ont "3 neste: iy on ak ¥ pk ic ; re a ie Peete . co phe > = Cs pats a ete aie ee Ake, stats tata ne x. reekekeN Se % - far gt tof eketicks oat, pat > 2 e XY aa a9 ss apes Wr Pi ete 2 os sie PLESE A. Stitat: wy pea eh SF t2% = >, a: ~ re ~~ ye: i PPS Pin a, Xb" co ody Vi $7Bi¥ 7 hd be 44 on | ) i is 5 oe ae : €°. Pe (tm 5 * ad! + ~ a5% :. ies . Prat eS oe Be tuty $18 t 5 j THE JOURNAL OF EXPERIMENTAL ZOOLOGY EDITED BY Wiuuiam E. Caste Jacques LoEB Harvard University The Rockefeller Institute Epmunp B. WILSON Columbia University Epwin G. CoNKLIN Princeton University Tuomas H. Moraan CHARLES B. DAVENPORT ; Columbia University Carnegie Institution H g GrorGE H. PARKER ERBERT S. JENNINGS Harvard University Johns Hopkins University RAYMOND PEARL FRANK R. LILLIE Maine Agricultural University of Chicago Experiment Station and Ross G. HARRISON, Yale University Managing Editor VOLUME 23 1917 THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA. COMPOSED AND PRINTED AT THE WAVERLY PRESS By rae Witurams & WILKINS COMPANY Bavrimorg, Mp., U.S.A. CONTENTS No. 1. JANUARY Harutey N. Goutp. Studies on sex in the hermaphrodite molluse Crepidula plana. I. History of the sexual cycle. Eighty-five figures........... Henry LAvuRENS AND J. W. WiuitaAms. Photomechanical changes in the retina of normal and transplanted eyes of Amblystoma larvae. Three TORGei eure srAG” ONS: Plate. cd. amen. se sian es ecie Seok eala feet aryl FRANKLIN PEARCE REAGAN. The role of the auditory sensory epithelium in the formation of the stapedial plate. Ten figures.................... / Epwin Carteton MacDowe tu. Bristle inheritance in Drosophila. II. Selection epee me noe ses, vsses «i 04 ches SRA noe oe coves tice eee as JoHun N. Lowe. The action of various pharmacological and other chemical agents on the chromatophores of the brook trout Salvelinus fontinalis Mitehill: . Lhree text figures and one plate... ...052.5..0.025.8s%enese-s Henry Laurens. The reactions of the melanophores of? Amblystoma tigrinum larvae to light and darkness.. Six figures.....................- Carey Pratt McCorp anp Fioyp P. ALLEN. Evidences associating pineal gland function with alterations in pigmentation. Seven figures......... No. 2. JULY Hariey N.Gouup. Studies on sexin the hermaphrodite molluse Crepidula planas “El, Influence of environment, on sexe... bogie aac. one oss BravLeEy M. Patrren. Reactions of the whip-tail scorpion to light. Four LAUT GSU ase ee a eet os 3s. 5 « «c's AMEE 5 OS PRRRER MEMS Oo Mee ake ic cl i> ANTE cy FRANK C. Mann anp Dexia Drirs. The spleen during hibernation. Four TUTE rte Sc ery Rei ae... AST 5 SERS oh hE: RAO teat Sew a Se Ropert T. Hance. Studies on a race of paramoecium possessing extra’ contractile vacuoles. I. An account of the morphology, physiology, genetics and cytology of this new race. Three plates and twelve CGT shat ee ach yen cpt ae re: se MEH lM PEER OA ye ERR S. O. Mast. Conjugation and encystment in Didinium nasutum with especial reference to their significanceses. 2.2 40.2. 20) tes cd te wsaws Sones Witi1am H. Cote anp Carteton F. Dean. The photokinetic reactions GEsiTEeeC AC DOLOSs 4; sb sa aero: Heaewien Sami Meri Re JAR ik mec a 5 Frank R. Linum. The free-martin; a study of the action of sex hormones in the foetal life of cattle. Twenty-nine figures....................... CATHERINE L. Cuapin. A microscopic study of the reproductive system of foetal free-martins. Sixteen figures iil 251 207 iv CONTENTS No. 3. AUGUST RosperT CHAMBERS, JR. Microdissection studies. IJ. The cell aster: a reversible gelation phenomenon. One plate.........................0:: 483 Wiui1aM L. Douuey, Jr. The rate of locomotion in Vanessa antiopa in intermittent light and in continuous light of different illuminations, and its bearing on the ‘‘continuous action theory”’ of orientation....... 507 Wm. A. Kerner AND A.M. Fosuer. Effects of light and darkness on the eye of Prorhynchus applanatus Kennel. Three text figures and one plate... 519 W. H. Lonetey. Studies upon the biological significance of animal colora- tion. Eight figures (one plate) STUDIES ON SEX IN THE HERMAPHRODITE MOLLUSC CREPIDULA PLANA I. HISTORY OF THE SEXUAL CYCLE HARLEY N. GOULD Department of Biology, Princeton University EIGHTY-FIVE FIGURES , CONTENTS IGA HOKE HOLS Be ORR OAS oD, c han Get ORG Soneete minus eae 1 IM GReTL taal raat A aVoye Wtyeusy teen eo Reet cr! Shay Ak eee aoe ss 2 A ela ee dace 3 @arevolelinjesima terials sts. shee ec ot Seam ies hie Tk) IRE kN Fea ee oe 3 Biers IGA ORES TAOCLIOVCTIS. (0 yon. Shaped tcc c neePR NG Dk baem alate amare eae sia Br aeeny se 3 Natural history of Crepidula plana with discussion of former work on hermaphroditism in Calyptraeidae elacionewitiemennaltsGrabs:......0 scapes oe cees «ce aoe ote oa «eee oe 4 Hormetion ol tlie shell ahs. 2): sc aROees 2. Aicc, laa eer oe Tete. (iments ND 4 ZBnwdconmental qolymiOrphismi’ same se 82 acct Seiten store ae ears <2 Meee. o 5 Pee Vig tiliiyrotemales eee: «35: eee eco Sah. ae me aka cos”) men nemetae 8 WMarittions in‘sexual ‘developmiembGs: +. 2. +. que tet. erin ees se cee 8 Genital organs of the Calyptracidae™. .. a2 .J8. o4ss 05. sabes ss eee ee 10 Whservations: oni Grepidila, plamassegn as. |:\.ceueabeariie ex coe rlate ae ease aes wate Lit -Establishment of the gonad and efferent duct.........................: 11 Ab PeStOm Cera COL Ss s.. + as. NRE en si eR MM os Phatce dae eet tee eee 12 Holitclencellan ss Mew rat a. 5 4. ieee Oe ee I RR es. S Mean nade 15 Male developmentim the gonads). 214 poate bien ne tena hae as eek eee 17 Development of the Apyrene spermatozoa in Crepidula plana...... 21 STP Ney 270 GIR A ESR AS ae ge, GL = ee A NR Re ae 28 iHistory of the gonad from male to female phase....................... 30 2 Regression: OLaGhe Gestis:) 3... \ 681. Length of specimen, 11 mm. 45 Section through strand of degenerate male gonad. Nuclei at periphery distorted. X 681. Length of specimen, 9 mm. 46 Section through different part of same gonad as figure 43. Sperm heads embedded in wall. > 1024. 47 Section through peripheral wall of gonad during transition period. Specimen from which figure was drawn was taken as adult male (15 mm.) from a colony; during thirty-seven days after transferring to a second hermit shell, penis entirely degenerated, and gonad was found inactive, when sectioned. x 1024. 48 Later stage. Part of syncytial wall becomes dissolved, while part con- denses about follicles nuclei. Cytoplasm of germ cells becomes more clearly defined. X 1024. Length of specimen, 13 mm. 62 STUDIES ON SEX IN CREPIDULA PLATE 5 HARLEY N. GOULD 63 PLATE 6 EXPLANATION OF FIGURES 49 Section through part of gonad showing first indications of female de- velopment, i.e., presence of many primordial egg cells surrounded by follicle cells. Gonad consists of almost solid strands. X 681. Length of specimen, 10 mm. 50 Section through wall of gonad in neuter specimen 10 mm. long with thin, smooth shell. 50a and 50b drawn at same place, but at different foci. 51 Section through an immature female gonad during later oogonial divi- sion and early nuclear changes of oocytes. X 681. 52 Section through immature female gonad during later nuclear changes and formation of growing oocytes. Follicle nuclei become very much enlarged as oocytes develop, and in adult ovary many more are present than in this figure. In the larger of the two resting oocytes in the figure, there is very little matter in the nucleus which takes a chromatic stain. The basichromatin at least is limited to a few granules disposed here and there on the linin threads. Some of these granules lie at the very periphery of the nucleus, and just outside in the cytoplasm there are many granules which have a similar appearance. Whether chromatin is escaping into the cytoplasm, or whether chromidia are appearing in the cytoplasm, must be determined by specific stains for chromidia. < 681. Length of specimen, 17 mm. ; 53 to 59 Apyrene spermatosomes which are degenerating during meta- morphosis. All X 1024. 53 Beginning of degeneration shortly after formation of nuclear vesicles. The cell has several large vacuoles. Two centrioles appeared at extreme edge. 54 Nuclear vesicles breaking up. Cell outlines irregular. 55 Degeneration while the axial fibres are growing out. The cytoplasm is full of large vacuoles. 56 Spermatosome in which all the nuclear matter has disappeared, but no fibres have grown out. 57 Degeneration during outgrowth of the axial fibres. 58 Degeneration during outgrowth of the axial fibres. This spermatosome resembles Kuschakewitsch’s figure of the nearly adult apyrene spermatozoon of Vermetus. 59 Degeneration after the apyrene spermatozoon has become nearly adult. The cytoplasm is in small globules and the axial fibres are thus left close together in a strand. 60 Adult eupyrene spermatozoon. 61 Adult apyrene spermatozoon. 64 STUDIES ON SEX IN CREPIDULA PLATE 6 HARLEY N, GOULD 65 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 1 PLATE 7 EXPLANATION OF FIGURES All figures X 681 62 Primordium of goniduct in specimen 1} to 14 mm. long. Figure drawn at proximal end of strand, where latter joins gonad. Optical section. 63 Slightly later stage, showing appearance of lumen in middle of strand. Optical section. 64 Higher focus at same place as figure 63, showing surface of duct. 65 Later stage, showing the flattening of the walls and the nuclei within them. Gonad of specimen contained spermatogonia, some of which have got into the goniduct. 66 Proximal part of the goniduct of specimen where rapid spermatogonial multiplication was taking place in gonad. Duct enlarged and slightly twisted upon itself. Cilia appearing. Length of specimen, 8 mm. Penis partly de- veloped. 67 Section through one turn of seminal vesicle in specimen having nearly mature testis. A few sperm have been formed and reached the seminal vesicle. Length of specimen, 5 mm. 68 Distal part of goniduct (vas deferens) in same specimen as above. 69 Section of small part of wall of seminal vesicle in specimen with de- generate testis, showing entrance of sperm head into wall. Length of specimen, 11 mm. 66 STUDIES ON SEX IN CREPIDULA PLATE 7 HARLEY N. GOULD 68 66 67 PLATE 8 EXPLANATION OF FIGURES 71 Showing an apyrene spermatosome, which has abnormally developed (see text) entering wall of seminal vesicle. X 681. Length of specimen, 14 mm. 72 Showing eupyrene sperm heads (esp) and apyrene sperm (asp) in wall of vesicle. Cytoplasm has extended out along apyrene sperm. One eupyrene sperm head has begun to break up, assuming beaded appearance. Length of specimen, 11 mm. Gonad reduced to small strands. X 681. 73 to 76 From asingle specimen a large animal (17 mm.) which had been a male when first collected, but which during 48 days under experiment had lost the male condition. All sperm in seminal vesicle had been absorbed. 73 Section through distal part of goniduct; somewhat oblique section. X 85. 74 Enlargement of small part of same. X 681. 75 Section through degenerate seminal vesicle. X 85. 76 Enlargement of small part of same. X 681. 77 to 80 Sections through goniduct of large sexually inactive specimen, formerly male. No oocytes yet present in gonad. 77 Section through distal part of goniduct. X 85. 78 Enlargement of small part of same. X 681. 79 Section through proximal part of goniduct (former semial vesicle). X 85. 80 Enlargement of small part of same. X 681. 81 to 84 Drawn from the goniduct of an immature female 20 mm. long, with yolked oocytes in ovary (fig. 83). 81 Distal part of goniduct (now oviduct) showing great growth and infold- ing of walls. X 85. 82 Enlargement of small part of epithelium of same. X 681. 83 Proximal part of oviduct in same animal. Somewhat oblique section. x 85. 84 Enlargement of small part of epithelium of same. X 681. 85 Section through epithelium of oviduct of adult female, to show unicellular gland. STUDIES ON SEX IN CREPIDULA HARLEY N. GOULD PLATE 8 69 = etal ae ar indid et oie os , + PHOTOMECHANICAL CHANGES IN THE RETINA OF NORMAL AND TRANSPLANTED EYES OF AMBLYSTOMA LARVAE HENRY LAURENS AND J. W. WILLIAMS Osborn Zoélogical Laboratory, Yale University THREE TEXT FIGURES AND ONE PLATE INTRODUCTION The changes in form and position of the visual cells and their nuclei and of the pigment in the retinal epithelium is a subject concerning which a vast literature has been accumulated. There are, however, points here and there concerning which our knowledge is incomplete. One of these is the influence of the central nervous system upon the photomechanical changes. The work which has been done has been recently reviewed by Det- wiler (16) and by Arey (16b). From this experimental evi- dence it appears that photomechanical changes may take place after the optic nerve has been cut, 1.e., independently of central control. Amblystoma larvae, normal, eyeless, and individuals with transplanted eyes, were being used extensively in experiments on the physiology of the melanophores and it was decided to make a study of the reactions of the retinal pigment and of the visual cells of these animals to light and darkness. It occurred to us that the investigation of the effects of light and darkness on transplanted eyes would be a method devoid of certain ob- jections that might be raised against the sectioning of the optic nerve which involves shock and degeneration. Such a trans- planted eye is not only under no normal nervous control but has never been so. There is a chance that nerve fibers, both spinal and autonomic, may grow into the eyeball, but the chance is indeed slight, and still less that they could exert any influence on the movements of the retinal elements. 71 72 HENRY LAURENS AND J. W. WILLIAMS Eyes were transplanted in frog tadpoles (Rana palustris) as well as in Amblystoma larvae, the stage of development used for the operations being that when the tail bud is just beginning to be perceptible (Laurens ’14). The optic vesicle was removed and transferred to the slightly enlarged cavity made by re- moving the auditory vesicle. All transplants were made on. the left side, the right eye being left to serve as a control. For some reasons all but two of the tadpoles died, before they reached a length of 30 mm. Sections of the transplanted eyes of the two survivors, killed just before metamorphosis, showed essentially normal conditions. It is planned to repeat the experiments on tadpoles for the reason that they will in all probability show even greater reactions and more extensive changes, as indicated by the results on normal eyes, than are to be reported now on the eye of Amblystoma. It is known from the work of Lewis and others that the trans- planted optic vesicle of the Amphibian will develop into essen- tially a normal eye. Uhlenhuth (18, 713 a and ’13 b) has shown that the same thing is true of the eye when it is transplanted in larval stages after its differentiation is far advanced. We have found in Amblystoma that the eye developed from the trans- planted optic vesicle is essentially normal, all the elements being “present with the same relative number of rods and cones. There are certain differences, however, to be noted. In the first place the characteristic form (to be described later) of the rod nuclei is lost, and secondly the number of ganglion cells is much less than in the normal eye. The optic nerve also has never been observed to be present. Moreover the orderly arrangement in two separate rows of the rod and cone nuclei is sometimes, though not always, disturbed. In figure A a view of the anterior end of an Amblystoma larva, 31 mm. long, is given to show the general position and appear- ance of the transplanted eyes. In figure B a section of such an eye is shown. CHANGES IN RETINA OF EYES OF AMBLYSTOMA 73 HISTORICAL Before proceeding to a description of results a brief review of the literature so far as it concerns photomechanical changes in the Urodele retina will be given. Fig. A The anterior end of a 31 mm. larva to show the general appearance and position of the transplanted eyes. 74 HENRY LAURENS AND J. We. WILLIAMS First as regards changes in the position of the pigment in the epithelial cells. Angelucci (’78) and van Genderen Stort (’86 and ’87 b) both describe it as taking place in the eye of Triton. Garten (’07, p. 20, figs. 7 and 8) shows quite a decided dif- ference in the position of the pigment in light and dark eyes, although he states that it is much less extensive than in many other vertebrates, and that the pigment, even in the dark eye Vig. BA section of a transplanted eye. Note the small number of ganglion cells. reaches down to the ellipsoids (p. 70). Howard (’08) observed pigment migration in the eye of Necturus which Arey (’16 a) found to amount to about 8 uz. As regards the inner segment of the cones van Genderen Stort (87 a) described and figured a shortening caused by light in the eye of Triton, and Angelucci (’94) claimed that in the eye of the salamander the cones stretched in darkness, an obser- vation which Garten (’07, p. 32) was not able to substantiate. CHANGES IN RETINA OF EYES OF AMBLYSTOMA 75 Changes in form and position of the rods have also been re- ported. Angelucci (94) found that the very large rods of Salamandra clearly show a decrease in the length of the outer segment after illumination. Garten (’07, p. 49) also observed that the rods of the illuminated retina of the Salamander were a little thicker and shorter. Van Genderen Stort (87a) has further reported that in the dark eye of Triton the nuclei of the rods extend over the external limiting membrane, resulting in making the rod longer. In the light eye the rod nuclei are de- scribed as all lying below the external limiting membrane. ‘This change in the position of the nuclei of the rods is supposed to be brought about by the contractility of the connection between the rod nucleus and the nuclei of the granular layer. Angelucci (94) reports the same thing as happen ng in the eye of Salaman- dra maculata. Garten (’07, p. 21 and 50) was able to confirm these observations for Triton (see his figs. 7 and 8) but owing to variations in his results on Salamandra he leaves the matter in doubt. ANATOMICAL FEATURES The larvae were exposed to light or darkness for varying lengths of time. ‘The entire animal was killed by throwing it into sublimate-acetic. After being hardened in alcohol, the upper jaw, containing the eye balls was cut off, and sectioned after imbedding in paraffin or the piece conta’ning the eyes was cut into two, right and left, parts and imbedded and cut sepa- rately. The sections were 10 y» in thickness, and were stained in eosin and toluidin blue, or in Ehriich’s hematoxylin and eosin. The visual cells of Amblystoma consist of both rods and cones in the approximate proportion of 4: 3. The most striking thing about the rods is the short length of the ‘myoid’ and the peculiar shape of the nuclei (see figs.). Instead of being round or oval these nuclei are angular, and show an extension on the internal side by means of the continuation of which the con- nection between the nuclei and the internal granular layer is effected. The external limiting membrane lies just below the lower (internal) angles of the rod nuclei, so that the greater part 76 HENRY LAURENS AND J. W. WILLIAMS of the nuclei is outside the membrane (fig. C). In young adults the general shape of the nucleus is the same as in the larvae, though the corners are slightly more rounded. The large oval nuclei in the