant Professor of Zodlogy, University of Missouri, Columbia, Mo. Do.tey, Jr., Witt1aAm Lex, A.B., A.M. (Randolph-Macon), Ph.D. (Johns Hop- kins), Professor of Biology, Randolph-Macon College, Ashland, Va. Drew, Gitman A., Ph.D. (Johns Hopkins), Assistant Director, Marine Bio- logical Laboratory, Woods Hole, Mass. Epmonpson, Cuartes Howarp, Ph.B., M.S., Ph.D. (Iowa University), Assist- ant Professor of Zoédlogy, University of Oregon, Eugene, Ore. Epwarps, CuHarutes Lincoun, B.S. (Lombard and Indiana), A.M. (Indiana), Ph.D. (University of Leipzig), Director, Dept. Nature Study, Los Angeles City Schools, 1032 West 39th Place, Los Angeles, Cal. EIGENMANN, Cari, H., Ph.D., A.M., A.B. (Indiana), Professor of Zodlogy; Dean of the Graduate School, Indiana University, Bloomington, Indiana. Extrop, Morron Joun, B.A., M.A., M.S. (Simpson), Ph.D. (Ill. Wes. Univ.), Professor of Biology, University of Montana, Missoula, Mont. MEMBERS 559 Enpers, Howarp Epwin, B.S. (Lebanon Valley College), M.S., B.S. (Michi- gan), Ph.D. (Johns Hopkins University), Associate Professor of Zoélogy and in charge of Biology, Purdue University, Summer School Staff, Dept. Zodlogy, Indiana University, 107 Fowler Avenue, West Lafayette, Ind. ErpMANN, Ruopa, Ph.D. (Munich), Lecturer in Biology, Yale University and Associate, Rockefeller Institute; Yale University, New Haven, Conn. Eyciesuymer, AtBert C., B.S. (Michigan), Ph.D. (Chicago), M.D. (St. Louis), Professor and Head of Dept. of Anatomy, University of Illinois; Univer- sity of Illinois Medical College, Honore and Congress Streets, Chicago, Ill. Fasten, Naruan, B.S. (College of City of New York), Ph.D. (Wisconsin), In- structor of Zodlogy, Science Hall, University of Washington, Seattle, Wash. Ferris, Harry Burr, B.A., M.D. (Yale), E. K. Hunt, Professor of Anatomy, Medical Department, Yale University, 395 St. Ronan, New Haven, Conn. Foor, KatuHarine, 955 Park Avenue, New York City. Fox, Henry, B.S., M.A., Ph.D. (Pennsylvania), Entomological Laboratory, Clarkesville, Tenn. GaGe, Stwon Henry, B.S. (Cornell), Emeritus Professor of Histology and Em- bryology, Cornell University, 4 South Avenue, Ithaca, N. Y. Gatitoway, THomas W., A.B., A.M., Ph.D. (Cumberland), A.M. (Harvard), Litt. D. (Missouri Valley) Professor Biology, Beloit College, Beloit, Wis. Garman, Harrison, Head of Division of Entomology and Botany, Kentucky Agricultural Experiment Station; State Entomologist, Lexington, Ky. Geer, Witson, B.S. (University of S. C.), Ph.D. (University of California), Pro- fessor of Biology, Emery University, Oxford, Ga. GerouLp, Joun H., Litt.B. (Dartmouth), A.B., A.M., Ph.D. (Harvard), As- sociate Professor of Biology, Dartmouth College, Hanover, N. H. Guaser, Orro Cuarues, A.B., Ph.D. (Johns Hopkins), Junior Professor of Zoology, University of Michigan, Ann Arbor, Mich. Gouprarp, A. J., B.S. (College City of New York), Ph.D. (Columbia), Professor of Biology, College of City of New York, 251 West 112th Street, New York City. Gotpscumipt, Ricuarp B., Ph.D. (Heidelberg), In charge of the Department of Genetics, Kaiser Wilhelm Institut fiir Biologie, Daalem bei Berlin, Ger- many (Present address, Zoélogical Laboratory, Yale University, New Haven, Conn.). Goopate, Husert Dana, Ph.D. (Columbia), Research Biologist, Massachusetts Agricultural Experiment Station, North Amherst, Mass. Goopricu, Husert Baker, B.S. (Amherst), M.A., Ph.D. (Columbia), Instructor in Zodlogy, Wesleyan University, Middletown, Conn. GRAHAM, JoHN Youna, Ph.D. (Munich), Professor of Biology, University of Alabama, University, Ala. Grave, Bensamrn H., B.S. (Earlham), M.S. (Carleton), Ph.D. (Johns Hopkins), Professor of Biology, Knox College, Galesburg, Ill. Grave, CasweELt, B.S. (Earlham College), Ph.D. (Johns Hopkins), Associate Professor of Zodlogy, Johns Hopkins University, Baltimore, Md. Grecory, Emity Ray, A.B. (Wellesley), A.M. (Pennsylvania), Ph.D. (Chicago), Sweet Briar College, Sweet Briar, Va. 560 AMERICAN SOCIETY OF ZOOLOGISTS Grecory, Louise H., A.B. (Vassar), A.M., Ph.D. (Columbia), Instructor in Zoology, Barnard College, New York City. . Grirrin, LAWRENCE Epmonps, A.B., Ph.B. (Hamline), Ph.D. (Johns Hopkins), Professor of Zodlogy, University of Pittsburgh, Pittsburgh, Pa. Gross, AtFrrep O., A.B., (Illinois), Ph.D. (Harvard), Assistant Professor of Zodlogy, Bowdoin College, Brunswick, Maine. Gupacer, E. W., B.S., M.S., (Nashville), Ph.D. (Johns Hopkins), Professor of Biology, State Normal