second row belong to the cones. The cones are of three kinds. On the edges of the retina particularly, but scattered here and there through it, one finds large, stout cones, with a very short myoid and a bellied out Fig. C A portion of the retina of a light eye, showing the visual cells with their nuclei, four rods, three single small cones, and one double cone. inner segment with a clear ellipsoid and an oil drop. The outer segment is short and conical. The typical cone is much smaller, and has a longer, more slender myoid, a clear ellipsoid and an oil-drop in the inner segment. This type of cone is always single. The third type is one that is always, as far as can be ascertained, united with one of the first type to form a double cone. It differs from the small cone described in having a much onger myoid. The nuclei of the larger element of the double CHANGES IN RETINA OF EYES OF AMBLYSTOMA 77 cones are slender and long and tapering, instead of roundly oval, and stain more intensively. They are to be seen in several of the figures. EX PERIMENTAL Pigment migration. In a preliminary communication (Lau- rens and Williams 716) it was stated that although there was a decided forward movement of pigment when the eye was illumi- nated, it was impossible to measure the extent of this move- ment because of the fact that the distance from the external limiting membrane to the nearest pigment needle, or from the choroid edge of the epithelial cells to the farthest pigment needle, is practically the same in both light and dark eyes. In darkness, however, most of the pigment was found to be massed near the base of the epithelial cells, while only a comparatively small number of needles extended into the protoplasmic processes between the visual cells. In light, on the other hand, a decid- edly greater amount of pigment was found toward the external limiting membrane resulting in the basal layer being thinner. As a result of further study involving the examination of additional material it was found that this statement did not altogether fit the case. In figures 1 and 2 a light and dark eye are illustrated. A glance will show that these two figures, as far as pigment position is concerned, do not represent a typical light and dark eye respectively. For in the light eye the pigment is actually more retracted than in the dark eye. Nevertheless more pigment is forward in figure 1 (light eye) than in figure 2 (dark eye) although the basal layer of pigment in figure 2 (dark eye) is hardly any thicker than in figure 1 (light eye). These two figures represent extreme cases such as led us to the con- clusions stated in our preliminary communication concerning the inability of making measurements of the amount of migra- tion. That figures 1 and 2 are really light and dark eyes respec- tively is shown by the comparative lengths of the cone myoids. Figures 3 and 4 are two other figures of a light and dark eye respectively. These are, in contradistinction to figures 1 and 2, characteristic of light and dark eyes in general as far as the 78 HENRY LAURENS AND J. W. WILLIAMS position of pigment is concerned. In figure 3 the pigment is further forward than in figure 4, and in addition the basal layer of pigment is thinner. When figure 3 (light eye) is compared with figure 2 (dark eye) the condition mentioned in our prelimi- nary communication is very apparent. If it were not for the relative thicknesses of the basal layer of pigment it would be very difficult (disregarding the cones) to say whether the one or the other was a ight or a dark eye. In a few cases the pigment in light eyes has been observed to occupy an extreme position, the pigment needles being as far forward as the ellipsoids of the rods. The average of measure- ments, however, of light and dark eyes give a distance of 36 u from the choroidal edge of the pigment epithelial cells to the farthest pigment needle in light eyes, and of 29 » in dark eyes, so that we have an average pigment migration of about 7 u. In all these cases, whether the pigment showed the expected light or dark position or not, the characteristic difference in the length of the cones was present. Why pigment migration did - not take place although cone contraction did must remain un- answered. It may have been due to the fact that optimal conditions were not offered in the length of time of exposure, etc. In the transplanted eyes the pigment migration is even greater in extent than in the normal eye. Figures 5 and 6 are of a light and dark transplanted eye respectively. The average of all measurements made of transplanted light and dark eyes, gives a distance of 12 uw as the extent of the migration of pigment. Why the extent of the migration should be greater in trans- planted eyes than in normal is not clear. Detwiler (16) found that in the eye of the turtle there was what he called a loss of tone when the optic nerve was cut. Both the pigment cells and the cones seemed to relax in that the pigment extended down further (partial light position) and the cones stretched (partial dark position) though there was no great difference in the amount of movement occasioned by light and darkness as compared with that in normal eyes. Arey (’16b) has obtained some very interesting results with the retina of Ameiurus. He found that when the optic nerve only is severed that the char- CHANGES IN RETINA OF EYES OF AMBLYSTOMA 79 acteristic photomechanical responses fail to take place, also that after hemisection of the nerve, movements occur only in that region of the retina adjacent to its intact side. When the eye was left attached to the body by the optic nerve alone, or when it was excised, essentially normal responses take place. After further experimentation involving the cutting of muscles, etc., he concludes that in association with the muscles innervated by the oculomotor nerve there is an inhibiting mechanism the effect of which is evident when the optic nerve is cut, and that there are functional efferent nerve fibers in the optic nerve of Ameiurus, the impulses in these fibers blocking the tonic inhibi- tion exerted by the inhibiting system. The retinae of Abramis and Fundulus were found not to show this condition. Contraction of the cones. Regarding the influence of light and darkness on the myoid of the cones it is hardly necessary to say more than a word since the figures speak very clearly for themselves. Figures 1 and 3 are of light eyes, 2 and 4 of dark eyes. The differences between them are evident. The trans- planted eyes show the same condition of affairs (figs. 5 and 6). Measurement of normal light and dark eyes gives an average of 7.4 was the extent to which the length of the cone can be changed by light and darkness. The length of the cone inner seg- ment in the dark eye, that is extended condition, is about 25 u (not the entire length of the cone as was erroneously stated in our preliminary communication). ‘The extent of movement of the cones of the transplanted eyes is about 14 u. Movements of the rods. No differences in the diameter or in the length of the outer segments of the rods of light and dark eyes could be clearly demonstrated. Nor could any very con- stant differences be found in the length of the rod myoid. The red myoid in Amblystoma larva is very short, never longer than 2u. Figure 1 shows them particularly clearly. In most of the rods of light eyes they are of maximum length, though in some cases the myoid is so short that the ellipsoids of the rod seems to be in contact with the nucleus. On the other hand in dark eyes the myoid is in most rods of minimal length, between $ and 1 u long, though here again in the dark eyes the length of the rod 80 HENRY LAURENS AND J. W. WILLIAMS myoid may measure the maximum 2 y». All that can with cer- tainty be said is that there seems to be a slight shortening of the rods in darkness, a shortening, however, which is only be- tween 1 and 13 wu in extent. There is, however, a very evident difference in the shape of the rod nuclei, according as to whether the eye is a light or a dark one. In the light eye there is a distinct rounding off of the angular corners of the nuclei particularly of the external ones, so that the boundary of the nuclei on the myoid side is a curve of regular outline, instead of running for a time almost parallel with the internal boundary of the ellipsoid as it does in the dark eyes. This is a result which might happen if the nuclei were subjected to a pull on the internal side (see figs. 1 and 3 as com- pared with figs. 2 and 4). The changes in the position of the rod nuclei described by van Genderen Stort and Garten in Triton are supposed to be due to the contractility of the connections between the rod nuclei and the granular layer. Perhaps con- tractility is not so highly developed in the eyes of Amblystoma so that when the pull takes place all that can happen is for the nuclei to change their shape in response. Certainly they do not change their relative position with regard to that of the cone nuclei or of the external limiting membrane, which passes, as mentioned above, just below the lower angles of the rod nuclei (fig. C). SUMMARY 1. Pigment migration and cone contraction take place in the transplanted eyes of Amblystoma larvae as well as in normal eyes, and to a greater extent. 2. The extent of pigment migration in the normal eye averages 7 u. In the transplanted eye 12 u. 3. The extent to which the myoid of the inner segment of the cone contracts in the normal eye is 7.7 ». In the transplanted eye 14 u. 4. The rod myoid may lengthen in the light, but if it does so, it is only to the extent of between 1 and 1.5 u. CHANGES IN RETINA OF EYES OF AMBLYSTOMA 81 5. There are no differences in the length or diameter of the outer segments of the rods of light and dark eyes, nor do the rod nuclei change their position. 6. The rod nuclei, however, do change in form. The angles of the nuclei in the light becoming rounder as if in response to a pull. LITERATURE CITED Ancetuccr, A. 1878 Histologische Untersuchungen tiber das retinale Pigment- epithel der Wirbeltiere. Arch. f. Physiol., 8. 353-386. 1894 Untersuchungen iiber die Sehtitigkeit der Netzhaut und des Gehirns. Moleschott’s Untersuchungen zur Naturlehre, Bd. 14, S. 231-357. Argy, L. B. 1916a The movements of the visual cells and retinal pigment of the lower vertebrates. Jour. Comp. Neur., vol. 26, pp. 121-202. 1916 b The function of the efferent fibers of the optic nerve of fishes. Jour. Comp. Neur., vol. 26, pp. 213-246. DETWILER, 8. R. 1916 The effect of light on the retina of the tortoise and lizard. Jour. Exp. Zo6l., vol. 20, pp. 165-191. GarTEN, S. 1907 Die Verinderungen der Netzhaut durch Licht. Graefe- Saemisch Handb. d. ges. Augenheilk. Teil 1, Bd. 3, Kap. 12, Anhang, S. 1-130. VAN GENDEREN Srort, A. G. H. 1886 Uber Form und Ortsveriinderungen der Elemente in der Sehzellenschicht nach Beleuchtung. Ber. d. 18. Versamm. d. Ophthal. Ges. S. 43-49. 1887 a Uber Form- und Ortsveriinderungen der Netzhautelemente unter Einfluss von Licht und Dunkel. Arch. f. Ophthalmol., Bd. 33, Teil 3, S. 229-292. 1887 b Mouvements des élémente de la Rétine sous l’influence de la lumiére. Arch. Neerlan. des. Sci. ex. et. nat., T. 21, p. 316-3886. Howarp, A. D. 1908 The visual cells in vertebrates chiefly in Necturus macu- losus. Jour. Morph., vol. 19, pp. 561-631. Laurens, H. 1914 The reactions of normal and eyeless amphibian larvae to light. Jour. Exp. Zodél., vol. 16, pp. 195-210. LauRENS, H. anp WriuuiaAms, J. W. 1916 Changes in form and position of the retinal elements of normal and transplanted eyes of Amblystoma larvae occasioned by light and darkness. Proc. Soc. Exper. Biol. and Med., vol. 13, pp. 183-184. Untennutu, W. 1912 Die. Transplantation des Amphibienauges. Arch. f. Entw.-mechan. d. Org., Bd. 33, 8. 723-747. 1913.a Die synchrone Metamorphose transplantierter Salamander- augen. Arch. f. Entw.-mechan. d. Org., Bd. 36, 8S. 211, 261. 1913 b Der Einfluss des Wirtes auf das transplantierte Amphibie- nauge. Arch. f. vergl. Ophthal., Bd. 3, S. 343-355. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 1] PLATE 1 EXPLANATION OF FIGURES All figures on this plate were photographed at a magnification of 1000. They have been reduced one-half in reproduction. 1 A light eye of a 45 mm. larva showing the typical light or contracted con- dition of the cone myoid. The pigment, however, is in the retracted or dark position. 2 A dark eye of a 42 mm. larva, showing the typical dark or extended condi- tion of the cone myoids. The pigment, is again, however, not typical but shows the forward or light position. 3 A light eye of a 39 mm. larva showing the typical condition of both cones and pigment. 4 A dark eye of a 41 mm. larva showing the typical condition of both cones and pigment. 5 A transplanted light eye of a 43 mm. larva showing the typical light con- dition of contracted myoids and forward pigment. 6 A transplanted dark eye of a 41 mm. larva showing the typical dark condi- tion of extended cone myoids and retracted pigment. 82 CHANGES IN RETINA OF EYES OF AMBLYSTOMA PLATE 1 HENRY LAURENS AND J. W. WILLIAMS 83 THE ROLE OF THE AUDITORY SENSORY EPITHE- LIUM IN THE FORMATION OF THE STAPEDIAL PLATE FRANKLIN PEARCE REAGAN Department of Comparative Anatomy, Princeton University TEN FIGURES Certain structures which seem to have undergone transforma- tion in the process of transition in vertebrate life from aquatic to terrestrial conditions have always been of great morpho- logical interest. Structures of this sort are abundant in the pharyngeal region, whieh, especially in earlier times, furnished a most productive field of study. Such study was often concerned with the homologies among the nervous, muscular, vascular, skeletal and epithelial derivatives of this pharyngeal region. Between the intermittent communications of the pharynx with the exterior there are developed mesenchymatous visceral arches in which chondrification takes place, forming the so-called ‘visceral ribs;’ these may ossify and retain their original position as components of the pharyngeal skeleton, or they may become greatly modified in the higher forms, acquiring a special function very unlike that of their more primitive homologues. In the lower vertebrates the pharyngeal ribs or components of the visceral skeleton tend to preserve their original resemblances to each other. Particularly striking here, also, are the great independence and the wide separation of the visceral skeleton from the skeleton of the central nervous system—the cranial skeleton. In some of the primitive elasmobranchs, for in- stance, the mere cutting of three ligaments may be sufficient for the complete separation of these two skeletal complexes (..e., visceral and cranial). But even in forms as low as the holo- cephali, the visceral skeleton has become immovably articu- 85 86 FRANKLIN PEARCE REAGAN lated with the brain-case. Here the palatoquadrate is firmly fused with the cranial wall, and the upper end of the hyoid arch (hyomandibula) takes no part in this union; this is a distinct advance towards the differentiation of a definite quadrate. The skeletal complex of the first visceral arch forms the car- tilage of the jaws. The antero-dorsal cartilage is known as pterygoquadrate. The ventral element comprises Meckel’s cartilage. In selachians these two are suspended from the skull by the hyomandibular cartilage, the latter having been derived from the dorsal portion of the second visceral arch. During the ontogeny of the higher vertebrates the pterygoquadrate and hyomandibular homologues become very closely associated with the developing cranial skeleton, either losing entirely their identities, or becoming greatly transformed. Certain it is that the jaw articulations. of the lower forms are not homologous with those of higher ones. The hyomandibular, or at least the dorsal portion of the second visceral cartilage, is believed by many to be represented by that columnar bone in the series of ear-ossicles which be- comes the most intimately associated with the otic capsule. In amphibia, sauropsida, and monotreme-mammalia, this homo- logue has been called by various names such as ‘columella,’ ‘columella auris,’) and ‘columella cranu.’ The cartilaginous plate fitting into the fenestra ovalis has been designated in amphibia as the ‘operculum.’ In higher forms the cartilaginous plate occupying the fenestra ovalis is known as the stapedial plate. The terms ‘columella’ and ‘columella cranii’ have been em- ployed with a great deal of confusion, as, for instance in the writings of Gegenbaur and Schimkewitsch. Gegenbaur (Vergl. Anat. der Wirb., p. 374) interprets the columella of amphibia as follows: In urodeles there is a ligamentous process stretching from the oper- culum to the cartilaginous quadrate. In the anura the operculum is continued as an elongate ossified staff, the columella, which is to be regarded as a part of the auditory apparatus. These are two skeletal units which have taken the place of the hyomandibulare. FORMATION OF STAPEDIAL PLATE 87 He regards (p. 380) the columella of reptiles as a homologue of the upper end of the hyoid arch, but derives the operculum from the chondrocranium. On p. 386 he states: To the labyrinth region belongs still another bone which springs up from the pterygoid to the parietal—the columella. It has a cartilaginous Grundlage (Leydig) which is laid down on a process of the chondro- cranium (Gaupp) and which is also distinguishable in amphibia and evolves itself in lacertilia into a columnar form which is characteristic Glib: It is difficult to see by what line of reasoning this latter colu- mella could be regarded as belonging to the labyrinth region. It is much farther removed from the labyrinth region than a great deal of the pterygoid itself from which it is derived. This evident error would be of minor interest if it had no counterpart in the writings of more recent observers. Schimkewitsch, (Lehrb. d. vergl. Anat., p. 121) describing the conditions in Sphenodon, states: ‘“‘From each pterygoid there reaches up a ‘platten formig’ bone (anti-epipterygoideum or columella cranii) which represents a process of the palatoquadrate which has already existed before in anuran amphibians.’ Although he deals with the homologies of this bone in almost all groups of reptiles, he neglects its description in the skull of the snake (figures from Boas) where (figs. 184 and 135) the term columella cranil is applied to the ear-ossicle whose internal end fits into the fenestra ovalis. If these two columellae are homologous it is puzzling that both should exist in the same reptilian form. In higher mammals the auditory ‘columella’ seems to be rep- resented by the staff-like portion of the stapes. There seems at present to be little doubt that the latter arises as a chondri- fication in the mesenchyme of the second visceral arch; our ob- servations concerning this point are not, however, in complete unison. Concerning the origin of the plate-like cartilage which forms the distal (distal from the point of view of the visceral skeleton) portion of the stapes homologue which closes the fenestra ovalis there is very great diversity of opinion. Accord- ing to one view, the entire stapes including the stapedial plate arises as a chondrification in the second visceral arch. Among 88 FRANKLIN PEARCE REAGAN the many observations supporting this view may be mentioned those of Baumgarten, Broman, Huxley, Keibel, Kingsley, Peters, Rabl, Reichert, Schenk, Schafer, Zittel, and Zondek; closely allied to this view we have phylogenetically the hyo- mandibular origin of the stapes, supported by Baraldi, Claus, Gegenbaur, Hasse, R. Hertwig, Kiikenthal, and Wiedersheim, and Parker. On the other hand, there are observations which tend to show that the entire stapes arises from the otic capsule, constituting then, a portion of the cranium and in no way re- lated to the visceral skeleton. Among these observations may be mentioned those of Fuchs, Koélliker, Marshall, Parker and Bettany, and Wiedersheim. Minot derived the stapedial plate as an independent chondrification in the fenestra ovalis. These interpretations have been combined into a third view according to which the stapes is formed partly from the otic capsule (1.e., ‘the stapedial plate), and partly from the hyoid arch. This view of the mixed origin of the stapes has been supported by Grade- nigo, van Norden, and Schultze. Ginther described the stapes as developing from the mandibular arch. Dreyfuss thought it developed from either the first or second arch. Cope and Frazer described the origin of the stapes as ‘peri-arterial.’ O. Hertwig regards its origin as uncertain. Finally one might mention the view of Siebenmann who would dismiss as immaterial the possibility of a definite relation of embryonic visceral cartilage and adult ear ossicles. One must admit with Keibel (1912, p. 281) that “‘there is great difficulty in tracing back a skeletal structure to the early — pre-chondral stages, and it carries with it the danger of sub- jective interpretation.” It seems desirable temporarily to set aside considerations of phylogeny and attack the problem from the point of view of the mechanics of development involved in a single ontogeny. It is a well known fact that the crania of all vertebrates pass through a common phase of development which may be regarded as a ground-plan of vertebrate cranial formation. Surrounding the anterior end of the notochord there is found a parachordal cartilage, and anterior to this are located the trabeculae. In the FORMATION OF STAPEDIAL PLATE 89 meantime there have arisen three pairs of bilaterally symmetri- cal sensory epithelia—those of the nostrils, eyes, and ears. Kach of these sensory epithelia becomes more or less completely surrounded by a prechondral cytoblastema which subsequently becomes cartilaginous. So striking is the intimacy with which each cartilaginous capsule adjusts itself to the contour of its respective epithelium, that it seems not unreasonable to suppose that each epithelium in some way furnishes the stimulus which effects the chondrification of the surrounding mesenchyme. If this inference be correct, it is evident that the removal of a given sensory epithelium would inhibit the development of the corresponding cartilage. If this also be true, the early removal of the auditory epithelium would furnish a means of testing to what extent the stapes homologue together with its stapedial plate can develop in the absence of a cartilaginous otic capsule, or in the absence of the stimulus to which the latter owes its formation. The work of W. H. Lewis (’04) is of great interest and im- portance in this connection. Lewis found that if the otocyst of an anuran be transplanted to the mesenchyme of a urodele, there develops around the transplanted otocyst a cartilaginous capsule which is typically urodelan in character. Unfortunately, experimentation of this sort is at present impossible so far as mammalian development is concerned. Avian development does, however, lend itself to this sort of procedure. REMOVAL OF THE OTOCYST Chick embryos of from thirty-five to sixty hours constituted the material for experiment. The experiments were of two types: 1) one of the otocysts was completely or incompletely removed by insertion into it of a very warm fine-pointed plati- num needle, for a sufficient length of time to coagulate the liquid contents of the otocyst, whereupon the seasory epithelium would adhere to the needle when it was removed: 2) the otocyst was transplanted. After being subjected to this sort of treatment the eggs were sealed and allowed to incubate for various lengths of time— 90 FRANKLIN PEARCE REAGAN generally to an age at which the normal stapes on the uninjured side was well differentiated. A distinct advantage of this method is that the normal side serves as a control. In general the results of the removal of the otocyst have been made known through a previous communication (Reagan, 14). Int. Int.car, Fig. 1 Frontal section through the otic region of a five-day chick. On the left side an otocyst is present. On the right none is seen. The section is other- wise almost symmetrical. All sections figured in this account are viewed from their posterior faces so that the embryo’s left corresponds to the reader’s left. P. EK. C1 No. 1120. Int car., internal earotid; Int. 7., internal jugular; Oicst., otocyst. A preliminary study was made of embryos at an age in which the mesenchymatous tissue surrounding the normal otocyst was still in a prechondral or membranous stage. Figure 1 shows a section through the otic region of a chick embryo which had been incubated for five days. On the unoperated side there is a 1 Princeton Embryological Collection. FORMATION OF STAPEDIAL PLATE 9] typically developed otocyst, around which the mesenchyme has begun to condense, staining rather deeply. On the operated side of the same embryo (right side of figure 1) it will be noticed that the otocyst is entirely lacking. The mesenchyme occupy- ing the former region of the otocyst resembles in every way the other surrounding, lightly staining mesenchyme, and shows no evidence of condensation into an otic capsule. There was no attempt at regeneration of the removed auditory epithelium. This embryo had not yet developed visceral cartilages. We may now consider cases in which the embryos which had been operated upon were allowed in each case to live until the stapes of the normal side was well developed. Eight and nine- day embryos were found to be most favorable for study. Figure 2 represents a frontal section through the otic region on the normal side of an eight-day embryo from which the right otocyst was almost completely removed at the forty-fifth hour of incubation. The columella is seen lying between the external auditory meatus and the fenestra ovalis. Fused to the inner end of the columella is a flange-like ring of cartilage; but the two are everywhere separated by a distinct perichondrium which seems to be shared in common by the periphery of the mesial end of the columella and by the lateral internal surface of this flange of cartilage, the stapedial plate. A large part of the stapedial plate, or more correctly, the stapedial flange—projects freely into the mesenchyme. A portion of it, however, is continuous with the cartilage of the otic capsule, and seems always to have been a part of that cartilage. Situated inside the otic capsule is the auditory epithelium. ‘The plane of section lies transverse to the transitional portion of the epithelium between sacculus and lagena. Mesial and dorsal to this epithelium is a portion of the acoustic nerve. A small branch of the internal jugular vein projects into the otic capsule. Dorsal to the capsular region, the facial nerve is seen emerging from its foramen. 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ERATION te’ : Sent RaTioN (ao cy MS 21S SMA ers = 5 BRISTLE INHERITANCE IN DROSOPHILA ens means. This fact, together with the warmth of the constant temperature room, renders the means in this period (generations 32 to 49) obviously uncomparable with the means in earlier EXTRA BRISTLES MEANS MALES ' ‘ 8 n 4 4 it " A i ot vs : = . . oe a Wott tae od ’ ~ . Ait a 7 . Pil are Te NP a ar Dias ata S ead vegapre: s Hy of L ' ee ee eae Sys. ele : : H : Spacoie Hop’ HY ee? eer H 7 FAL OTA + : Nee etree eae on Ha : . y ‘ . Sie ottl oak um amac late ye : . ' a Ni 1 ' Mi ye eth ig 77 A a ‘ ‘ ef, \ \ t ay Py ’ . ' ‘ ‘ . ‘ ‘ 7 ‘ . oe rie ‘> : ‘ & See Gey : ae or ‘ See? ue M) ae rH ‘ ‘ : : ' : ree as He , y at : : PARENTS SINGLELINE ‘*% A L On 5 Ge Wan tt wou ut : on . ' : a ‘ ‘ ‘ SONS SINGLE LINE SONS TOTALGENERATIONS ——— 2 GENERATIONS 26 28 30 32 34 36 38 40 42 44 46 48 lI4 16 18 20 22 24 A EXTRA BRISTLES TOTAL GENERATIONS ——— SINGLE LINE © GENERATIONS 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 B Fig. 2 Means in the line of single families compared with means in the total Since there was only one family each in generations 24, 25 and 26 In ‘A’ the sons are compared 14 16 generations. there is only one curve through these generations. in ‘B’ the daughters are compared. The mean grade of the two parents in each When single families generation of the single line is shown with the ‘A’ curves. are examined it is found that fluctuations in the parental grades are not accom- panied by corresponding changes in the average grades of the offspring. 114 EDWIN CARLETON MACDOWELL periods. The progress of selection must be sought within this period and within the earlier period. With these facts in mind the curves showing the means of the sons and daughters in the high selected race are to be examined (fig. 3). The means for generations 2 to 11 differ somewhat from those given previously. In order to increase the rigidity of selection, families with certain lower grade parents were MEANS : EXTRA BRISTLES HIGH RACE : fa ss : ; Hoi | es moe Pe H PARENTS f\ H Har \ i Sen nies foe oi i DAUGHTERS: DAUGHTERS 7 f/ GENERATIONS 24 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 Fig. 3 Means of the parents, sons and daughters in the total generations of the high selected race. The means of the parents have been weighted according to the numbers of their offspring. The curves of the sons and daughters are much alike in their fluctuations; except in the early generations the curves of the parents and offspring do not show parallel fluctuations. excluded together with all their descendants. As here given, the means include all such families. The resulting changes in the means are insignificant from the standpoint of a general conclusion, since they do not modify the general form of the curve. In the earlier report, the initial rise in the means was emphasized. It would be a difficult matter to determine just where this initial advance was stopped. Were certain striking BRISTLE INHERITANCE IN DROSOPHILA 115 and evident responses to environment not present, such as are found in generations 26 to 30, the curves might be treated math- ematically, and a theoretical curve fitted, but since such clear evidence of the irregular influence of environment is present, this mathematical treatment seems out of place. The critical period is between generations 11 and 23. In this period it seems possible that the ideal condition is represented by the series of high points at grade 4? for the females, that the long gradual decline is due to less and less favorable conditions, which later are improved till the high point is again reached. It may be somewhat problematical whether the high or the low points (at 33) in this period should be considered ideal, yet it does seem probable that an intermediate position would not be so considered. Moreover there can be found no tend- ency for the later generations in this period to be higher than the earlier ones. The irregularities in generations 24, 25 and 26 may be discounted, since, as explained, these include very small families. Although the high peak in generations 29 to 31 may not be directly due to the conditions of the constant temperature room, yet its occurrence immediately upon the introduction of the flies into that room, and the absence of any comparable high region in all the twenty months of the experi- ment, strongly suggest a causal connection between the two. However it is evident that whatever stimulus the constant humidity and temperature may have been at first, the effective- ness of this stimulus became weakened long before the flies were removed from this room. The portions of the curves of the sons and daughters following this high region show general fluctuations, but one can- not detect a tendency for the earlier generations to have lower means than the later ones. Between generations 36 and 38 there is a continuous rise, but this rise does not exceed the limits of fluctuation shown by the later generations. To conclude, it may be said that there is a rise at the beginning of selection; that this is followed by a period in which no rise is discernible; that the irregularities in generations 24, 25 and 26, and the sudden rise in generations 29 to 31, are not due to the 116. EDWIN CARLETON MACDOWELL selection; and finally that in the last period (generations 32 to 49) no permanent rise in the means can be found. Extremes of variation The range of variation gives further light on the nature of the changes during selection. If the race as a whole is being changed, upper and lower extremes should change as well as the means. In this race the lower limits show very s ight varia- tion. In all the generations (excepting the 24th and 25th) the low limit for the male is either grade 0 or 1; in 33 generations it is 0; in 12 generations it is 1. The females are a little higher; EXTRA BRISTLES HIGH RACE HIGHEST FEMALES “a A 1 \a—_—-=-=- Nesey VSssen \ 4 NE NC See HIGHEST MALES “MV = “ A. , See -——--.7 7 Be eee \- | | yee OW es —— MEANS OF MALES GENERATIONS 2 4 6 8 10 1l2 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 Fig. 4 Highest grade son and daughter in each of the generations of the high selected race, compared with the means of all the sons and daughters. Selection has not resulted in the raising of the high limit of variation. in 20 generations the low limit is 0; in 18 generations it is 1; in 7 generations it is 2. From this one may say that the low ex- treme is a relatively fixed point. The high extremes are more variable. These are shown in figure 4. The highest grade recorded (16) was found in generation 8; the highest grade re- corded (13) in the 41 generations of selecting that followed this, appeared in the first generation raised in the constant tempera- ture room. Throughout the whole series there appears a tend- ency for the highest female to be grade 9 and the highest male, grade 7. Selection then has not called into being any new grades that were not obtainable very near the beginning of the experiment. BRISTLE INHERITANCE IN DROSOPHILA EEF Standard deviations If the extremes in a frequency distribution are stationary, the movement of the mean will tend to modify the standard deviation. If the mean and the mode of a curve are close to one end of the distribution, their movement towards the middle of the range will have the tendency to raise the standard devia- tions. There is a fairly close approximation to these conditions in the frequencies of the different generations. The early gen- erations show means and modes close to the lower end of the scale of grades; in later generations the means rise toward the middle of the scale, but the extremes are not greatly changed. This leads one to expect the increase in the standard deviations that actually has been found to follow the increase in the means. Moreover the standard deviations fall when the means fall. In figure 5 A and B the standard deviations for the sons and daughters are separately compared with their respective means. Attention’ must be called to the fact that the means and the standard deviations are plotted in these curves on different vertical scales, and that the coincidence of the lines does not in- dicate fluctuations of equal magnitude, but rather, fluctuations in like directions. The close parallelism between these two sets of constants disappears soon after the 32d generation. This is apparently due to the fact that, as explained, the complete yield from each bottle was not recorded. This incomplete sampling slighted the lower part of the distributions so that the means were raised, but the standard deviations were reduced and rendered irregular. Besides rising and falling with the means in each sex, the standard deviations of the sons are lower than those of the daughters, in the same way as the means of the sons are lower. The general conclusions to be drawn from the study of the standard deviations may be stated as follows: The standard deviations of the frequencies with lower means are lower than the standard deviations of the frequencies with higher means. The generations between the 32d and the 49th of course form an exception to this statement, but in accord with the expla- 118 EDWIN CARLETON MACDOWELL nation of this exception is the claim that the higher averages in this period are due to incomplete counts and not to a modi- fication due to selection. SCALE OF MEANS EXTRA BRISTLES SCALE OF STANDARD DEVIATIONS HIGH RACE 16 1.4 as \ be ! " / Soe s oN Nees 1.2 1.0 DIP A Nome MOM mI4Nn iG Niels omapNad 28 30 32 34 36 38 40 42 44 46 48 A SCALE OF MEANS EXTRA BRISTLES SCALE OF STANDARD DEVIATIONS . HIGH RACE FEMALES 2.0 1.8 5S 1.6 4 A 7, \ 1.4 3 Ry i i \ STANDARD DEVIATIONS | L2 2 LO 2A ear Ss) OMI2 14-16 IS a20r 22 28 30 32 34 36 38 40 42 44 46 48 B Fig. 5 Standard deviations in the high selected race compared with the corresponding means. It must be noted that the standard deviations are plotted on a scale five times as large as that of the means. Generations 24, 25 and 26 have been omitted on account of their small numbers. ‘A’ is based on the sons, ‘B,’ on the daughters. A close parallelism is demonstrated between these con- stants. All flies from each bottle were not counted in generations 33-49 in- clusive; this probably accounts for the break in this relationship that appears in those generations. BRISTLE INHERITANCE IN DROSOPHILA 119 Discussion of the distributions The study of the frequencies of the 49 high selected genera- tions, whether by the means, the extremes, or the standard deviations, has failed, except in the first few generations, to indicate any advance that may be attributed to selection. It seems that breeding only high grade parents has not succeeded in producing increasingly higher distributions, that new ex- treme grades have not appeared after the continued selection of the highest flies as parents. Other than genetic factors seem to influence the character of the distributions, yet it remains possible that a part at least of the fluctuations in the means may be due to corresponding variations in the parents selected. This possibility is considered in the following section. Comparison of parents and offspring The point of primary theoretical interest is not the ability, or inability, to advance the means and raise the whole dis- tribution of a race by selection. ‘The crux of the selection prob- lem is whether abmodal parents have abmodal children. Spe- cifically it is, do parents with higher bristle grades produce children with higher bristle grades? When the means of the offsprmg, which have already been considered by themselves are compared with the means of the parents that produced them, important evidence is forthcom- ing. Referring again to figure 3, this comparison will be con- sidered. The dotted line in this figure shows the means of the parents selected. These have been weighted according to the numbers of offspring produced by the various grades of parents