School, Greensboro, N. C. Guticx, Appison, A.B. (Oberlin), A.M. (Harvard), Ph.D. (Wiirzburg, Germany), Assistant Professor in Physiology, University of Missouri, Columbia, Mo. Guyer, Micuaen F., B.S. (Chicago), A.M. (Nebraska), Ph.D. (Chicagv). nO: fessor of Zoslony. University of Wisconsin, Madison, Wis. Hatt, Maurice Crowruer, 8.B., M.A., Ph.D., D.V.M., Parasitologist, Re- search Laboratory, Parke, Des & Go. Dees Mich. HaMAKER, JOHN Irvin, A.B. (Kansas), A.B., A.M., Ph.D. (Harvard), Professor of Biology, Randolph-Macon Woman’s College, 12 Princeton Street, Lynch- burg, Va. Haraitt, CHARLES W., Ph.D. (Ohio University), Professor of Zodlogy, Syracuse University, Syracuse, N. Y. Harcirt, GrorGe Tuomas, Ph.B. (Syracuse), A.M. (Nebraska), Ph.D. (Har- vard), Associate Professor of Zodlogy, Syracuse University, 909 Walnut Avenue, Syracuse, N. Y. Harmon, Mary Tueresa, A.B., M.A., Ph.D. (Indiana), Assistant Professor of Zodlogy, Kansas State Agricultural College, Manhattan, Kan. Harrer, Evcenrt Howarp, A.B. (Oberlin), A.M. (Harvard), Ph.D. (Chicago), Bedford, Va. Harrison, Ross GRANVILLE, Ph.D. (Johns Hopkins), M.D. (Bonn), Bronson Professor of Comparative Anatomy, Yale University, 142 Huntington Street, New Haven, Conn. Hart, Cuarues A., Systematic Entomologists, Illinois State Laboratory of Natural History, University of Illinois, Urbana, Ill. Hartman, Cart G., B.A., M.A., Ph.D. (Texas), Adj. Prof. of Zodlogy, Univer- sity of Texas, 1908 University Avenue, Austin, Tez. Heatu, Haroup, A.B. (Ohio Wesleyan), Ph.D. (Pennsylvania), Professor of Invertebrate Zoélogy, Leland Stanford University, 231 Walnut Street, Pacific Grove, Cal. HEeGNER, Rosert W., B.S., M.S.,(Chicago), Ph.D. (Wisconsin), Assistant Pro- fessor of Zodlogy, University of Michigan, 1430 Hill Street, Ann Arbor, Mich. Hertprunn, L.V., Ph.D. (Chicago), University of Illinois Medical School, Congress and Honore Streets, Chicago, Il. HENCHMAN, ANNIE P., Box 84, Jaffrey, N. H. HensHaw, SAMuEL, Life Member, Directorof Museum of Comparative Zodélogy, 8 Fayerweather Street, Cambridge, Mass. Herrick, CHARLES Jupson, Ph.D. (Columbia), Professor of Neurology, Anatomi- cal Laboratory, University of Chicago, Chicago, Ill. Herrick, Francis Hospart, A.B. (Dartmouth), Ph.D. (Johns Hopkins), Se.D. (Pittsburgh), Professor of Biology, Western Reserve University, Adelbert College, Cleveland, Ohio. MEMBERS 561 Hitron, Wiiuram Arwoop, B.S., Ph.D. (Cornell), Professor Zoédlogy, Pomona College, Claremont, Cal.; Director Laguna Marine Laboratory; Editor Journal of Entomology and Zoélogy, Claremont, Cal. Hoar, Mttprep Aspro, A.B. (Goucher), A.M., Ph.D. (Columbia), Instructor in Zodlogy, Indiana University, Bloomington, Ind. Hoaur, Mary Jane, A.B. (Goucher), Ph.D. (Wiirzburg), Instructor in Zodlogy, Wellesley College, Wellesley, Mass. Houtmes, Samueu J., B.S., M.S. (California), Ph.D. (Chicago), Associate Pro- fessor of Zodlogy, University of California, Berkeley, Cal. Hooker, Davenport, B.A., M.A., Ph.D. (Yale), Assistant Professor of Anatomy, Yale University, School of Medicine, 846 Orange Street, New Haven, Conn. Houser, Gitspert Loaan, B.S., M.S. (lowa), Ph.D. (Johns Hopkins), Professor of Animal Biology and Director of the Laboratories of Animal Biology, State University of Iowa, Iowa City, Towa. Howarp, Arruur D., B.S. (Amherst), M.S. (Northwestern), Ph.D. (Harvard), Scientific Assistant, United States Bureau of Fisheries, Fairport Biological Laboratory, United States Biological Laboratory, Fairport, Iowa. HuntsMAN, ARCHIBALD GowANLock, B.A., M.D. (Toronto) Lecturer in Biology, Biology Department, University of Toronto, Toronto, Canada. Hussaxor, Louts, B.S. (City College of New York), Ph.D. (Columbia), Curator of Ichthyology, American Museum of Natural History, 77th Street and Cen- tral Park West, New York City. Huxtry, JuLIaAN Soretu, B.A. (Oxford), Assistant Professor of Biology, Rice Institute, Houston, Tex. Hyper, Roscor Raymonp, A.B., A.M. (Indiana), Ph.D. (Columbia), Assistant Professor of Zodlogy and Physiology, Indiana State Normal School, 636 Chestnut Street, Terre Haute, Ind. Issen, Heman Lawritz, B.S., M.S., Ph.D. (Wisconsin), Assistant in Experi- mental Breeding, University of Wisconsin, Madison, Wis. IsELy, FrepericK B., B.S. (Fairmount), M.S. (Chicago), Professor of Biology, Central College, Fayette, Mo. Jacops, MERKEL Henry, A.B., Ph.D. (Pennsylvania), Assistant Professor of Zodlogy, University of Pennsylvania, Philadelphia, Pa. JENNINGS, HerBert S., B.S. (Michigan), A.M., Ph.D. (Harvard), LL.D. (Clark), Henry Walters