included in each mean. The means of the parents in each gen- eration are plotted on the same vertical line as the means of their sons and daughters. The basic numbers for these curves will be found in table 1. One finds on making this comparison that, excepting the first few generations, the means of the parents and their children vary with great independence. The high peaks of the parents curve are not accompanied by high peaks in the curves of their MACDOWELL CARLETON EDWIN 120 6c0 0+ 688° T/E0' 0+ 96§' 1/ZF0' 0-822 'S/€E0' 0886'S} ¢ vy {1&6 |Z08 | $20 06962'9 |00'8i8¢'s} |T G IV IZ IE |Z 62 660 0+ £69 1|9Z0 0+ €6E' T|IF0 O=ELZF F/9E0 OF FIT’ §) ¢ & |082 |909 | 280 0S829'9 |€8 LITT 9 G Ic |Z |€ IP 86 $60 0+ F99 1/120 OF OSE T|\FEO0 0 FOF F\0E0 0 E96 Z| F G |9GOL/886 | 0€0 OF ZF8C'E |FF 199 'Z @ |G |G |P IS [22 920°0* 62I 1/790 0+ 298 O|SOT OF 08E' E080 OF EEE 2} § | Z |OE ler 000¢°¢ |00'S\00°9 We ite 9% 690 0+ 8SP OCF O= €F6 0/860 0 002 F\10Z 0006 €) F € |OL OF 0000°¢ |00°S\00°¢ G GS 660 0+ 896 '0/€80 0+ I8Z O|OET 0089 FISTI 0008 | ¢ € |S¢ 0G 000¢°S |00°9)00°S T {I FG 0S0 0+ 0LF T\0F0 0+ FS 1/020 0 ZFL F\990 0 LEZ | ¢ € |86I |PIe | 680 0+88z9'°9 |G2 9)0¢ 9 JG |G |€ IT &% S€0 0 STS 1/620 0+ 006 1|6F0 0 S8FS FIFO OF TIT | F € |Scr |6Z8 | 610 0+02480'9 |0¢'9/00'9 GaISAIG GG 0€0 0+ 699 1/SZ0 0+ 16G 1|ZF0 0 GFF F/9E0 OO0T | F € |669 |c8S | [€0 O= TOIT 9 |€€ 9/E8'¢ I tes: Wa Wiz 1Z 8€0' 0* £99 1/080 OF F8E'TIFS0 OF Z8L E/9F0 0=729'Z| & | Z |SIF |668 | 9F0' 0=8286'S [08 9/09'°¢ I |Z |G |2 0G 120’ 0* 66F T/810' 0+ 992 T/0€0 00S '€/620 OF FES "Z| € G |SIIT/ESIl] 910 OF O0FSO'9 |SE 9IF9'¢S & |G |I1|6 61 £60 OF FSF 1/610 OF GLT T/ZE0 0 SOF €/220 OF 9LE Z| G |206 |S¥8 | 120 0 2028'S |9E 9/20 '¢ I |@ |% |F SL SI 960° 0+ O€& 1/220 0+ G0 1/90 O= LEP §|ZE0 OF ZIF Z| € G |909 |Z299 | SIO OF GE0S'S 18 GizG'¢ I |? |6 LT 080 0+ G9 T/FZ0' OF 6ES 1\ZF0 OF OFF €\680 OF FES ‘Z| F G \69P |Z9V | 400 OF €LFE'9 |22°9199'S G IG |9 |S 91 960° 0+ 969 T/Zc0 OSES 1\0F0 OF F8Z°E/180 OF SLE Z| G |ZéZ \IZ | 680 0=ZE90'9 |S¢'9Iee T {I I |S jO1 GI GVO 0+ 689" T/8E0' 0+ 08 1\090 0= Z2I F\FS0 0 G08 Z| F & |09€ |€6¢ | 220 0 €869'S |99 9/99 '¢ I |G |G al 620 0+ €82 1/020 0+ 62E' T/TIL 0 8E8 F/660' 0+ 2FZ | F (VALUES |e} CFI 0*ZO8Z'S |0G Gize'¢ os I rea €P0 0 €29 T\SF0' O=OFS 1/090 0 FST F/E90 9+ E8L ‘Z| F G |00€ |89¢ | 980 02S7Z8'¢ |09 9/02 ¢ iE HIE G 9 ai Se BUA, soley So[BUIOT Sole 5 a E = 5 = or IT JOT }6)8)/2)9)S & > > sejeuloy pue sopeu | > © Bare {SUBOUI Pay Sto oy SUOT}EIAEp prvpurzg suBoy sepoy vod a suBoyy srequinu SER Give anne neq DNINdSdAtO SLINGUVd ‘suaqybnop pup suos 4Vay} pup suoynsauab quasaf{rp ay) Ur pajoajas sjuaid ay, waanjaq sdiysuoynzas ay? moys 0} pabunssm aove pajoajas ybry ay) woLf DIDG T HTaVoL 121 BRISTLE INHERITANCE IN DROSOPHILA 0S0'0= 196° 170 0+ FES" 670 O= LE9° 960 0+ POF 970 OF ESE — LE0 OF FES 080 0 ZL9° 1¢0 0+ €18" 960 O= LEG" 640 0-897 Ses oSsoSs nsw we aan 090 0+ 879° 880 0 L6P- 660 OF GIG LE0 O= TSG" SFO OF GEG 1&0 O= 166" GeO O= IGP GVO O97 G60 OF CLT 840 0 826" 0€0 0+ Z8€ET/1E0 0+ 99E" 820 0+ PIE 1/$c0 0+ TOT 920 0+ 89 1/F20 OF €6T LE0 OF CEP 1\GE0 OF G9" 1¥0' 0+ 067 T/€E0 OF 96T L€0' 0 LLG 1/660 0 026" £90 0+ 029 T/€F0 0+ 696° 860 ' 0+ 999 1/220 0 066" S¥O 0+ €€8 T|ZE0 0 968" 6€0 0+ STS T/F€0 OF OFF 120 0+ SCE F 690 0+ 6¢0 ¢ 040° 0= 090° ¢ 790 0-976 7 990 0+ 9S1 9 690 0+ 068 F vO O= 8h1 ¢ GLO 0+ 98E F OVO TOgieg 0240’ 0 S68 GvO OF PZT ¢ OFO 0+ 696 ¢ 9€0 0+ €80 ¢ 690 0+ 62° 890 0+ 606° G90 O=GIS F 1L0 0+ 269 F 660 0219 F 790 O= T@L'¢ GSO 0+60€ 9 71690 OF 68E 040 0+990'€ 1S0 O-PV8 € G90 0 Z0Z°€ €90 0 G86" VFO OF GPS G80 O= 079 90 0+ LF 970 0+ 190° 190 OF 91S" vPO 0+ 9S9" 80 0+ 9T8" 80 OF OTL 976 OF LLG 670 OF LLG" 170 0+ 190° 190 O= I7¢ TE0' 0 096 '€ 650 0+ 980 F OO st OO OD OD OD OD Se) OD OD OD OD OD OD 640 OF GPE F TR 19 29 SH DH SH 19 19 29 19 SH 1D HH 19 19 © 19 © CO 19 HOS OOD 09 09 OD OD OD SH SH SH SH NI OD OD SH OOD SH SH OD PPE cry OSG 86E c6L E8E GOL 066 F9G 006 €8P O6F Z§9 LVE 266 GIP 802 OTL &Zé 88P 880 0 9S9E 4 GGO O* L896 & 8c0 O= T€99 ¢ 260 0 1169 9 170 0+9Z8F 9 990 0+ 8982 9 00’ 0+ Sc80 9 9F0 0 GPS8 9 £70 O= LE8P 9 960 0+ L9T9'9 660 0 G98E 9 [60 0+ 69€6 9 S20 OF L6LV 9 OF0 0 GS9F 9 840 06269 9 €60 OF GI9T 9 FEO 0+ 90I8 9 £80 0 8996 ¢ 050 0+ $F86'8 €€0 0+ 2292 9 tr 8 89°9 og 9 (0.6) N Ot ty et OY eb Re) st rt id 290 20 re ~KOooronn nt S We oO =a) 1D © 19 19 19 © O 19 © 19 19 19 19 19 19 S ~~ N 29 Oo O29 © I N ON N N a THN OOO N SS 19 tH OD mM NS NNMIWD NTN oD Noo torIioatan N S I bee Aanmnmr~eott tear er) nN =) N 0 Oniogt st X & Ta Oo tH o> © rr % CO ol asd OD NS = oD D a>aNm> ore alcol A STANDARD DEVIATIONS EXTRA BRISTLES FEMALES nM \ I 1 \ U 8 ry ‘ r \ \ 4 I \ Iy , 7X 1 \ TRAN 16 i\ Lae \ Lia \ - ye \ HIGH RACE \ Ve i * \ ii ans \ ; 1 1.4 \ | ! | 12) 1.0 EXTRACTED RACE GROUPS | 18 20 22 24 26 28 30 45 (6, Be Ome aiseIS B Fig. 10 Standard deviations of the extracted race compared with the stand- ‘ard deviations of the high race. ‘A’ based on sons, ‘B,’ on daughters. As the similarity of the standard deviations of the return and high races indicates sim- ilarity of the races, so the differences in the variability of the extracted and the high races indicate dissimilarity in these two races. duced lower children. But after this, the means of the off- spring were completely independent of the means of their parents. Although this extracted race responded to the same environment in the same general way as the high race, there was some sort of a restriction that inhibited the production of the higher bristle grades. BRISTLE INHERITANCE IN DROSOPHILA 139 DISCUSSION The role of environment When families raised at the same time are compared, the means of the return, extracted and the main high race show a parallelism. Of these three races there is one with high parents and high means, one with low parents and high means, and one with low parent and low means, yet the means of all the off- spring rise and fall with striking similarity. Differences in par- entage, similarity in environment, similarity in variation; it is an evident conclusion that these variations are due to some- thing other than differences in the germ plasm. There seems to be considerable justification for the belief that the greater part of the variations found in the curves is due to differences in the environments of the developing flies. An inverse rela- tionship has been found to hold, in general, between the age of a culture and the number of extra bristles on the flies hatching; the older the bottle the smaller the flies are apt to be. The number of extra bristles appearing can be controlled to a certain extent. At any time small flies can be obtained by allow- ing the larvae only a small amount of food; this may be done either by allowing the bottle to dry, to mould, or to have too small an amount of banana. Optimum conditions for the pro- duction of small flies are the optimum conditions for the produc- tion of low bristle grades. When a unique set of atmospheric conditions are encountered, as in the constant temperature room, there appears a unique modification of the means (generations 29 to 31, high race) It seems hardly possible to escape the conclusion that the controlling factor in producing the varia- tions in bristle numbers is environment and not heredity. ‘This may incline some readers to immediately claim that, since the influence of environment is so strong, it would be im- possible to discover any changes in the germ plasm, and forth- with condemn any conclusions that may be drawn from these data. At this point it will be well to consider the basis for our suppositions that we can know the character of the germ plasm by the condition of its somatic bearer. 140 EDWIN CARLETON MACDOWELL It would be difficult to conceive of an organism that could mature free from all forces except those conditioned by the germ plasm. Every stage of an individual’s growth is shaped by the reaction of the germinal to the extra-germinal, the hereditary to the environmental. In the case of the flies, the role of the environment in controlling the bristle numbers is obvious; we see changes in one, we see changes in the other. In some other cases of variation it is not possible to see, in this clear and simple way, changes in the environment associated with changes in somatic structures. Failing to find such a relationship, it is tempting to conclude that the variations in the soma do not indicate environmental changes but rather ger- minal variations. Can it rightly be supposed that in the ab- sence of correlation between obvious fluctuations in temperature and food and such like, that there are no other controlling influences than those originating in the germ plasm? ‘The an- ticipated criticism of this work is that the environmental influ- ences are too potent for the germinal changes to appear. But suppose the power to influence bristle numbers, that seems as a matter of fact to lie in the amount of food eaten, etc., rested in some unknowable condition outside the germ plasm, such as the age of the sperm before fertilization, or the temperature of the mother’s body during the maturation of the egg, then this objection would probable not be considered. The individual may be considered to be a reaction between the germ plasm and the environment but does it not sometimes seem that students of selection are so intent upon their consideration of the germ plasm that there is a tendency to ignore the scope of the influence of environment? In this way one of the most essential tools in attacking the problem is neglected. The point to be empha- sized is that there is very little support for the supposition that the soma mirrors the germ plasm in all cases except when obvious environmental relations are found. It seems hardly possible that one éan look forward to ever establishing firmly an exact relationship between soma and germ plasm, at least in bi-sexual multicellular animals. In the present case the difficulties are all to clear too permit the statement that the germ plasm did not BRISTLE INHERITANCE IN DROSOPHILA 141 change in any way during the many generations of unsuccessful selection. However, in spite of the strong influence of environ- ment, some conclusions that do.bear on ‘the problem of the germ plasm are to be drawn. It may be concluded that environment was not responsible for the initial rise in the high race. Environment was as potent at this time as at any other, yet only at this time were the curves of the parents and offspring parallel. At the beginning low selection was successful, showing that genetic differences between low and high grade flies did exist. After this rise, the race as a whole was changed. This is shown by the failure of the return selection; at first, selecting these same low grades of parents established a low race. Crosses result in more modification after this rise than before. The analysis of indi- vidual families shows that before this rise the parents with higher grades produced children with higher mean grades, while after this rise, similar analysis shows that the highest grade parents were no more likely to produce offspring with especially high than with especially low mean grades. It is certain that there were differences already present in the germ plasma of the flies first bred; that these same sorts of dif- ferences were not found among the flies whose ancestors had been selected for several generations; in other words, in regard to those germinal differences that account for the parallelism be- tween parents and offspring in the early generations, that deter- mine the difference between the successful low selections at the beginning and the unsuccessful return selections later on, that make the difference between an uncrossed selected race and a selected race that has been recovered from the dominance of a cross by normal, in regard to all such demonstrable differences, one finds that selection has sorted out a uniform race. Such a conclusion will stand however completely the environment may be proved to control the fluctuations in all but the early gener- ations of the high race. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 1 142 EDWIN CARLETON MACDOWELL Accessory factors vs. one variable factor Although the evidence was against it, the possibility was ad- mitted‘ of applying to the first eleven generations of selection an alternative hypothesis involving one ever-varying factor, similar to the hypothesis adopted for the selection experiments of Castle. The hypothesis of accessory factors has formerly been discussed in full; its application to all the data will now be made, and then the impossibility of so applying the alternative hypothesis, without numerous subsidiary hypotheses, will be shown. The germ plasm of the original pair of flies, besides having a factor that permitted more than the four normal bristles to appear, had accessory factors that influenced the numbers of extra bristles that developed. Some of these accessory factors were in a heterozygous. condition so that, due to their segre- gation, the germ cells of these flies were of different kinds. The effect of these gerrninal differences was strong enough to make the somas of the offspring fairly good relative indices of their ger- minal constitutions, even though the vitiating influences of en- vironment were active. Selection of extreme variates moved the means in the direction of the selecting, raising them or keeping them low. Generation by generation the selecting reduced the heterozygosity of the accessory factors, so that the segregation was reduced and the germ cells that were produced were less varied. Shortly the differences between germ cells in an individual disappeared or at least became so small that their influence was overpowered by the influence of environ- ment. Variations in bristle number then had no relation to the germinal condition. As long as brother by sister matings were made, the high and low grade flies produced similar offspring; no longer was it possible to move the means either up or down by selection. But as soon as these flies were crossed with normals, the distribution of the extra bristles was lowered; the 4 MacDowell: loc. cit., p. 91. ® Castle, W. E.: Experiments in mass selection. Amer. Nat., vol. 49, no. 588, pp. 722-723. BRISTLE INHERITANCE IN DROSOPHILA 143 heterozygosity of these accessory factors was regained in F,, and in F, the segregation of these factors resulted in the lowered bristle averages, and again offered a chance for selection to modify the means. The differences between germ cells had become great enough to influence the somas in spite of the uncontrollable variations in the environment. If a single Mendelian factor that varied in potency be assumed, the production of a high and a low race would be the expected result of selection. Furthermore, it would be expected that the transformation of the high race into a low race would be just as easy. Since this was not the fact, it becomes necessary to as- sume further that, because the factor had varied a certain amount in one direction, it was hindered from varying in the opposite direction—a supposition for which one would have difficulty in devising a mechanism. If one varying factor be made the basis of the explanation, a special hypothesis must be made to explain why the advance shown in the early generations of selecting was not continued. The physiological limit in bristle numbers had evidently not been reached; some limitation on the higher variations of this factor must be assumed. The last two hypotheses leave a variable factor that, after a series of selections is limited in its further variations in both directions. It becomes a matter of the number of generations before the variability of this factor no longer exists, before the end of the cul de sac is reached. Further subsidiary hypotheses would have to be added to account for the results of crosses with normals. It would be necessary to assume that the variations found in the germ cells of an individual heterozygous for this factor, are dif- ferent from those in the germ cells of a homozygous individual. The ‘Contamination theory’ affords an explanation for this. The cross brought this factor and its allelomorphic mate to- gether in the same nucleus. In order to explain any difference due to this heterozygosity, it must be assumed that there is an intimate fusion between these two members of the pair, and then that this fusion weakens the power of that factor for form- ing extra bristles. When the high race was selected longer, the modification of the means and modes found in F, was greater, 144 EDWIN CARLETON MACDOWELL yet the extremes were as high as in the uncrossed race. To explain this one may assume that when the extra factor is more potent, it is more easily weakened by its contact with the normal factor, but that in some cases it is not weakened at all. If there be free variations in the factor it is difficult to under- stand the rigidity with which the normal four bristles are held as the. lower limit of variation. There may be flies with only the normal four bristles in almost any generation of selection, yet, no matter how small a fly may be, the four normal bristles are inevitably found. ; There can be no question as to which of the two main hypo- theses is more fully supported by the interpretations that have been made of the facts. The supporting hypotheses required to make the hypothesis of a single varying factor fit the case are so numerous and, in some cases, so unthinkable as to render the main hypothesis of very slight value. It may be claimed that, in time, in a hundred instead of fifty generations, selection could have accumulated enough ger- minal variations to bring the germ plasm again in contro of the numbers of extra bristles. But if there are differences in the germ plasm already present or continuously occurring, a long series of generations is not required to prove their existence. It would seem rather a forced conclusion to claim that the changes that might finally appear in a line that had been se- lected without success for a long time, were due to the long selection. It would not be hard to believe that if one waited long enough, without any selection at all, mutations could be found in almost any material. The point at issue is whether the changes in the germ plasm are continuous or spasmodic, whether they are like the continuous breakers on an ocean beach or the storm-caused splashings of an inland pond. It does not take long to find the breakers at the sea shore; one may spend weeks beside a little lake without seeing a single wave dash against the bank. In these experiments germinal differences were found to exist; their presence was found almost immediately. But after selection had separated the different kinds,no further differences were found. BRISTLE INHERITANCE IN DROSOPHILA 145 As stated, this paper has not attempted to deal with the problem of absolute stability of germ plasm; it has questioned the theoretical possibility of a solution for that problem; but it has attempted to show that none of the various phenomena herein described require the assumption of any internal, or spontaneous, change taking place during the course of these experiments. SUMMARY I. A race of flies with extra bristles has been selected for 49 generations for the production of high numbers of extra bristles. From the study of this race the following points have been determined: 1. In any generation after the early ones the distribution of a single family is similar to that of the distribution of al the families taken together in that generation. 2. For about 8 generations the means rose; following this were two periods not comparable with each other, within neither of which was any evidence of further advance to be found. 3. Continued selection did not produce any high extremes that were not. obtainable near the beginning of the experiment. The range of variation changed only very slightly; the low limits being most frequently at 0 or 1, the high limit at 9 for the females, at 7 for the males. 4. The standard deviations rose and fell together with the means; as the means of the females are higher than those of the males, so the standard deviations of the females are higher than those of the males. These relationships do not hold true when the complete yields of the bottles are not included (generations 33-49). 5. Changes in the means of the parents are not accompanied by changes in the means of their offspring, except at the begin- ning of the experiment. II. By selecting low grade parents from the second generation of the extra bristled race a race of flies was established which had markedly lower means than the high selected race. III. By selecting low grade flies from the 15th generation of the high race and. continuing to select for low grades, it was 146 EDWIN CARLETON MACDOWELL impossible in 8 generations to establish a race that was distin- guishable from the high race. This attempt was repeated, start- ing from the 26th generation of the high race, and continued for 6 generations with similar results. Return selection does not reverse the progress made by the advance selection. Flies with high and low bristle grades appear to have very similar offspring. IV. By selecting low grade parents from the F, of a cross between normals and flies from the 16th generation of the high race, a low race was established (extracted low) The following points were derived from the study of this race: 1. One selection was sufficient to establish this race as dis- tinct from the high race. 2. For 4 generations the curves of the parents and off- spring are parallel. After this, the two curves are completely independent. 3. For 4 generations the low selection continued to lower the means. 4. Except in the first few generations, the curves of the prog- eny rise and fall in harmony with the curves of the high race, when families raised at similar times are compared. 5. Besides being lower than the high race the variability of this race is less than that of the high race; in response to the same improvement in conditions this extracted race does not advance as far. V. Comparing the different races it is found that no matter what the parentage, they all exhibit high points and low points at the same times. Environment is accountable for the varia- tions in most of the generations. The initial rise in the high race however, was not due to environment, as this rise resulted in a genetic change in the race. VI. The supposition of a single varying factor to explain the above results can not be justified, as it would require num- erous further assumptions. All these results are simply ex- plained on the assumption that there were genetic differences present among the original flies with extra bristles, that these genetic differences, or genes, are entirely independent of the main factor that occasions the monohybrid ratio in crosses with normal flies. THE ACTION OF VARIOUS PHARMACOLOGICAL AND OTHER CHEMICAL AGENTS ON THE CHROMA- TOPHORES OF THE BROOK TROUT SAL- VELINUS FONTINALIS MITCHILL JOHN N. LOWE From the Department of Zoélogy, University of Wisconsin THREE TEXT FIGURES AND ONE PLATE CONTENTS Materialgancdeme th ods tery. tc. cack s 5.0 cee Oe od eee 148 RveENC OOS) UO a2 STS 08 a a Be ce) yy la & 150 UAC Qi ielilss oo AA Ad ook ees aeeye cok ig aes Tee 150 PCA LHORSEGIOXIGG:4. ta... os ae eee ee 150 Bicctyomersiiled water >. 5045-0... ...2.cne tee ee 151 HEA CHES HOUSER...) cs, Sea ete... . ccna I le: A 1152 Reiheces ainpatassium, saltg.....:....<. 2 sere se beet: soe) ats.) Sas 153 Zo Lite cts Omsodium: saltseaks... .. Saye ee ice basin 154 3 DISCUSSLONES Wee ....,... oan: 5: A ne eager oo eras 156 ReaActionsstoralCoholsss.:........ sty. + .. ae a he. oe oe 163 devMiethylbalecholee os ..4:.ookaaee 5 5 ke ee eres 164 Pd Diode Se ce SN . ae! ae 164 GemErO pW AlCOHOl nin. Ss <,eRRae. ©. . 5. Ae eee pe en Tee 167 Reactionsstoralicalords:c:..... 0. eos...’ ne tee Ee eee SE 169 LOTSA (GLO AVI ee I SOA ee PU ae 170 NC EE UO ero Sacegdithe «An 1 ee, ee 172 De OET AUD ChE Tey eee s « . 2 fe sche. «2 EE ree eE coc ees 173 oh. AORN TEN 5 oes a see es lhe Se ea 174 bh (CURD 3 208 SO 2 At ne ORE RR eR o> Wy ie hs Pama a ane 176 Oe INTOC TIE SER ki Ge i ene he eee Re | ha a eae 178 (fo! SASSO) UNLESS a, Ap Ca AI cP icc. Sand 0 179 Se OOCAITICNE ES fe 78.2 oso \o > bea es et dle he 180 OMMVCRAUGITMC emer leo! oy 3s. Sete Raa Gem MULE RNs" nS 8 ALE 181 MU) unre eagle es) diss ea ei IG Pee Oe Soh gs EY 182 ro JMET AA, oisc'e MEMS AcE ep er ORR Se 28 We ORS, SR rr 183 LEMON OF 6100 [5.0m seamen Sires) iF Se a ee 187 The reactions of melanophores (pigment cells) to pharmaco- logically active agents have been but little investigated. In the 147 148 JOHN N. LOWE majority of the physiological researches upon the melanophores, the experiments have only included the study of such physical agents as light, heat, etc. The problem here undertaken was to determine the reactions of the melanophores of young trout em- bryos in response to changes in their chemical environment. The trout embryos that were used in these experiments were too young to react to a change in the light conditions, and through- out the work gave no evidence of any psychic influence of the pigment cells. MATERIAL AND METHODS Young brook trout embryos, from two days to two weeks after hatching, were used. The melanophore of such young individuals are dark, much branched cells with deep black or brown pigment granules. These are the only kind of pigment cells present at this time. The xanthophores, the yellow or reddish pigment cells, appear after or a little before the yolk is absorbed. All the experiments were performed before the xanthophores appeared. After the yolk is absorbed the fish begin to react to the back ground. When placed in a dark dish, they become dark; when placed in a white dish, light in color. Microscopical examina- tion shows that the pigment cells (melanophores) are expanded in the dark colored individuals and contracted in the light ones. The very young, two-day or two-week old embryos do not respond to changes of the back ground. ~ This constant condition is taken as a known factor. The contraction of the pigment cells was used as the criterion for determining stimulation, and their expansion (relaxation) as a mark of depression. The expansion of the pigment cells is char- acterized by the peripheral migration of the pigment granules within the processes of cell, and in contraction the movement is centripetal. My reason for considering contraction as stimula- tion and expansion as a depression is that certain reagents, alka- loids for example, if used in high concentrations produce no ob- servable change in the pigment cells which under normal con- ditions are expanded. Small or ‘therapeutic’ doses produced a CHEMICAL AGENTS ON CHROMATOPHORES 149 contraction. Large doses produced an expansion of all-the cells which had contracted in the weak solution. Inasmuch as it has been shown by various investigators that large doses of pharma- cologically active agents produce a depression, and small doses incite a stimulation in other tissues, it is inferred that the condition is essentially the same with the melanophores. All the chemicals used in these experiments were of Merck’s, Kahlbaum’s and Baker’s manufacture. The solutions were made up with oxygenated distilled water. Chemically pure oxygen was bubbled through the water before it was used. This precaution was taken because the distilled water was very low in oxygen content and in it the pigment cells contracted. When oxygen was added no such contraction occurred. The details of the way in which the solutions were prepared are given under the respective heads. The experiments with the salts and the alkaloids were carried on in Syracuse watch glasses, which were kept covered to prevent excessive evaporation. They were uncovered only when actual observations were made. The amount of the solution used was about 10 ec. Experiments were performed in stender dishes of 50 cc. capacity as a check on the Syracuse watch glasses. There was no difference in the results. The experiments with volatile substances were carried on in wide-mouthed, glass stoppered bottles, with a capacity of 50 cc. All precautions were taken to prevent evaporation. Most of the experiments were carried on at room temperatures which varied between 69° and 72° F., although some were per- formed at the fish hatchery where the temperatures were from 46° to 50° F. The experiments with the alkaloids and alcohols were started in solutions of 0.0001 per cent. The concentrations were in- creased in multiples of ten. The experiments were repeated ten to fifteen times for each solution tested. In many cases the experiments were repeated double the number, in order to eliminate all possible individual variation and errors. 150 JOHN N. LOWE I wish, here, to express my chief indebtedness to Prof. M. F. Guyer, for his kindly criticism and suggestions during the progress of the work. To Prof. A. 8. Loevenhart, I wish to acknowledge my appreciation of many courtesies extended. For the privilege and use of the fish hatchery and trout embryos, I desire to express my appreciation of the favor to Dean E. A. Birge and Superintendent James Nevin of the Wisconsin Fish Commission. Reactions to gases 1. Oxygen. The oxygen used in these experiments was chemi- cally pure. The pigment cells remained expanded in an atmos- phere of oxygen, and the fish lived indefinitely. The hydrogen used in these experiments was obtained by the action of chemically pure hydrochloric acid on Merck’s highest purity zinc. The gas was passed through two towers of KOH and then through two towers of distilled water, of which one had red litmus, and the other blue litmus. The trout were in the fifth tower. The pigment cells contracted completely in four to six min- utes when the embryos were exposed to hydrogen. If oxygen was substituted before the fish died the pigment cells expanded. If the oxygen was again replaced by hydrogen the pigment cells contracted. The results of these experiments’show (1) that the absence of oxygen caused a contraction of the melanophores; (2) that the oxygen is necessary for the maintenance of the expanded pigment cells. ; 2. Carbon dioxide. The carbon dioxide was generated through the interaction of chemically pure hydrochloric acid on marble. The gas was purified by being passed through a tower of sodium bicarbonate and then through a tower of acidified lead acetate, and lastly through two towers of distilled water. The fish were exposed to water through which the carbon di- oxide was bubbling in a steady slow stream. The carbon dioxide produced a complete contraction of the pigment in two and one- half minutes. The time of contraction was the same for all the CHEMICAL AGENTS ON CHROMATOPHORES 151 experiments performed. If an intense stream of oxygen was bubbled at the same time with the carbon dioxide, the pigment cells remained expanded. The proportion of the two gases which maintained the expansion of the melanophores was not de- termined. Briefly summarized the results prove that carbon dioxide produces a contraction of the pigment cells of trout embryos. The presence of oxygen antagonized the action of the carbon dioxide. Effects of distilled water The first experiments that were performed were to determine the effect of distilled water on the pigment cells of trout embryos. The normally expanded pigment cells contracted in ten to twelve minutes and the fish died usually in about twenty minutes— differing somewhat with the individual lots of fish. After an in- terval of ten to thirty minutes, following the initial contrac- tion, the pigment cells began to expand. This secondary ex- pansion of the melanophores in no way equaled the normal ex- panded condition. The processes of the cells were short and blunt. This expanded condition lasted for a short period; then the walls of the melanophores began to break down and the cell contents, viz., the pigment granules migrated into the inter- spaces of the epidermal layer. Often the pigment cells disinte- grated without a previous expansion. Spaeth (13) obtained essentially the same results with isolated scales of Fundulus in which the chromatophores (1) expanded, (2) contracted, (3) expanded a second time with a final degeneration. He did not try oxygenated distilled water. If 2 cc. of boiled tap water were added to 8 ec. of distilled water the results were the same. Then boiled tap water was tried and the pigment cells con- tracted in fourteen and twenty-two minutes. In distilled and boiled tap water through which oxygen had been bubbled the melanophores remained expanded and the fish lived indefinitely. The conclusion was obvious. It was oxygen want and not the absence of salts in the distilled water that caused the contrac- tion of the pigment cells and the death of the fish. 152 JOHN N. LOWE Reactions to salts The problem of salt action is one of the most interesting within the scope of physiology and has wide applications. The relation of various salts to heart beat is a long debated question. Howell (’98), p. 49, is of the opinion ‘‘that the inorganic salts of the blood and liquids of the heart tissues especially of the calcium compounds, stand in a peculiar and fundamental rela- tion to the initiation of the inner stimulus of the heart con- tractions.”’ Loeb (’00 a, 00 b) believes that the sodium cations acting on the striped muscle to be the stimulating agents being counteracted by the ions of potassium and calcium. The posi- tion of Loeb is supported by Lingle (’00). Benedict (’05 and ’08) is of the opinion that the anion probably plays an important ~ role in the action of salt solutions upon heart beat. Mathews (04a, ’04b, ’05, 06) maintains that in the action of salt solutions on motor nerves, colloids, and sea urchin eggs, the ionic potential of the salt, which is the reciprocal of the solution tension, is an important factor in ionic action. R. 8. Lillie (11, ’12a, ’12b) working with the larvae of Arenicola and eggs of starfish, and McClendon (’10) on sea urchin eggs put forth the hypothesis that ionic action is due to the modification of the permeability of the plasma membrane. Loeb (’00 b) holds that ionic action is due to the formation of ion protein com- pounds, that is that the ions of the salt combine directly in some way with the protein molecules of the living protoplasm. True and Kahlenberg (’96) working with plants (Lupinus albus) be- lieve that the anion is unimportant in the toxic action of the salt. Spaeth (13) working on the chromatophores in isolated scales of Fundulus heteroclitus concludes that the anion in potassium salts is of no importance in causing the initial contraction of the chromatophores, but that in the secondary expansion of the chromatophores the action of potassium is modified by the anions. On the other hand, the duration of the sodium expan- sion varies with the nature of the anion. CHEMICAL AGENTS ON CHROMATOPHORES 153 The above opinions tend to show that the part played by ions in stimulation is by no means a settled question. In an attempt to gain further insight into the subject brook trout embryos were subjected to solutions of pure potassium and sodium salts. The results have been so promising that the work is being extended to numerous other salts. The salts used were of the purest of Merck’s, Kahlbaum’s and Baker’s manufacture. The solutions were made up in a 0.2 molecular concentration with oxygenated distilled water. The solutions of the iodides which readily undergo decomposition were never older than thirty-six hours when used. The experiments were carried on in Syracuse watch glasses in about 10 ce. of the solution. At times small dishes of 25 to 50 ec. capacity were used. 1. Effects of potassium salts. When the trout embryos are immersed in a 0.2 M. KI solution a rapid contraction of the nor- mally expanded chromatophores results within two or three minutes. They then appear as minute dots with no peripheral processes. In placing a similar lot into a 0.2 M K.SO, equiva- lent solution the change does not occur as rapidly, being com- pleted in fifteen to twenty minutes. This at once suggested that there is a specific difference in the rate of contraction for potas- sium, varying with the anion. The experiments were extended to include the following neutral salts of potassium, viz., K.SOu, KCl, KBr, KNO; and KI. Practically the first experiment showed that there was a distinct difference in the rate of con- traction varying with the anion. The rate and intensity of the contraction was most rapid in the order given (figs. 1, 2, 3, 4 and 5). iS NO; > Br> Cl> SO.> In KI the contraction was complete before it had even begun in KCl or K.SO,. The experiments were repeated many times and as a check several of my colleagues were asked to come in and arrange the sets showing the greatest change. In all cases their arrangement was in the above order. This clearly indicates that if contraction in the melanophore is specifically induced by the 154 JOHN N. LOWE cation of potassium, it is unqualifyingly modified by its anion or the residual part of the undissociated molecules. Another interesting feature observed was that after a longer or a shorter interval after the first contraction there followed a peripheral expansion of the pigment cells (figs. 6, 7, 8, 9 and 10), that is, the pigment cells put out processes which became longer and longer as time went on but which never reached the original size they had before treatment with the potassium salt solutions. This expansion set in earlier in KI where the contrac- tion took place first, evidently the secondary expansion or pa- ralysis is reciprocal of the first contraction. The expansion is in the order of the first contraction (figs. 6, 7, 8, 9 and 10). ts NO; > Lycos Be we) ies SO, This peripheral migration of the pigment is in the nature of a paralysis. The paralytic state (depression) is soon followed by death of the pigment cell. The walls of the pigment cell dis- integrate and the pigment granules flow into the interspaces of the body tissues. Death of the cells takes place often before the expansion is complete, and then premature disintegration of the pigment cells occurs. The condition or extent of the de- generation is dependent upon the ‘physiological state’ of the melanophores and the individual fish. The maintenance of the irritability of the melanophores fol- lowed the same order, correlated with this was the longevity of the fish. The fish lived the longest in K,SO; and KCl. They died very rapidly in KI. The reactions varied with the concentration of the solutions, for in solutions of 0.1 M or less the changes were slightly slower. Molecular solutions gave no results but killed the fish imme- diately. 