Professor of Zodlogy and Director of the Zoélogical Labora- : tory, the Johns Hopkins University, Baltimore, Md. JOHANNSEN, Oskar Auausrus, B.S. (Illinois), A.M. (Cornell), Ph.D. (Cornell), Professor of Biology, Cornell University, College of Agriculture, Ithaca, N. Y. JOHNSTON, JoHN B., Ph.D. (Michigan), Professor Comparative Neurology, University of Minnesota, Minneapolis, Minn. JONES, ORREN Luoyp, B.S., M.S., Ph.D. (Wisconsin), Associate Professor, Ani- mal Husbandry, Jowa State College, Ames, Iowa. JorDAN, Harvey Ernest, AB., AM. (Lehigh), Ph.D. (Princeton), Professor of Histology and Embryology, University of Virginia, University, Va. Jupay, Cuauncey, A.B. and A.M. (Indiana), Biologist, Wisconsin Geological and Natural History Survey; Lecturer in Zodlogy, University of Wisconsin, Madison, Wis. 562 AMERICAN SOCIETY OF ZOOLOGISTS KampmMetrer, Orro F., Ph.D., Instructor in Embryology and Comparative Anat- omy, School of Medicine, University of Pittsburgh, Pittsburgh, Pa. Kewuicotr, Wm. E., Ph.B. (Ohio State University), Ph.D. (Columbia), Pro- fessor of Biology, Goucher College, Baltimore, Md. Kepner, WitiiamM Auuison, A.B., A.M. (Franklin and Marshall College, Lan- easter, Pa.), Ph.D. (Virginia), Associate Professor of Biology, University of Virginia, University, Va. Kincaip, Trevor, M.S., Professor of Zoélogy, University of Washington, Seattle, Wash. Kina, Heten Dean, A.B. (Vassar), A.M., Ph.D. (Bryn Mawr), Assistant Pro- fessor of Embryology, The Wistar Institute of Anatomy and Biology, The. Wistar Institute, Thirty-sixth and Woodland Avenue, West Philadelphia, Pa. Kinessury, BENJAMIN FREEMAN, Ph.D. (Cornell), M.D. (Freiburg), Professor of Histology and Embryology, Cornell University, 2 South Avenue, Ithaca, NY: KINGSLEY, JOHN StTeruiinG, A.B. (Williams), Se.D. (Princeton), Professor of Zodlogy, University of Illinois, Urbana, IIl. Kirxuam, WILLIAM Barri, B.A., M.A., Ph.D. (Yale), Instructor in Biology, Sheffield Scientific School, Yale University, 103 Everit Street, New Haven, Conn. Kite, Greorce Lester, M.D. (Virginia), Ph.D. (Chicago), Hoods, Va. Knower, Henry McE., A.B., Ph.D. (Johns Hopkins), Professor of Anatomy, Department of Anatomy, University of Cincinnati, Cincinnati, Ohio. Koroip, Cuartes Atwoop, A.B., Sc.D. (Oberlin), M.A. (Harvard), Ph.D. (Harvard), Professor of Zodlogy, University of California, and Assistant Director Scripp’s Institution of Biological Research, Berkeley, Cal. KorNHAUSER, SIDNEY I., Ph.D., A.M. (Harvard), A.B., (Pittsburgh), Assistant Professor of Zoélogy, Northwestern University, 718 Clark Street, Evanston, Tl. KReECKER, Freperic H., A.B., Ph.D. (Princeton), A.M. (Cornell), Assistant Professor of Zoédlogy, Ohio State University, Columbus, Ohio. Kriss, Hersert Guy, A.B. (Oberlin), Ph.D. (Pennsylvania), B.A. (Union), Assistant in Zoédlogy, University of Pennsylvania, Philadelphia, Pa. KUNKEL, BEVERLY WauGH, Ph.B., Ph.D. (Yale), Professor of Zodlogy, Lafayette College, Easton, Pa. Kuntz, ALBert, Ph.D. (State University of Iowa), Associate Professor of Biology and Histology, St. Louis University School of Medicine, St. Louis, Mo. Lamsert, Avery E., B.S., Ph.D. (Dartmouth), Burr Professor of Natural His- tory, Middlebury College, Middlebury, Vt. Lanpacre, Francis Leroy, A.B. (Ohio), Ph.D. (Chicago) Professor of Anatomy, Ohio State University, Columbus, Ohio. Lane, Henry Hiaartns, Ph.B. (De Pauw), A.M. (Indiana), Ph.D. (Princeton), Professor of Zoédlogy, University of Oklahoma, Norman, Okla. La Rue, Greorce R., B.S. (Doane), A.M. (Nebraska), Ph.D. (Illinois), Assist- ant Professor of Zodlogy, University of Michigan, Ann Arbor, Mich. LasHuLey, Kart Spencer, A.B. (West Virginia), M.S.(Pittsburgh), Ph.D. (Johns Hopkins), Johnston Fellow, Johns Hopkins University, Baltimore, Md. MEMBERS 563 Laurens, Henry, A.B., A.M. (Charleston), Ph.D. (Harvard), Assistant Pro- fessor of Biology, Yale College, Osborn Zoélogical Laboratory, Yale Uni- versity, New Haven, Conn. Leg, Tuomas G., B.S., M.D. (Pennsylvania), Professor of Comparative Anatomy, University of Minnesota, Institute of Anatomy, Minneapolis, Minn. Lerevre, Georar, A.B., Ph.D. (Johns Jopkins), Professor of Zoélogy, Univer- sity of Missouri, Columbia, Mo. Litiin, Frank R., B.A. (Toronto), Ph.D. (Chicago), Professor of Embryology and Chairman of the Department of Zoélogy, University of Chicago; Direc- tor, Marine Biological Laboratory, Woods Hole, Mass. University of Chi- cago, Chicago, Ill. Linton, Epwin, A.B., 8.M. (Washington and