2. Effects of sodium salts. Here as in the potassium salts the embryos used had their melanophores expanded. It was ob- served that the neutral salts of sodium produced a contraction of the melanophores very slowly. In some instances the contrac- tion did not take place in 92 to 116 hours, especially in the solu- tions of NasSO,and NaCl. The contraction in Nal was complete CHEMICAL AGENTS ON CHROMATOPHORES 155 in five to forty-five minutes. It was confirmed by repeated ob- servation, that these contractions, slow as they may be for certain solutions (Na.SO, and NaCl), were in the following order: Iss NO; > Bre Cl> SO, A number of experiments were tried to determine if the sodium salts produced an expansion of the melanophores after the po- tassium salt contraction. The embryos were exposed to KCl from fifteen to twenty minutes when they were removed and rinsed in water to free them of the excess of KCl. They were now placed into the five neutral salts of sodium. The rate and degree of expansion was in the following order: S0.> Cl> Br NO; > I The expansion was most rapid and complete in Na,SO, and NaCl. In Nal there was no expansion. The experiments were repeated with embryos that were not rinsed with water. The result was the same as in those that were washed in water. If the melanophores are contracted with KI instead of KCl the results are the same. SO,> Cis Br> NO; > I It is interesting to note here that no expansion of the mel- anophores occurred in the Nal solution. Is this because the sodium cations are inhibited in permeating the cell membrane due to the presence of the dissociated iodine anions or some other factor? Are the cells permeable only to the iodine anions and not to the cations of sodium? Hamburger and von Lier (’02) claim that the blood corpuscles are permeable only for anions and are not permeable to the cations. If the expansion of the melanophore is specific for the sodium cation, it is overcome by the antagonistic action of the iodine anion, which produces a contraction. Nevertheless we must consider another factor, that is, the action exerted by the residual undissociated molecule which is present at all times in the solution. The expansion in- 156 JOHN N. LOWE duced by the sodium salts after a potassium salt contraction is followed by a contraction of the melanophores in the usual order. The position or order of the contraction was the same as for the expansion of the melanophores; but with one excep- tion where the NaNO; changed places with the NaBr. SO.> Ch> NO; > Bes I The extent to which the life of the fish and the irritability of the melanophores are preserved is possibly the function of the cation which is modified by the anion or the residual undisso- ciated molecule. 3. Discussion. All these results seem to lend themselves to the interpretation that salt solution having a common cation are modified by their anions or the residual undissociated mole- cule. This is clearly shown by the rate and degree of the con- traction of the melanophores by the potassium salts, where the contraction may be specific for the cation of potassium. Speath (13) p. 547 says in speaking of the action of potassium salts: “The time of this contraction (K) is the same for the five salts within the limits of the variation of the individual scales. Since there is this common cation K+ in all five salts it seems prob- able that the initial effect (contraction) is specific for the K+ ions.’”’ My own results in the case of pigment cells of trout embryos are contrary to this conclusion. If contraction is spe- cific for the positive cation of potassium (K*), it should be the same in rate and degree in all the salts of potassium. Since the rate and degree of the contraction are not the same for the five potassium salts (figs. 1, 2, 3, 4, and 5) it must depend on some other or some modifying factor which is responsible for this difference. A dissolved electrolyte conducts a current in proportion to the extent that it is dissociated or ionized. Its maximum conduc- tion will be at complete ionization which occurs at infinite dilu- tion. Therefore the degree of the dissociation or the coefficient of dissociations can be obtained from the conductivity of solu- tion. The conductivity of an electrolyte divided by its num- CHEMICAL AGENTS ON CHROMATOPHORES 157 ber of gram equivalents in cms. is the molecular conductivity’ of the substance written as A. However, the conductivity is at its maximum at infinitely dilute solutions, therefore the value Ac is taken as a measure of the total number of ions that are produced by the dissociation of one gram equivalent of the substance. Therefore d the degree of dissociation is directly proportional to the conductivity; thus we have the simple form- ula a = ae The equivalent conductivity at infinite dilution for KCl is calculated to be 130.10. The equivalent conductivity of a two-tenth molecular KCl is 107.96 A o2 m. The degree of che ad: TX O20M. § 107.96 dissociation at 18°C. is the ratio Fess) OY 13919 82.98 per cent. The values obtained in this way may be regarded only as approximate. The values are given in the following table. TABLE 1 SALT % KeSOs KCl KBr KNOs KI infinite dilution A « Equivalent conductivity at 0.2 M dilution A 0.2 M Equivalent conductivity at \ 192.8 130.10} 132.30] 126.50! 131.10 } 87.76 107.96 | 110.40] 98.74] 111.2 tiona = A0.2M 82.98 83.44 78.05 84.82 Per cent or degree of dissocia- 66.03 A« A study of the table leads one to believe that the rate and the degree of the contraction are in some way correlated with the degree of dissociation of the salts. The lowest rate and degree of . contraction was found in K,SO,, where the degree of dissociation is 66.03 per cent. The most rapid and complete contraction occurred in KI where the dissociation is 84.82 per cent. Potassium nitrate is out of place. It has a greater stimulating action than its degree of dissociation would indicate. It should fall between potassium sulphate and potassium chloride. The possible explanation for this break in the series may be that the THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 1 158 JOHN N. LOWE TABLE 2 SALT 4 Na2SOu NaCl NaBr NaNOs3 Nal Equivalent “conducunaoyer 23/5 eased | iow) 1105 -conlmnane infinite dilution A. « Equivalent conductivity at 3 0.2 M dilution A 0.2 M (14: 87.73 91.2 82.28 90.2 Per cent or degree of dissocia- tiona = A0.2M 64.03-| 80.49.) 81.43) 78.11 | ©8208 A & nitrate anion exerts an independent action or it may form nitrites which are more active. In table 2 are shown the equivalent conductivities and degree of dissociation of the sodium salts. The values were calculated in the same manner as those for the potassium salts. Here, as in the potassium salts, the reac- tion of the melanophores was correlated with the degree of dissociation. There are two reactions of the melanophores which are char- acteristic of the potassium salts: (1) a primary contraction, (2) an expansion which is the sign of death or degeneration of the cell. The cell wall breaks down and the pigment granules escape into the surrounding tissues. The degree of the cytolysis is directly proportional to the degree of dissociation of the salt. In sodium salts we have two specific reactions: (1) the expansion and maintenance of the expansion for a certain period of time, (2) a slow contraction. The two reactions of sodium salts occur in an inverse order to those of the potassium salts, where con- traction is followed by a cytolytic expansion. The contraction in sodium salts is not followed by a cytolytic expansion, but the disintegration takes place directly from the contracted pigment cell. This contraction in sodium salts is directly comparable to the cytolytic expansion observed in potassium salts, for both ° of these stages indicates the death of the pigment cell. A. P. Mathews (’06) suggested that it is the ionic potential of the ions, and not the difference of voltage between the plate of CHEMICAL AGENTS ON CHROMATOPHORES 159 a metal and any solution of its salts, but rather the difference in pressure between a single ion and a single atom of the metal that determines the chemical action of the ions. Since solution tension is a measure of the difference in potential between the solution which contains a known amount of the ions of the metal and the metal itself, it is also the difference between the tendency of an atom of the plate to become an ion. When applied to living protoplasm the metal plate is replaced by the protoplasm. The yalue varies with the amount of electrolytic dissociation and the kind of plate present. The solution tensions in volts of elements in normal ionic solutions. CS ae RPS es es 3 ee 1.694 Gaps n tenes, 2.92 1B eT bios Ce eae eee Re 1.270 Na 2.54 LB raced cree a Re 0.797 INO See yacter wee Weeds saves 580 2.229 The ionic potential is the reciprocal of the solution tension. Ionic potential is the tendency of any ion in any concentration of solution to change into an atom of its metal. The ionic potentials of the ions of metals in volts are: Cl.2 ee eee en) 1604 (2) RGU Cle ies eh Br> Cis SO, In sodium salts there were two characteristic reactions, (1) an expansion, (2) a contraction. The rate and degree of the ex- pansion of the melanophores was greatest in Na.SO, and least in Nal. The rate of contraction was rapid in Nal and least in Na.SO,. The order of the expansion was SO, > cls Br> NO; > I The contraction rate of the pigment cells was inverse to the above. | ess Br> NO;> (Ci SO The cationic action was modified by the nature of the anion. This anionic order was observed by Paul and Kronig (’96) on the disinfecting power of mercuric salts of chloride bromide and cyanide. Mathews (’06) has shown for the eggs of Fundulus heteroclitus that the fatal dose varied with the anion. Loeb and Cattell (15) have shown that the hearts of Fundulus embryos, previously poisoned by KCl, and recovered by sodium salts was an anion effect inasmuch as it increased with the anion, apparently in agreement with Hardy’s rule (ion effect = ex- ponential function of the valency) for the acetate was much more efficient than the chloride. 2. That it is the cation of potassium or of sodium that causes the reaction of the pigment cells of trout embryos. Loeb (10, 712) and Loeb and Wasteneys (lla and ’11 b) maintain that there is an antagonism between the sodium cation 162 JOHN N. LOWE Na + and the potassium cation K + and not between the po- tassium cation and the chlorine anion K + Cl—. This is sup- ported in part by the foregoing experiments on the pigment cells of trout embryos. The pigment cells are expanded in sodium salts after a potassium salt contraction. But this is not true of all the salts of sodium. If the pigment cells are con- tracted in KCl or KI and are now placed in Nal there is no ex- pansion. Apparently there is an antagonism between the dissociated anions of (Cl — and I —) and the sodium cation (Na +) for from the conditions of the experiment we should get an expansion. It is probable that Loeb underestimated the antagonism between the positive ions of K + and Na + and their negative ions Cl —. The longevity of the fish is better protected in sodium salts than in potassium salts. But again some of the sodium salts are more protective (Na.SO, or NaCl) than others (Nal). That the potassium and sodium cations do exert some such modifying action is undeniable, but to say that it is independent of its anion is not warranted by the facts at our command. 3) That it is the residual undissociated molecules in the solution that modify the action of the salt. ; In 0.2 M, KI the degree of dissociation is much greater than in an equivalent 0.2 M, solution of K,SO,. Correspondingly KI initiates more intense responsiveness of the pigment cells than does K.SO,. The rate and degree of the reactions of the pigment cells decline as the number of the undissociated molecules in- creases. In the potassium salts the primary contraction and the expansion vary with undissociated molecule, thus, SO,> (Gili Bre> NOs I The degree of dissociation for 0.2 M solutions are 66.03 82.98 83.44 78.05 84.82 The fact that the nitrate is out of place was mentioned before. As already stated, this may be due to the independent activity of the nitrate, which may break down to form a nitrite. In sodium salts the dissociation percentages are slightly less than in potassium salts. The rate of contraction is much slower. CHEMICAL AGENTS ON CHROMATOPHORES 163 The fish live longer in sodium solutions, and the pigment cells retained their irritability longer. The pigment cells expand if transferred after they are contracted in potassium salts. Is this reaction specific for the sodium ion or is it due to the in- creased number of undissociated molecules in sodium solution? It cannot be positively concluded whether this difference in residual molecules is enough to account for the difference in the rate of contraction of the melanophores in sodium and potas- sium salt solutions. The ascribing of the principles of salt ac- tion to the anion or cation without the consideration of the residual undissociated molecule is just as out of proportion in the field of physiology as to say that the undissociated alkaloids and other substances have no action. Reactions to alcohols Whether or not alcohols have a stimulating action is a much debated question. The Schmiedeberg school of pharmatbdlo- gists maintains that alcohols produce no primary stimulation of the central nervous system. According to this view the giving alcohol to a mammal, if followed by an increased muscular ac- tivity, is said to be due to the depression of the cerebral centers, - thus removing the restraint from the motor areas. Binz and his followers hold to the view that alcohol first stimulates and then depresses the nerve cells. The literature on the pharmacological action of alcohol on the heart and other tissues is very extensive, but to my knowledge there are no records of any attempts to determine its action on the melanophores. Whatever action the alcohols exert on the melanophores will not settle the question whether alcohols stimulate or depress the nervous system, as the melanophores in the very psture of their origin and structure must be looked upon as specialized mesenchymal cells. While it is not at all improb- able that the general facts observed with melanophores may be 1 After these experiments were completed Spaeth 1916 published results, where he subjected isolated scales of Fundulus to vapors of alcohol, ether and chloroform and always obtained a contraction of the melanophores, and larger amounts of these vapors inhibited the contraction. 164 JOHN N. LOWE true also of other tissues, I refrain from applying the results to such an interpretation. The literature is used in a compara- tive way, but not in the sense that the results obtained with melanophores are directly comparable. Ten per cent stock solutions of methyl (Sp. G. O. 796), ethyl (Sp. G. O. 796-800) propyl (Sp. G. O. 8066) alcohols of Merck’s manufacture were made up with oxygenated distilled water. The dilutions were made from these stock solutions with oxygenated distilled water. The experiments were carried on in glass stop- pered bottles of 75 cc. capacity. All work was done at room temperature of 18° to 20°C. 1. Methyl alcohol. Overton (01) showed that methyl alco- ho} has a less powerful narcotic action on tadpoles than ethyl alcohol. Vernon (’11) confirmed that the same was true in the depressing action of methyl alcohol on the- heart muscle of a turtle’s heart. Young brook trout embryos of the same age and condition were subjected to the action of the respective alcohols of the various concentrations. The contraction of the pigment cells was taken as the criterion of stimulation, the relaxation (expansion) as that of a depression. Ethyl alcohol in solutions of 1.6 to 2.5 per cent produced a complete contraction of the pigment cells. Methyl alcohol of an equal concentration did not cause a contraction. In a 3.5 per cent solution there was a slight retraction of the pigment cells, but the contraction was not complete. A 4.5 per cent solution produced a complete contraction of the melanophores. Solu- tions of 5 per cent to 5.5 per cent produced a slight contraction of pigment cells. This partial contraction was followed by an immediate expansion. If embryos in which the melanophores were just contracted in a 0.005 per cent strychnine solution were subjected to 5 per cent to 5.5 per cent methyl alcohol the pig- ment cells expanded. In 7 per cent to 10 per cent solutions of methyl alcohol there was no visible change in the expanded melanophores. Thus it may be concluded that (1) methyl alcohol in high con- centration acts as a depressing agent, (2) in medium concentra- CHEMICAL AGENTS ON CHROMATOPHORES 165 tion it has a stimulating action, and (3) in very weak solution it has no effect on the melanophores of trout embryos. Methyl alcohol has a less pronounced stimulation action than ethyl alcohol on pigment cells of trout. It was necessary to double to concentration so as to bring about reactions in any way com- parable to those produced by ethyl alcohol. The action of methyl alcohol was less striking and the stages of stimulation and relaxation were slower in appearing than in ethyl alcohol. 2. Ethyl alcohol—When trout embryos were exposed to weak solutions (0.01 per cent to 0.8 per cent) of ethyl alcohol, no change took place in the pigment cells. The embryos did not show any signs of depression and appeared perfectly normal. In solutions of 1 per cent to 1.5 per cent the embryos became more restless and the pigment cells exhibited a partial contraction. In con- centrations of 1.6 per cent to 2.5 per cent of ethyl alcohol the fish became more active, the pigment cells showed a complete contraction; while in solutions of 3.0 per cent to 4.5 per cent they showed a transitory contraction, followed by an expansion. This result could be very easily overlooked. In 6 per cent to 10 per cent solutions the trout embryos died rapidly in from fif- teen to twenty-five minutes, and there was no contraction of the pigment cells. If embryos that had their pigment cells con- tracted in the 2 per cent solution were transferred to a 7 per cent the pigment cells expanded rapidly. If the embryos in which the pigment cells were contracted were transferred to a 4.5 per cent to 6 per cent solution an imme- diate expansion resulted. This expansion was due to the de- pression caused by the high concentration of the alcohol, which was far beyond the maximum threshold of stimulation. When the fish which had their melanophores contracted in a 2 per cent solution were placed in a 0.5 per cent solution they expanded. Here the dilution of the alcohol was below the threshold stimu- lus. If embryos that were exposed to 7% per cent solution for . an interval of four to six minutes, were placed in water or very weak alcohol, there was observed a contraction of the pigment cells which was of a very short duration. This result was no doubt due to the washing out or the dilution of the alcohol within 166 JOHN N. LOWE the tissues, to the threshold stimulus and as the process of dilution continued the point was reached where the concentration fell below the threshold and a relaxation (expansion) of the melanophores occurred. After a complete recovery of the em- bryos from the effects of the alcohol the pigment cells reacted normally to other stimuli. These results show clearly that very weak solutions of ethyl alcohol do not have any effect on the pigment cells of the trout embryos. This is in harmony with the work of Kobert (’82) on the frog’s muscle, Lee and Salant (’02) on the gastrocnemius muscle of the frog, and Carlson (’06) for the heart muscle and heart ganglion of Limulus, all of whom observed that weak or very weak solutions of ethyl alcohol had no stimulatory action. Ethyl alcohol in contractions of 1.3 per cent to 2.5 per cent water shows a decided stimulatory action on the pigment cells of brook trout embryos. This is in accord with results of others on the primary stimulation of ethyl alcohol. Pickering (95) has shown that alcohol excites the embryonic heart muscle of the chick. Scheffer (’00) has observed that in the frog’s gastrocnemius when it was treated with alcohol the capacity for work was increased. If the muscle was curanized the stimulating effect of alcohol was nil. ©. Loeb (’05) has noted that in solutions of 0.13 to 0.3 per cent that the action of the isolated mammalian (cat) heart was augmented. Wood and Hoyt (’05) have shown that small amounts of ethyl alcohol increased the force of the heart beat in the frog, snake, tortoise, and turtle. Lee and Salant (02) have demonstrated that in medium concentrations of ethyl alcohol there was an increased rate of contraction and relaxa- tion in frog’s muscle (gastrocnemius). Carlson (?06) has ob- served that for the heart muscle and heart ganglion of Limulus, alcohol stimulated. Vernon (’10) has shown that alcohol has an excitatory effect on the isolated heart of the turtle (Emys). The (02) observed a marked increase in the number of contractions of the bell of the Medusa Gonionema in ethyl alcohol of 0.5 to 0.25 per cent. In a strong concentration of 4.5 per cent there was a marked depression or an expansion of the pigment cells. In this con- CHEMICAL AGENTS ON CHROMATOPHORES 167 centration there was no primary stimulating period observed. If it is to be found, it may be so short as to be very easily over- looked. Alcohol in large amounts decreased the rate of contrac- tion in the gastrocnemius frog’s muscle, Lee and Salant (’02). Romanes (’77) found that strong solutions of ethyl alcohol pro- duced increased and spasmodic contraction of the medusa bells of Sarsia (sp.) and Tiaropsis (sp.). These were followed by a depression. Lee (02) observed that in solutions of ethyl alcohol of a greater concentration than 2 per cent the contractions of the bell of the medusa, Gonionema, were much reduced in volume and in number. Dogiel (’77) has shown a depression in the heart rhythm of Corethra plumicornis. Vernon (710) observed that large doses of ethyl alcohol depressed the rate and volume of the contraction of a turtle’s heart (Emys). 3. Propyl alcohol. Weak solutions of propyl alcohol 0.01 per cent to 0.04 per cent did not effect the melanophores. Ina 0.06 per cent there was a noticeable contraction of the pigment cells. Solutions of 0.08 per cent to 0.125 per cent produced a rapid and complete contraction. In 0.7 per cent to 1.3 per cent the contrac- tion was only temporary, and was followed by an immediate relaxation of the pigment cells. A 1.5 per cent to 2 per cent produced no visible change in the expanded melanophores, and when embryos with contracted melanophores were exposed to the solution the melanophores expanded. In these concentra- tions there was observed a marked disintegration (cytolysis) of the cells. Higher concentrations (2.5. per cent to 4 per cent) killed the embryos without inducing any change in the expanded pigment cells. Contracted cells exposed to these solutions ex- panded instantaneously and after this response gave no reactions to other stimuli. It is obvious from these results that the stimulation of the pigment cells by propyl alcohol begins in solutions of lower con- centrations than it does in ethyl and methyl alcohol. It will be seen that my results for methyl, ethyl, and propyl alcohols are in perfect agreement with the results on the toxicity of the above alcohols of other investigators. 168 JOHN N. LOWE Joffroy and Serveaux (95) studied the toxicity of alcohols on mammals by intravenous injections. Bear (’98) introduced the alcohol directly into the stomach of the mammals. Picaud (97) placed fish and amphibians in the solutions of the alcohols and in this way determined the toxicity of the alcohols. Brad- bury (99) and Cololian (01) used fish, Overton (’01) on tadpoles employed the same method in their investigations. Wirgin (04) determined the concentrations at which the various alcohols in- hibited the growth of Micrococcus pyogenes aureus. He also investigated the laking power of the alcohols on the red corpuscles of the rabbit. Vernon (’11) studied the depression of an iso- lated tortoise heart by the alcohols. In table 3 the toxicity of ethyl alcohol is taken as unity and the values given are the comparative toxicities of the other alcohols. The values are only approximate. TABLE 3 TROUT TROUT ALCOHOL TOISE HEART MENT CELLS MENT CELLS OF THE PIG- or MAMMALS MAMMALS FISH TADPOLES IRGIN, BACTERIA CORPUSCLES OF THE PIG- EMBRYOS EMBRYOS STIMULATION OF | JorrROY, PICAUD, FISH BRADBURY, COLLOIAN, FISH OVERTON, WIRGIN, RED VERNON, TOR- DEPRESSION - © oe | a BAER, | Ww Methylorre nee 0.8/0.67| 1.0] 1.1/0.73/0.73/0.84 | 0.72} 0.45 0.55 Bithyl 2 ein amen 3.0 The stimulation level is lowest in methyl alcohol (4.5 per cent); next is ethyl alcohol (1.6 per cent to 2.5 per cent); and lastly propyl alcohol (0.08 per cent to 0.125 per cent). This is in harmony with the results of other investigators on the toxi- city of alcohols where it was found that methyl was less potent in bringing about narcosis, and the potency increased for the other alcohols directly with the molecular weight. It was shown by Baer (’98) that the toxicity of the alcohols varied directly as their boiling points. Meyer (’99) and Overton (’01) discovered that the narcotic action of the alcohols varied with their solvent power for fats or lipoids. It may be suggested that in addition to the above physical factors involved in the action of the alco- hols, that the dielectric constant of the alcohols probably plays CHEMICAL AGENTS ON CHROMATOPHORES 169 an important part in their action. It was observed that the greater the dielectric constant of the alcohols used the lower the stimulating or depressing power, and conversely the lower the dielectric constant the more striking were the reactions. What- ever may be the relation of these physical factors of the alco- hols in stimulation or depression, their chemical structure must not be overlooked; for as the length and complexity of the chain in monohydric alcohols increases so does the strength of their action. STIMULATING & DIELECTRIC a ALCOHOLS Pee eat. 08s BOILING POINT, °C: oa) FAKE AS Methyl. 2.2... 32.03 B12 65.7 0.45 Bithylh ee) a 46.05 25.8 78.4 1.0 Bropylies sce 60.06 22.0 97.4 2.0 Reactions to alkaloids The study of the action of drugs on the pigment cells of trout was undertaken with a threefold purpose, viz., to compare the action of drugs on the pigment cells with that of other tissues; second, to determine if possible the controlling mechanism of the pigment cells, and third, to see if the drugs had a specific action on the pigment cells. The literature on the pharmacology of the pigment cells of fish is not very extensive. The earliest historical record of experi- ments on the action of drugs on the pigment cells is that of Redi (1664), who observed that eels which died in a tobacco decoction were light in color. Pouchet (’76) observed that Gobius niger changed in color when placed in strychnine. Morphine, qui- nine, and santonin had no effect. Lode (’90) concluded that curare destroyed the nerve endings of the pigments cells of trout (Salmo fario). von Frisch (11) found that chloral why- drate contracted the pigment cells of the minnow and erucion. He also concluded that the action of cocaine was through the central nervous system. The pigment cell may be stimulated or fegreeaed by the drug acting: 1) on the pigment cell in such a way as to increase or 170 JOHN N. LOWE decrease its irritability; 2) on the nerve endings leading from the ganglia controlling the pigment cells; 3) on the central nervous system. I have no direct evidence to offer which will enable us to determine which of these or combination of these three fac- tors are operative in the action of the drugs on the pigment cell, for I was unable to separate the nervous and pigment cell tissues for experimental purposes. It is obvious that large doses have no selective action. At certain optimal concentra- tions all the drugs show a selective action on the pigment cells or their controlling mechanism. ‘This selective action of drugs on the mechanisms of the pigment cells will further our knowl- edge as to their function. : Fig. 1 A normal brook trout embryo showing the general alignment of the body. In interpreting my results I have given special emphasis to their relation in a comparative way to the observations of other observers on various vertebrate and invertebrate tissues. This comparative method makes the results easier of interpretation and is less liable to lead to an erroneous conclusion. The drugs used were all of Merck’s manufacture. They were dissolved in oxygenated distilled Water. The stock solutions were made up from 0.25 per cent to 0.5 per cent. Dilutions were made from these solutions. The experiments were carried on in Syracuse watch glasses in 10 ec. of the solution. These re- sults were checked by experiments in small stender dishes of 50 ce. capacity. The conclusions are based on experiments re- peated ten times in 1913 and again in 1914 another series of ten was tried. Five to ten animals were used at one time in each dilution. The trout embryos used were from four days to two weeks after hatching. In no case were the individuals of the different ages mixed. 2 Figures 1, 2, and 3 were drawn by Miss H. J. Wakeman. CHEMICAL AGENTS ON CHROMATOPHORES icra Other experiments are in progress to determine the action of drugs on pigment cells isolated from the nervous system. 1. Strychnine. In 0.5 per cent oxygenated solution death re- sulted without primary stimulation of the pigment cells. In 0.05 per cent strychnine solution the results were the same. Solutions of 0.005 per cent strychnine caused a contraction of the pigment cells rapidly, the contraction was complete in five min- utes. There was a remarkable thing observed in this concen- tration of strychnine. The irritability of the fish was increased to a high degree. The fish went into typical strychnine spasms. The head was thrown backward and the tail curved upward and forward, describing a half circle (as shown in text fig. 2). A Fig. 2. Showing a brook trout embryo in a typical opisthotonos response in 0.005 per cent strychnine. passing shadow over the disk brought on anew spasm. Shadows in rapid succession increased the concavity backward. If the dish was tapped very lightly the same responses occurred. ‘This period of heightened excitability lasted from eight to twelve minutes. During this interval the pigment ceils remained con- tracted (fig. 13). As this convulsive period disappeared, the pig- ment cells expanded (fig. 14). This expansion showed that the depression and paralysis of the pigment cell controlling mecha- nism had occurred. In 0.0005 per cent the pigment cells were contracted in ten minutes. No convulsions were observed in this concentration. In weak solutions of 0.00005 per cent to 0.000025 per cent no contractions of the melanophores was produced. 172 JOHN N. LOWE Pouchet (’76) observed that the pigment cells of Gobius niger contracted in strychnine solutions. Romanes (’77) noted that in the medusa Sarsia (sp.) the swimming motions were much accelerated by strychnine, also that convulsions occurred in this and three other forms—Cyanaea capillata, Tiaropsis indicans, and Tiaropsis diademta. Hedborn (’99) has. shown that strong doses of strychnine augment the beat of the isolated mam- malian heart (cat). Dogiel (’77) demonstrated a slight increase in the rate of the heart beat of Corethra larvae. Pickering (’93) observed that weak solutions of strychnine had a primary stimu- lating action on the heart muscle of an embryonic chick. Carlson (06) has found that strychnine in very weak concentrations had a distinct stimulatory action on the heart ganglion of the Limu- lus heart. Stronger solutions produced augmentation followed by paralysis. He was unable to note any primary stimulation on heart muscle. Laurens (715) observed that if a drop or two of a 1 per cent solution of strychnine was injected into the body cavity of Amblystoma larvae the pigment cells contracted. All the above experiments on other tissues show that strych- nine has a primary stimulating action and especially on the motor ganglia. From the evidence of Ballowitz (’93) who dem- onstrated that the pigment cells of fish have a connection with the nervous system, and from the fact that strychnine stimu- lates the nervous system, we are warranted in concluding that strychnine acts directly on the nervous mechanism controlling the melanophores of trout embryos, rather than on the melano- phores themselves. The seat of strychnine poisoning is in the spinal cord, therefore, the melanophores of trout embryos are in all probability controlled in part by the spinal nervous system. 2. Picrotoxin. Picrotoxin is used as a fish poison. It pro- duces a medullary stimulation and ultimately results in death. When trout embryos are exposed to a 0.25 per cent solution of picrotoxin the pigment cells contract rapidly. The contraction is complete in two to five minutes. The contraction remains for forty-eight to sixty-four hours, if the fish are kept in this solution. The fish live in 0.25 per cent solution for one hundred CHEMICAL AGENTS ON CHROMATOPHORES 173 and twenty-six hours. There is no convulsive period. In a weak solution of 0.025 per cent of pictoroxin the contraction is slightly less rapid, and lasts indefinitely (fig. 11). When the tail is cut away the pigment cells in the tail portion. expand (fig. 12). They remain expanded for six hours and then degeneration sets in. ‘The melanophores in the anterior or head end remain contracted. The contraction continues for eight to twelve hours and then disintegration of the pigment cells occurs. This justifies the conclusion that the reactions of the pigment cells of trout embryos are in some way controlled by the higher nerve centers. If the pigment cells that are contracted in picrotoxin are ex- panded in 0.2 M. NaCl and are now placed in picrotoxin the contraction is much slower than the first time. The sodium chloride seems to counteract the action of the picrotoxin. 3. Morphine. In embryos exposed to 0.5 per cent solution of morphine hydrochloride the pigment cells remain expanded. In a 0.12 per cent most of the pigment cells were expanded but there were a few isolated areas that showed a contraction. After an exposure of three hours these isolated areas of contracted pigment cells had increased. In a 0.06 per cent solution of morphine the result was the same. In a 0.012 per cent solution no change occurred, all the pigment cells remained expanded. There was no contraction of the pigment cells in a 0.005 per cent solution. Pigment cells contracted by picrotoxin, potas- sium iodide or strychnine were expanded by morphine- Br> Cls SO, If the contraction or degeneration of the melanophores is specific for the potassium cation, it is unqualifiedly modified by its anion, or the residual part of the undissociated molecules. 6.- The neutral salts of sodium, Na.SO,, NaCl, NaBr, NaNOs, and Nal, caused a slow contraction of the melanophores. The contraction was most rapid in Nal and slowest in Na.SO, and other salts were intermediate as is NO;> Br> Clie oS, Degeneration appeared first in Nal and last in Na.SO, and varied in this order i NO; > Br> Cis SO, The irritability of the chromatophores and life of the fish was maintained longest in Na,SO, and NaCl from (118 to 132 hours) in Nal from one to two and one-half hours. 7. The pigment cells that were contracted in potassium salts, when placed in sodium salt they expanded. The order of ex- pansion was SO.> Cle Br> NO; > if There was no expansion in Nal. 8. The results obtained in the experiments on the action of the salts on the pigment cells of trout are probable to be ex- plained on one or more of three assumptions: (1) That it is due to the antagonistic action between anion and cation; (2) that it is the independent action of the cation; (3) that reaction of the melanophores is likely modified by the undissociated molecule. CHEMICAL AGENTS ON CHROMATOPHORES 185 9. The narcosis or depression of the pigment cells of trout by the homologous alcohols corresponds very closely to their nar- cotic action as determined by Overton and numerous other investigators. 10. Very dilute solutions of methyl, ethyl, and propyl alcohols exert no action on the pigment cells of trout. 11. The pigment cells of trout embryos respond to alcoholic stimuli. Their mode of reaction is comparable to the reaction of other tissues to alcohols inasmuch as they are stimulated by small doses and depressed by large doses. 12. Strychnine in moderate doses causes a primary con- traction of the expanded melanophores. Large doses cause a depression without a primary stimulation (contraction). The action of the strychnine is on the nervous system rather than on the pigment cells directly. 13. The action of picrotoxin causes a rapid contraction of the pigment cells. The mechanism controlling the pigment cells is in the higher centers, because if the spinal cord is severed the, pigment cells expanded. 14. Morphine induces a contraction of the melanophores in isolated areas. This is probably due to the selective action of morphine upon the nervous system. Large doses produce no change in the expanded melanophores. Morphine expands the pigment cells that were contracted in picrotoxin, KCl, and strychnine. 15. Curara causes a mixture of responses, that is, there are areas of expanded and contracted melanophores. This is likely due to the unequal action of the curara on the peripheral nervous mechanism of the melanophores. 16. Medium solutions of nicotine cause a contraction of the pigment cells. Strong nicotine solutions have no effect on the pigment cells. The action of nicotine is directly on the nervous controlling mechanism of the pigment cells. 17. Atropine in all concentrations has no stimulating action on the pigment cells of trout. Atropine paralyzes the fine nerve connections of the pigment cells. 186 JOHN N. LOWE 18. Cocaine has a primary stimulating action on the pigment cells of trout. This action is probably on the nerve endings of the pigment cells that connect them with the reflex center. 19. Veratrine causes a primary contraction of the pigment cells which is followed by a rapid depression (expansion). The action of veratrine is through the reflex center of the spinal cord and medulla rather than local. 20. Quinine exhibits no primary stimulating action on the pigment cells. The drug has no selective action on tissues, therefore it is a general ‘protoplasmic poison.’ CHEMICAL AGENTS ON CHROMATOPHORES 187 BIBLIOGRAPHY Barr, G. 1898 Beitrag zur Kenntniss der acuten Vergiftung mit verschiedenen Alkoholen. Arch. f. (Anat. u.) Phys., Leipz., S. 283-296. 