Jefferson), Ph.D. (Yale), Professor of Biology, Washington and Jefferson College, 400 Fast Maiden Street, Wash- ington, Pa. Lirrie, C. C., A.B., §.D. (Harvard), Research Fellow, Cancer Commission of Harvard University, Boston, Mass. Locy, WiiurAmM ALBERT, Ph.D. (Chicago), Se.D. (Hon.) (Michigan), Professor of Zoélogy and Director of the Zoélogical Laboratory, Northwestern Univer- sity, Evanston, Ill. Lona, Josepn A., 8.B., A.M., Ph.D. (Harvard), Assistant Professor of Embryol- ogy, University of California, 1534 La Loma Avenue, Berkeley, Cal. Lonetey, WiiutaM H., M.A., Ph.D. (Yale), Professor of Botany, Goucher Col- lege, Baltimore, Md. Lunp, Etmer J., Ph.D. (Johns Hopkins University), Assistant Professor of Zodlogy, University of Minnesota, Minneapolis, Minn. Lutz, Frank E., A.B. (Haverford), A.M., Ph.D. (Chicago), Assistant Curator of Invertebrate Zodlogy, American Museum of Natural History, 77th Street and Central Park West, New York City. McCuiung, C. E., Ph.G., A.B., A.M., Ph.D. (Kansas), Professor of Zoédlogy and Director of the Zodlogical Laboratory, University of Pennsylvania, Phila- delphia, Pa. McCuvre, Cuartss F. W., A.B., M.A. (Princeton) D.Se. (Columbia), Pro- fessor of Comparative Anatomy, Princeton University, Princeton, N. J. MacCurpy, Hansrorp M., A.B. (Ohio Wesleyan), A.M., Ph.D. (Harvard), Professor of Biology, Alma College, 701 Center Street, Alma, Mich. MacDowe ti, EpwiIn Carueton, A.B. (Swarthmore), S.M. Zoél. (Harvard), S.D. (Harvard), Research Investigator, Station Experimental Evolution, Car- negie Institution of Washington, Cold Spring Harbor, Long Island, N. Y. MacGILiivrRay, ALEXANDER Dyer, Ph.D. (Cornell), Associate Professor Sys- tematic Entomology, University of Illineis. 603 West Michigan Avenue, Urbana, Ill. McGreoor, JAMES Howarp, B.S. (Ohio State University), A.M., Ph.D. (Colum- bia), Associate Professor of Zodlogy, Columbia University, New York City. McInpdoo, Norman Evucens, A.B., A.M. (Indiana), Ph.D. (Pennsylvania), Research Student on Senses of Honey Bee, Bureau of Entomology, Washing- ton; Ds CG: 564 AMERICAN SOCIETY OF ZOOLOGISTS Matt, FRANKLIN Payne, M.A., M.D., Sc.D. (Michigan), LL.D. (Wisconsin), Professor of Anatomy, Johns Hopkins University, Baltimore, Md. MarcHAND, GRACE B., .28 Mercer Street, Princeton, N. J. Mark, Epwarp L., A.B. (Michigan), Ph.D. (Leipzig), LL.D. (Michigan), LL.D. (Wisconsin), Hersey Professor of Anatomy and Director of the Zodlogical Laboratory, Harvard University, 109 Irving Street, Cambridge, Mass. MarsuHaLu, Ruts, B.S., M.S. (Wisconsin), Ph.D. (Nebraska), Lane Technical School, Chicago, Ill. MarsHALL, WILLIAM STANLEY, B.S. (Swarthmore), Ph.D. (Leipzig), Associate Professor Entomology, University of Wisconsin, 139 East Gilman Street, Madison, Wis. : Mast, Samuet Orrmar, B.S. (Michigan), Ph.D. (Harvard), M.Pd. (Michigan Yormal College), Associate Professor of Zodlogy, the Johns Hopkins Uni- versity, Baltimore, Md. Mayer, ALFRED GotpssporouGH, M.E. (Stevens Inst. Tech.), Se.D. (Harvard), Life Member, Director Department Marine Biology, Carnegie Institution of Washington, Lecturer in Biology, Princeton University, 276 Nassau Street, Princeton, N. JJ. Meap, Apert Davis, A.B. (Middlebury), A.M. (Brown), Ph.D. (Chicago), Se.D. (Pittsburgh), Professor of Biology, Brown University, 283 Wayland Avenue, Providence, R. I. Metcatr, Maynarp Mayo, A.B., D.Sc. (Oberlin), Ph.D. (Johns Hopkins), Oberlin, Ohio. Merz, Cuares W., B.A., Ph.D., Station for Experimental Evolution, Carnegie Institution of Washington, Cold Spring Harbor, Long Island, N. Y. Meyer, ArTHuR WiuutAM, B.S. (Wisconsin), M.D. (Johns Hopkins), Professor of Human Anatomy, Stanford University, 121 Waverley Street, Palo Alto, California. MippLETON, AuSTIN Raupu, A.B., Ph.D. (Johns Hopkins), Assistant Professor of Biology, University of Lousiville, Louisville, Ky. Moenxknuavs, WiiiiaM J., A.B. (Indiana), Ph.D. (Chicago), Professor of Physiol- ogy, Indiana University, 501 Fess Avenue, Bloomington, Ind. Mooptr, Roy Leg, A.B. (Kansas), Ph.D. (Chicago), Instructor in Anatomy, University of Illinois, Chicago, Congress and Honore Streets, Chicago, Ill. Moopy, Jutta ELeanor, B.S., M.A. (Mt. Holyoke), Ph.D. (Columbia), Instrue- tor in Zoélogy, Wellesley College, Wellesley, Mass. Moorr, J. Percy, Ph.D. (Pennsylvania), Life Member, Professor of Zodlogy, University of Pennsylvania, Philadelphia, Pa. Moraan, Ann Haven, A.B., Ph.D. (Cornell), Associate Professor of Zoélogy, Mt. Holyoke College, So. Hadley, Mass. Morean, Tuomas Hunt, B.S. (Kentucky), Ph.D. (Johns Hopkins), Professor of Experimental Zodlogy, Columbia University, New York City. Morauiis, Sererus, A.M. (Columbia), Ph.D. (Harvard), Creighton University Medical School, Omaha, Neb. Morritx, ALBRo Davin, B.S., M.S. (Dartmouth), Professor of Biology, Hamil- ton College, Clinton, Oneida County, N. Y. MEMBERS 565 Morrity, Cuaries V., A.M., Ph.D. (Columbia), Instructor in Anatomy, Cor- nell University Medical College, 28th Street and First Avenue, New York City. Mosuer, Epna, B.S. (Cornell), Ph.D. (llinois), Instructor in Entomology, University of Illinois, Natural History Building, Urbana, Ill. Mutuenrix, Rouurn Cirarken, A.B., A.M. (Wheaton), Ph.D. (Harvard), Professor of Biology, Lawrence College, 461 Washington Street, Appleton, Wis. Motter, Herman J., A.M., Ph.D., Instructor in Zoélogy, Rice Institute, Houston, Texas. Nasours, Rosert K., Ph.D. (Chicago), Professor of Zoédlogy, Kansas Agricul- tural College, Manhattan, Kan. Nacurries, Henry Francis, B.S. (Minnesota), Professor of Animal Biology and Head of the Department, Curator of the Zodlogical Museum, Univer- sity of Minnesota, Minneapolis, Minn. Nea, Hersert Vincent, A.B., A.M., Ph.D. (Harvard), Professor of Zodlogy, Tufts College, Tufts College, Mass. Netson, JAMES ALLEN, Ph.B. (Kenyon College), Ph.D. (Pennsylvania), Expert Bee Culture Investigations, Bureau of Entomology, United States Depart- ment of Agriculture, Washington, D. C. Newman, Horatio Hackett, B.A. (McMaster), Ph.D. (Chicago), Associate Professor of Zoélogy, and Dean in College of Science, University of Chicago, 5712 Dorchester Avenue, Chicago, Ill. Norris, Harry Waupo, A.B., A.M. (Grinnell), Professor of Zodédlogy, Grinnell College, Grinnell, Iowa. Nurtinc, CuHarLtes CLievenanp, M.A. (Blackburn), Professor and Head of Department of Zodlogy and Curator of Museum, State University of Iowa, 922 Washington Street, Iowa City, Iowa. Ossorn, Henry FarrFietp, A.B., Sc.D. (Princeton), LL.D. (Hon.) (Trinity, Princeton, Columbia), D.Sc. (Hon.) (Cambridge University), Ph.D. (Hon.) (University of Christiania, Upsala), Research Professor of Zoélogy, Colum- bia; President Board of Trustees, American Museum Natural History; Cura- tor Emeritus Dept. Vertebrate Paleontology, Vertebrate Paleontologist, United States Geological Survey. American Museum of Natural History, Seventy-seventh Street and Park West, New York City. Ossorn, Henry Lesxiiz, A.B. (Wesleyan), Ph.D. (Johns Hopkins), Professor of Biology, Hamline University, 1500 Hewitt Avenue, St. Paul, Minn. Osporn, Herpert, B.Sc., M.Sc. (Iowa State College), Professor of Zodlogy and Entomology, Ohio State University, Director Lake Laboratory, Director Ohio Biological Survey, Columbus, Ohio. Ossurn, Raymonp C., B.Sc., M.Sc., (Ohio State University), Ph.D. (Columbia), Professor of Biology, Connecticut College for Women, New London, Conn. PackarD, Cuarues, M.S., Ph.D., Instructor in Zoology, Columbia University, Schermerhorn Building, Columbia University, New York City. PAINTER, THEOPHILUS SHIcKEL, A.B. (Roanoke), A.M., Ph.D. (Yale), Adjunct Professor of Zodlogy, University of Texas, Austin, Texas. Parker, Georce Howarp, S.B., S.D. (Harvard), Professor of Zodlogy, Har- vard University, 16 Berkeley Street, Cambridge, Mass. Patcu, Enira M., B.S. (Minnesota), M.S. (Maine), Ph.D. (Cornell), Ento- mologist, Maine Agricultural Experiment Station, Orono, Maine. 566 AMERICAN SOCIETY OF ZOOLOGISTS Patrren, BrRapLEY Merritu, A.B. (Dartmouth), A.M., Ph.D. (Harvard), In- structor in Histology and Embryology Western Reserve Medical School, 1353 East Ninth Street, Cleveland, Ohio. Parren, WruitaM, B.S. (Harvard), M.A., Ph.D. (Leipzig), Professor of Zodélogy, Dartmouth College, Hanover, N. H. Patrerson, JonN Tuomas, B.S. (Wooster), Ph.D. (Chicago), Professor of Zodl- ogy, University of Texas, University Station, Austin, Texas. Payne, Fernanpus, A.B., A.M. (Indiana), Ph.D. (Columbia), Associate Pro- fessor of Zodlogy, Indiana University, Bloomington, Ind. Peart, Raymonp, A.B (Dartmouth), Ph.D. (Michigan), Biologist and Head of Department of Biology, Maine Agricultural Experiment Station, Orono, Maine. ; Pearse, ARTHUR SpEeRRY, B.S., A.M. (Nebraska), Ph.D. (Harvard), Associate Professor of Zoédlogy, University of Wisconsin, Madison, Wis. PEEBLES, FLORENCE, A.B. (Goucher), Ph.D. (Bryn Mawr), Newcomb College, New Orleans, La. Perkins, Henry F., A.B. (Vermont), Ph.D. (Johns Hopkins), Professor of Zoél- ogy, University of Vermont, 205 South Prospect Street, Burlington, Vt. PETRUNKEVITCH, ALEXANDER, Ph.D. (Freiburg), Assistant Professor of Zodl- ogy, Sheffield Scientific School, Zodlogical Laboratory, Yale University, New Haven, Conn. Puiuuirs, Everett FRANKLIN, A.B. (Allegheny), Ph.D. (Pennsylvania), In charge of Bee Culture Investigations, Bureau of Entomology, United States Department of Agriculture, Washington, D. C. PIERSOL, GEORGE ARTHUR, M.S. (Pennsylvania), Sc.D. (Pennsylvaina College), Professor of Anatomy, University of Pennsylvania, 4724 Chester Avenue, Philadelphia, Pa. Pike, Frank H., A.B. (Indiana), Ph.D. (Chicago), Assistant Professor of Physiology, Columbia University, 437 West 59th Street, New York City. Pratt, Henry SHERRING, A.B. (Michigan), A.M., Ph.D. (Leipzig), Professor of Biology, Haverford College, Haverford, Pa. Ranp, HERBERT WitpBur, A.B. (Allegheny, Harvard), A.M., Ph.D. (Harvard), Assistant Professor of Zodlogy, Harvard University; Museum of Comparative Zoélogy, Cambridge, Mass. RANDOLPH, Harriet, A.B. (Bryn Mawr), Ph.D. (Zurich), Life Member. 1310 South Forty-seventh Street, Philadelphia, Pa. Ransom, Brayton Howarp, B.Sc., M.A., Ph.D. (Nebraska), Chief, Zoological Division, Bureau of Animal Industry, United States Department ot Agri- culture, Bureau of Animal Industry, Washington, D. C. Reep, Hues Danie, B.S., Ph.D. (Cornell), Assistant Professor of Zodélogy, Cornell, 108 Brandon Place, Ithaca, N. Y. Reese, ALBERT Moorsg, A.B., Ph.D. (Johns Hopkins), Professor of Zodlogy, West Virginia University, Morgantown, W. Va. ReIGHARD, Jacop Ettswortu, Ph.B. (Michigan), Professor of Zoélogy; Director of Zodlogical Laboratory and Biological Station, University of Michigan, Ann Arbor, Mich. Reinke, Epwin Eustace, M.A. (Lehigh), Ph.D. (Princeton), Assistant Pro- fessor of Biology, Vanderbilt University, Nashville, Tenn. MEMBERS 567 Rice, Epwarp Loranvus, A.B. (Wesleyan), Ph.D. (Munich), Professor of Zoédlogy, Ohio Wesleyan University, Delaware, Ohio. Ricwarps, A., B.A. (Kansas), M.A. (Wisconsin), Ph.D. (Princeton), Wabash College, Crawfordsville, Ind. Rrppue, Oscar, A.B. (Indiana), Ph.D. (Chicago), Research Associate, Carnegie Institution of Washington, Cold Spring Harbor, Long Island, N. Y. Riney, Witu1amM Apert, B.S. (DePauw), Ph.D. (Cornell), Professor of Insect Morphology and Parasitology, Cornell University, Ithaca, N. Y. Rirrer, Wiui1aM E., A.B. (California), A.M., Ph.D. (Harvard), Director of Scripps Institution for Biological Research of the University of California, Professor of Zoédlogy, University of California, La Jolla, Cal. Rosertson, Auice, B.S., M.S., Ph.D. (California), Professor of Zodlogy, Welles- ley College, Wellesley, Mass. Ropertson, WriuiamM R. B., A.B. (Kansas), Ph.D. (Harvard), Assistant Pro- fessor of Zodlogy, University of Kansas, 1420 Ohio Street, Lawrence, Kans. Roaers, CHARLES GARDNER, A.B., A.M. (Syracuse), Ph.D. (California), Pro- fessor of Comparative Physiology, Oberlin College, Oberlin, Ohio. Rocers, Frep Terry, A.B., Ph.D. (Chicago), Assistant Professor of Zodlogy, Baylor University, Waco, Texas. RutHvEN, ALEXANDER G., B.S. (Morningside), Ph.D. (Michigan), Director, Museum of Zodlogy, Assistant Professor of Zodlogy, University of Michi- gan, Museum of Zoélogy, Ann Arbor, Mich. Scuarrrer, Asa Artuur, A.B. (Franklin and Marshall), Ph.D., (Johns Hopkins) Associate Professor of Zodlogy, University of Tennessee, Knoxville, Tenn. Scuiept, Ricuarp C. F., Ph.D. (Pennsylvania), Se.D. (Hon.) (Franklin and Marshall), Professor of Biology and Geology, Franklin and Marshall College, — 1043 Wheatland Avenue, Lancaster, Pa. Scott, Grorce G., A.B., A.M. (Williams), Ph.D. (Columbia), Assistant Profes- sor, Natural History, College of the City of New York, New York City. Scott, Joun W., A.B., A.M. (Missouri), Ph.D. (Chicago), Professor of Zoél- ogy, University of Wyoming, Laramie, Wy. Scorr, Wiii1am, Ph.D. (Indiana), Assistant Professor of Zoélogy, Indiana Uni- versity, Bloomington, Ind. SHEeLpon, Ratpew Epwarp, A.B., A.M. (Cornell), S.M. (Harvard), Ph.D. (Chi- cago), Assistant Professor of Anatomy and Director of Anatomical Labora- tories, University of Pittsburgh, School of Medicine, Pittsburgh, Pa. SHELForD, Victor Ernest, B.S., Ph.D. (Chicago), Assistant Professor of Zodl- ogy, University of Illinois, and Biologist of Illinois State Laboratory, 606 West Iowa Street, Urbana, Ill. SHepHerp, W. T., A.M., Ph.D., Professor of Zodlogy and Dean, Wasnestaurg College, Wargnestercy, ‘By. SHorey, Martian Lypia, A.M. Ph.B., (Brown), Ph.D. (Chicago), Hugenot Col- lege, Wellington, South Africa. SHutt, AARON Franxuin, A.B. (Michigan), Ph.D. (Columbia), Associate Pro- fessor of Zodlogy, University of Michigan, 520 Linden Street, Ann Arbor, Mich. SteeRFoos, CHar.es P., B.S. (Ohio State), Ph.D. (Johns Hopkins), Professor of Zodlogy, University of Minnesota, Minneapolis, Minn. 568 AMERICAN SOCIETY OF ZOOLOGISTS SmaLLtwoop, Wiiit1am Martin, Ph.D. (Harvard), Professor of Comparative Anatomy, Syracuse University, 525 Euclid Avenue, Syracuse, N. Y. Smita, Bertram GARNER, A.B. (Michigan), Ph.D. (Columbia), Assistant Pro- fessor of Zodlogy, Michigan State Normal College, 122 College Place, Ypsil- anti, Mich. Smits, Franx, Ph.B. (Hillsdale College), A.M. (Harvard), Professor of Zodl- ogy, University of Illinois, Urbana, Ill. Smita, Lucy Wricut, B.A. (Mt. Holyoke), M.A., Ph.D. (Cornell), Instructor in Zodlogy, Mt. Holyoke College, South Hadley, Mass. Smita, Sipney I., Ph.B., A.M. (Yale), Professor of Comparative Anatomy, Emeritus, Yale University, New Haven, Conn. SpanTuH, Reynowp A., Ph.D. (Harvard), Instructor in Biology, Yale University, Osborn Zoological Laboratory New Haven, Conn. Stites, Cartes W., A.M., Ph.D. (Leipzig), S.M., S.D. (Wesleyan), Life Mem- ber, Professor of Zodlogy, United States Public Health and Marine Hos- pital Service, Hygienic Laboratory. Twenty-fifth and E Streets, N. W., Washington, D. C. (October 1-May 1); Wilmington, N. C. (May-—October 1). STocKARD, CHARLES Rupert, B.S., M.S. (Mississippi Agricultural and Mechani- cal College), Ph.D. (Columbia), Professor of Anatomy, Cornell Univer- sity, Medical School, Cornell Medical College, First Avenue and Twenty- eighth Street, New York City. STROMSTEN, FRANK ALBERT, B.S., M.S. (Iowa), D.Sc. (Princeton), Assistant Professor of Animal Biology, State University of Iowa, 943 Iowa Avenue, Iowa City, Iowa. Strone, Oxtver §., A.B., A.M. (Princeton), Ph.D. (Columbia), Instructor in Anatomy, Columbia University, College of Physicians and Surgeons, 437 West Fifty-ninth Street, New York City. Srrone, Reusen Myron, A.B. (Oberlin), A.M., Ph.D. (Harvard), Vander- bilt University Medical School, Nashville, Tenn. SturTEVANT, ALFRED H., A.B., Ph.D. (Columbia), Cutting Fellow, Columbia University, New York City. Sumner, Francis B., B.S. (Minnesota), Ph.D. (Columbia), Biologist, Scripps Institution for Biological Research, La Jolla, Cal. Surrace, Frank M., A.B., A.M. (Ohio State), Ph.D. (Pennsylvania), Biologist, Maine Experiment Station, Orono, Maine. Swezy, Outve, B.S., M.S., Ph.D., (California), Associate in Zoélogy, Assistant Zodlogist, Scripps Institution for Biological Research, University of Cali- fornia, Hast Hall, University of California, Berkeley, Cal. TANNREUTHER, GrorcE W., A.B. (Manchester), A.M. (Antioch), Ph.D. (Chi- cago), Instructor in Zodlogy, University of Missouri, Columbia, Mo TasHIro, Surro, B.S., Ph.D. (Chicago), Instructor in Physiological Chemistry, University of Chicago, Hull Bro. Chemical Laboratory, Chicago, Ill. TENNENT, Davin H111, B.S. (Olivet), Ph.D. (Johns Hopkins), Professor of Biol- ogy, Bryn Mawr College, Bryn Mawr, Pa. THompson, CaroLIne Buruina, 8.B., Ph.D. (Pennsylvania), Associate Pro- fessor of Zodlogy, Wellesley College, Leighton Road, Wellesley, Mass. Torrey, Harry Beat, B.S., M.S. (California), Ph.D. (Columbia), Professor of Biology, Reed College, Portland, Ore. MEMBERS 569 Tower, Wittram Lawrence, 8.B. (Chicago), Assistant Professor of Zodlogy, University of Chicago, Chicago, Il. TREADWELL, AARON L., B.S., M.S. (Wesleyan), Ph.D. (Chicago), Professor of Biology, Vassar College, Poughkeepsie, N. Y. Tyzter, Joun Mason, Ph.D. (Hon.) (Colgate), Professor of Biology, Amherst College, Amherst, Mass. Van Cieave, Haruey Jones, B.S. (Knox College), M.S., Ph.D. (Illinois), Asso- ciate in Zoology, 300 Natural History Building, Urbana, Ill. Verritu, Appison F., 8.B. (Harvard), A.M. (Yale), Professor of Zoélogy, Emeri- tus, Yale University, New Haven, Conn. Waaner, Greorcr, M.A. (Michigan), Assistant Professor of Zodlogy, Univer- sity of Wisconsin, Biology Building, Madison, Wis. Waite, FrepericK CiayrTon, Litt.B. (Adelbert), A.M. (Western Reserve), A.M., Ph.D. (Harvard), Professor of Histology and Embryology, School of Medicine, Western Reserve University, 1353 East 9th Street, Cleveland, Ohio. Wattace, Loutse Barro, A.B. (Mount Holyoke), Ph.D. (Pennsylvania), Dean of Constantinople College, Constantinople, Turkey, South Hadley, Mass. Water, Herspert Evcenr, A.B. (Bates), A.M. (Brown), Ph.D. (Harvard), Assistant Professor of Biology, Brown University, Providence, R. I. Watton, Ler Barker, Ph.B. (Cornell), A.M. (Brown), Ph.D. (Cornell), Profes- sor of Biology, Kenyon College, Gambier, Ohio. Warp, Henry Batpwin, A.B. (Williams), A.M., Ph.D. (Harvard), Professor of Zoédlogy, University of Illinois, Urbana, Ill. Wetcn, Paut Smits, A.B. (James Millikin), A.M., Ph.D. (Illinois), Assistant Professor of Entomology, Kansas State Agricultural College, Manhattan, Kan. ‘ Wetts, Morris Miuter, B.S. (Chicago), Ph.D. (Illinois), Instructor Depart- ment of Zodlogy, University of Chicago, Chicago, Ill. Wenricu, Davip Henry, B.A., M.A., Ph.D., Instructor in Zoology, University of Pennsylvania, Zoological Laboratory, Philadelphia, Pa. WENTWoRTH, Epwarp N., M.S. (Iowa), Professor of Animal Husbandry, Kansas State Agricultural College, Manhattan, Kans. WERBER, Ernest I., Ph.D. (Vienna), Sessel Research Fellow, Yale University, Osborn Zoélogical Laboratory, New Haven, Conn. WHEELER, Witt1aM Morton, Ph.D. (Clark), Professor of Economic Entomology, Bussey Institution, Forest Hills, Boston, Mass. Wauitinc, Putneas W., A.B., M.S., Ph.D., Harrison Research Fellow, Univer- sity of Pennsylvania, Zoélogical Laboratory, Philadelphia, Pa. Wuitney, Davip Day, B.A. (Wesleyan), M.A., Ph.D. (Columbia), Department of Zoédlogy, University of Nebraska, Lincoln, Neb. Wieman, Harry Lewis, A.B., A.M. (Cincinnati), Ph.D. (Chicago), Associate Professor of Zodlogy, Head of Department, University of Cincinnati, Cin- cinnatt, Ohio. Witper, Harris Hawrsorne, A.B. (Amherst), Ph.D. (Freiburg), Professor of Zodlogy, Smith College, Northampton, Mass. Wiper, Inez WuippLe, Ph.B. (Brown), A.M. (Smith), Instructor in Zodlogy, Smith College, Northampton, Mass. 570 AMERICAN SOCIETY OF ZOOLOGISTS WitpMan, Epwarp E., B.S., M.S., Ph.D. (Pennsylvania), Head Department of Science, West Philadelphia High School-for Girls, 4331 Osage Avenue, Philadelphia, Pa. Wiarp, W. A., Ph.B. (Grinnell), A.M. (Tufts and Harvard), Ph.D. (Harvard), Professor of Histology and Embryology in Charge of Department, Univer- sity of Nebraska, College of Medicine, Omaha, Neb. Witurams, STEPHEN Rices, A.B., A.M. (Oberlin), A.M., Ph.D. (Harvard), Professor of Zodlogy and Geology, Miami University, 300 East Church Street, Oxford, Ohio. Witson, Cuarues Brancu, A.B., A.M. Sc.D. (Colby), Ph.D. (Johns Hopkins), Head Science Department, State Normal School, Westfield, Mass. Witson, Epmunp B., Ph.B. (Yale), Ph.D. (Johns Hopkins), LL.D. (Yale, Chi- cago, Hopkins), M.D. (Hon.) (Leipzig), Sc.D. (Cambridge), Da Costa Pro- fessor of Zodlogy, Columbia University, New York City. Witson, Henry Van Peters, A.B., Ph.D. (Johns Hopkins), Professor of Zodl- ogy, University of North Carolina, Chapel Hill, N. C. WopDsEDALEK, JERRY Epwarp, Ph.D., M.Ph., Ph.D. (Wisconsin), Professor of Zodlogy and Head of the Department of Zodlogy and Entomology, Uni- versity of Idaho, Moscow, Idaho. Wotcort, Ropert Henry, B.Sc., M.D. (Michigan), M.A. (Nebraska), Profes- sor and Head of the Department of Zoélogy, University of Nebraska, Lin- coln, Neb. Wooprurr, LoranpE Loss, A.B., A.M., Ph.D. (Columbia), M.A. (Yale), Pro- fessor of Biology, Yale University, Osborn Zoélogical Laboratory, New Haven, Conn. Wricut, Atpert Hazen, A.B., A.M., Ph.D. (Cornell), Assistant Professor of Systemic and Field Zodlogy, Cornell University, Cayuga Heights, Ithaca, Ney: Wricut, Sewatt G., 8.B. (Lombard), S.M. (Illinois), S.D. (Harvard), Senior in Animal Breeding Investigation, Animal Husbandry Division, Bureau of Animal Industry, Department of Agriculture, Washington, D. C. YerKeES, Ropert M., Ph.D. (Harvard), Assistant Professor of Comparative Psychology, Harvard University, Emerson Hall, Cambridge, Mass. Youna, Rosert T., B.S. (Pennsylvania), Ph.D. (Nebraska), Professor of Zodl- ogy, University of North Dakota, University, N. D. ZmLENY, Cuarues, B.S., M.S. (Minnesota), Ph.D. (Chicago), Professor of Zodlogy, University of Illinois, Urbana, Ill. Peta i, a eee eet = ee +m Roe / ih: TNE He, : en of ie Ve a a ‘oy eee tie ihe ed fe Wass dt Neviadein, SPs a eu eNhs | fara ho ee “a a ee Hae Cage hy Ate, coy Wesas ' ‘ rere Soa, Fin Age we Pes yee + ise i Wn he Ra Tey ae wo Lote Dap ess.: Suite SIR ey eS f ee Teta Pye iy At, PRD AE, BAER ARiuae “8 Mic ohn: Ks Tek 1 tipingtak ik et CARA, AE Wade A eile aan ene Fi ire, Ficgell? ic ciaee V0 schon RS gia aaoee a jen kore ee gt Pant Sheeley Sines WS Pee a i ; PEt Peta,

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