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PLATE 1 EXPLANATION OF FIGURES In figures 1 to 10 are shown five sets of brook trout embryos which were ex- posed to the action to 0.2 M solutions of Kl, KNO;, KBr, KCl, and K.S0Ou,, 1 to 5 for an interval of fifteen minutes, and figures 6 to 10 for a period of three hours. 1 The melanophores were completely contracted in Kl]. 2 The contraction was not as pronounced in KNOs3. 3 In KBr the melanophores had longer processes than in the two preceding solutions. 4 In KCl the processes were more distinct and showed the finer arboriza- tions. 5 An exposure of fifteen minutes to K,80. produced no observable change in the melanophores. 6. After an exposure of three hours to K1 the melanophores showed a distinct secondary expansion. 7 In KNO; an exposure of three hours produced a less extensive secondary expansion than Kl. 8 In KBr the processes were very much shorter than in Kl] and KNOs3. 9 After three hours in KCl the melanophores were still spherical, but there was a suggestion toward a peripheral migration of the pigment as indicated by the swollen condition of the cells. 10 After three hours in K.SO, there was no expansion of the melanophores. 11 All melanophores contracted, photograph taken after twenty-four hours of exposure to Picrotoxin. 12 The melanophores expanded after severing the tail in an individual which was exposed to Picrotoxin for twenty-four hours. Photograph taken five min- utes after cutting. 13 All melanophores contracted during the period of Strychnine con- vulsions. 14 Showing the expansion of the melanophores after the strychnine convul- sion had subsided. 15 The contracted and expanded melanophores as they occurred in 0.0025 per cent curara 192 CHEMICAL AGENTS ON CHROMATOPHORES JOHN N. LOWE z ' THE REACTIONS OF THE MELANOPHORES OF AMBLYSTOMA TIGRINUM LARVAE TO LIGHT AND DARKNESS HENRY LAURENS Osborn Zoélogical Laboratory, Yale University SIX FIGURES INTRODUCTION In two earlier papers (Laurens 715 and 716) the reactions to light and darkness of the melanophores of normal and eyeless larvae of A. punctatum and of A. opacum were described. These differed in some details from the corresponding reactions de- scribed by Babak (710) for normal and blinded Axolotl larvae. The present paper is concerned with the description of similar reactions of the melanophores of larvae of Amblystoma tigri- num, the tiger salamander, the so-called Axolotl. Owing to the close similarity of these results with those obtained with A. punctatum and A. opacum they can be briefly dealt with. A few observations were also made of the melanophores of larvae of A. microstomum.! These showed responses slightly different from the others, but owing to the fact that only a few larvae were available at the time the observations were made, not very much can be said about them beyond the statement that the melano- phores apparently do not show the secondary reactions which have been described for punctatum and opacum. Babak (710), it will be remembered (see Laurens 715, p. 620 and 716, p. 237), found that there was a difference between the reactions of the melanophores of normal and blinded Axolotl larvae to light and darkness. In bright diffuse light the melan- 1 The eggs of A. tigrinum were sent to me by Prof. C. Judson Herrick, of the University of Chicago, those of A. microstomum by Prof. G. E. Coghill, of the University of Kansas. It is a pleasure to thank both of these gentlemen for their kindness. 195 196 HENRY LAURENS ophores of the normal larvae contract, those of the blinded larvae expand. In darkness the melanophores of normal larvae ex- pand, while those of blinded larvae contract. This opposite reaction of the melanophores of normal and blinded larvae he found not to occur until the larvae were 17 mm. long, and he supposed that before this the retina had not acquired the pig- ment motor function which it later has, and the melanophores respond, therefore, merely to direct stimulation, the reaction to which is the same in normal and blinded individuals. After this time, by means of the control which the eyes gain through the central nervous system, the sense of the reactions of the melan- ophores is reversed, the effect of indirect stimulation through the eyes being opposite to that of direct. Babak explains this difference between direct and indirect stimulation by assuming that both phases of the movement of the chromatophores of normal Axolotl larvae are governed by the central nervous sys- tem, and that this double innervation is conditioned upon the retinae which have opposite influences upon the nervous system according as to whether they are illuminated or darkened. The darkened retinae are believed to exert a positive influence on the chromatophores through the nervous system just as the illuminated retinae do, but in the reverse direction. The de- struction of the retinae has an entirely different result from that obtained by darkening them. In other words, the retinae in -complete darkness are active and exert a positive influence which is directly opposite to that caused by illumination. Neither of these two opposite effects of the retinae upon the chromatophores are, according to Babak, inhibitory, but they are both tonic influences. The impulses bringing about the ex- pansion of the chromatophores originate in the darkened ret- inae, and are so strong that they overcome the tendency of the darkened chromatophores to contract and cause their expan- sion. On the other hand, the impulses for the contraction of the chromatophores originate in the illuminated retinae and are in turn so strong that they overcome the tendency of the illumi- ‘nated chromatophores to expand and bring about their contraction. REACTIONS OF MELANOPHORES 197 Pernitzsch (713) also found that when Axolotl larvae were kept in darkness they became dark, due to the expansion of the melanophores. The results which were obtained from the study of the reac- tions of the melanophores of A. punctatum and A. opacum larvae (Laurens 715) were such that Babak’s explanation could not be applied to them. They threw no doubt, however, on the assumption that both phases of the movement of the melan- ophores are normally, by means of the eyes, ‘under the control of the nervous system, although it seemed necessary to regard one of the influences of the retinae as inhibitory and opposite in effect to that which causes the pigment cells to contract. It was found that the melanophores of normal and eyeless larvae of A. punctatum and of A. opacum react in identically the same way, expanding in light and contracting in darkness, the only difference being that the reactions came about more quickly in the normal than in the eyeless larvae. Later the melanophores of normal larvae were found in the opposite con- ditions, in light and in darkness, to what they were in before, for after having been kept for from three to five days in light the melanophores are contracted, and after having been for five days or more in darkness, the melanophores are expanded. The melanophores of eyeless larvae did not show these secondary reactions. EXPERIMENTAL The same methods employed in my former work on the pig- mentation of Amblystoma larvae were carried out here. The optic vesicles of the eyeless individuals were removed at the tail bud stage (Laurens ’14, p. 197), and the eyeless and nor- mal larvae were kept in individual dishes. The melanophores were observed under the binocular microscope, attention having been already called to the inaccuracy of the method of simply observing the general coloration of the animals as an index of the contracted or expanded condition of the pigment cells. As in the case of A. punctatum only the subepidermal or corial melanophores are referred to in the following description. The 198 HENRY LAURENS epidermal pigment cells do not show sufficient regularity of re- sponse to light and darkness to fall in line with those of the corial melanophores. The xanthopores seem always to remain ex- panded. RESULTS The state of the melanophores of normal and eyeless larvae of A. tigrinum under different conditions are in complete agree- ment with those of A. punctatum and A. opacum. If normal seeing larvae are placed in bright diffuse light over an indiffer- ent background and in darkness, and kept there, it will be seen after several days, that the melanophores of the animals kept in the light are partially contracted ($ to $+ expansion) (fig. 5), while those of the animals kept in darkness are partially expanded (2 to 7 expansion) (fig. 6). Eyeless larvae show the reversed condition, the melanophores of those in the light being expanded, of those in darkness, contracted. Table 1, which is reproduced from my earlier paper (’15), summarizes these observations, the conditions of the melanophores over a white and a black back- ground being also included. TABLE 1 State of the melanophores after long continued illumination and darkness LIGHT BACK- GROUND DARKNESS White Black Indifferent Normal | Contracted | Expanded|Contracted (4 to + exp.)} Expanded (3 to 4) Eyeless | Expanded | Expanded|Expanded Contracted But the reactions, or primary responses, to light and dark- ness of the melanophores of the normal larvae are different (table 2), and do not agrée with the conditions of the melano- phores, which have just been described. When the melanophores of larvae, normal and eyeless, that have been in darkness for only a few hours, are examined, it is seen that they are completely contracted, appearing as fine black dots. If these larvae, over an indifferent bottom, are now exposed to bright diffuse light the melanophores soon (five min- REACTIONS OF MELANOPHORES 199 TABLE 2 The reactions of the melanophores of normal and eyeless larvae to light and darkness LIGHT INDIFFERENT BACKGROUND DARKNESS Normal I. reaction Expansion (7) (1 to 2 | Contraction (2 to 3 hours) hours) II. reaction Contraction (¢ to + exp.) | Expansion ({ to #) (5 days (4 days or more) or more) Eyeless I. reaction Expansion (2 to 3 hours) | Contraction (4 to 5 hours) utes) begin to expand, though it is sometime before expansion is completed, the time varying between one and two hours for the normal larvae, and between two and three hours for the eyeless (figs. 1 and 2), If, after the melanophores have expanded, they are transferred back to darkness, the melanophores contract (figs. 3 and 4). This reaction does not begin so quickly as that to light, and it takes longer to be completed, between two and three hours for the normal larvae, and between four and five hours for the eyeless. It is clear then that, as in A. punctatum, there is no differ- ence between the responses of the melanophores of normal see- ing and eyeless larvae of A. tigrinum. In light they expand (figs. 1 and 2), in darkness they contract (figs. 3 and 4). This reaction always take place, from the time the melanophores are first responsive to light and as long as they have been examined, the reactions of the melanophores having been observed in larvae ranging in length from 20 to 70 mm. It may be mentioned in passing that, as the larvae grow older and the number of melan- ophores increases, the reactions take longer and are less com- plete. Due to the fact that, although the reactions of the melano- phores of A. punctatum and A. opacum was to expand in light and contract in darkness, the state of the melanophores of see- ing larvae, when these were kept for some time in light or dark- ness respectively, was different, a distinction was made _ be- 200 HENRY LAURENS 2 4 6 Photographs at about 1} magnification of living animals, not anaesthetized, taken by Prof. Alexander Petrunkevitch, to whom it is a pleasure to express here my thanks. Figures 3 and 4 are unfortunately slightly out of focus. Larvae that have been in darkness for several hours when brought into the light are rather restive, and it is some little time before they can be quieted sufficiently REACTIONS OF MELANOPHORES 201 tween these conditions, the first being known as the primary reaction of the melanophores, the changed condition as the secondary reaction. It has been found that, as indicated above, such reactions take place also in the melanophores of the larvae of A. tigrinum. When these are placed in darkness the melanophores at first contract, but remain so for only a limited time, for after five or more days of darkness,—interrupted by illumination with very weak red light only long enough to clean out the dishes and to add food,—the larvae are dark in appearance, the melano- phores having expanded (fig. 6). This is a so-called ? to 7 expansion. EHyeless larvae never show this secondary reaction of the melanophores, for these remain contracted, no matter how long the larvae are kept in darkness. Very long tenure in darkness naturally affects the melano- phores, both of seeing larvae, where they are secondarily ex- panded, and of eyeless larvae, where they are contracted. The number of melanophores does not increase so rapidly as nor- mally occurs with the growth of the larvae, and the amount of pigment decreases, so that the animals eventually assume a golden, and then a transparent silvery appearance, due to the lack of pigment. When such individuals are brought into the light, the melanophores increase in number and eventually the larvae cannot be distinguished from those that have been always in the light. Light then seems to have more influence on the number of pigment cells than does the expanded condition to snap them. The melanophores therefore have usually expanded slightly before the pictures can be taken. Fig. 1 Normal seeing larva showing expanded melanophores after having been exposed to bright diffuse daylight for four hours. Fig. 2. The same for an eyeless larva. Fig. 3 Normal seeing larva showing contracted melanophores after having been in darkness for five and one-half hours (slightly out of focus). Fig. 4 The same for an eyeless larva (slightly out of focus). Fig. 5 Normal seeing larva showing the melanophores in their secondarily contracted condition (} to } expansion) after having been six days in bright diffuse light over an indifferent bottom. : Fig. 6 Normal seeing larva showing the melanophores in their secondarily expanded condition (? to 7) after having been six days in darkness. 202 HENRY LAURENS of the melanophores (see von Frisch, *11, and Babak ’13). This is supported by observations on the number of melano- phores in seeing larvae that have been kept permanently in light over white bottoms where the pigment cells are always contracted. That the expanded condition is ineffective in this regard is not claimed, for comparisons between larvae kept in the light over white bottoms and those kept over black bottoms show that the number in the latter is greater. In seeing larvae that have been kept in the light over indif- ferently colored bottoms for some time the melanophores also secondarily react. Continual or constant illumination does not seem to be necessary for this secondary reaction. Comparative observations have been made on larvae placed over an indiffer- ent bottom in bright diffuse daylight, on those that have been continuously illuminated in a dark room by an electric lamp placed above the dish, the light being passed through ground glass and thus diffused, and on those continuously illuminated by light from a nitrogen-filled Mazda lamp passed through ‘daylite’ glass. All show after a few days the melanophores, which expand at first when they are illuminated, to be con- tracted (fig. 5). This, as was before found for A. punctatum, is not a complete contraction, but what has been called a $ to + expansion. It takes, on the average, four days to come about. In a few individuals it sometimes takes a shorter time, in some it takes much longer, and in a few it never takes place. Table 2 summarizes all of these reactions of the melanophores. Normal seeing larvae of Amblystoma tigrinum kept in an aquarium with Elodea and other plants are when young a pale, dirty, greenish gray which as the larvae grow older becomes a rich brown color. Eyeless larvae, on the other hand, are much paler in general appearance, having been described as ‘anemic looking’ by some who have observed my animals. In such an environment as has been described the general background is a black or dark one, and it is to this that the rich dark brown appearance of the normal larvae is due. It is dependent upon the eyes as is shown by the pale appearance. of the eyeless individuals. REACTIONS OF MELANOPHORES 203 Seeing larvae when reared over a white bottom are very pale in appearance, due not only to the contracted condition of the melanophores, but also to the fact that the number of the melan- ophores is not so great. Eyeless larvae reared over no matter what bottom are darker than these, the melanophores being always expanded in the light. But, although the number of the melanophores is greater than the number in seeing larvae on white backgrounds, it is smaller than the number in seeing larvae under the general environmental conditions of the aver- age aquarium dark bottom described above, and over a black one. That the reactions of the melanophores are adaptive is shown by the above observations and also by the following. Seeing and eyeless larvae were placed when young (25-30 mm. long), in a large aquarium containing Elodea and other water plants. Coarse, brownish sand composed the bottom. As time went on, with the sinking to the bottom of decayed leaves and stems and the collected faeces of the animals, the bottom became a very dark brown, almost black, over which the seeing larvae were relatively inconspicuous, while the _ eyeless_ larvae were distinctly to be seen. Periodically, the aquarium was di- vided into two parts by a plate of glass placed diagonally across it and the nature of the bottom in one half changed by sprink- ling white sand over it. The seeing larvae promptly changed over the ‘white’ bottom, becoming markedly paler, due to the contraction of the melanophores. The melanophores of the eye- less larvae did not respond. When the dividing plate of glass was removed the difference between the seeing larvae that have been over the ‘white’ bottom and those that have been over the dark one became very apparent, while the eyeless were alike over both bottoms, and different from each of the kinds of see- ing larvae, being darker than the seeing larvae that had been over the ‘white’ bottom, and paler than those that had been over the dark bottom. No further evidence regarding the cause of the secondary re- sponses of the melanophores of seeing larvae to light and dark- ness is advanced beyond that which has been earlier expressed. 204 HENRY LAURENS In light it is thought that the constant illumination or stimula- tion of the retinae starts impulses by certain photochemical changes in the retinae, the end effects of which are opposite to those of direct stimulation, and in this way bring about the secondary reaction of contraction. In darkness the same kind of thing is supposed to take place, long continued absence of light producing impulses by chemical changes, the end effects of which result in the expansion of the melanophores. ‘That the secondary responses do not take place in the eyeless larvae shows that the seat of the causes of them must be sought in the retinae. The eyes through the nervous system cause the melanophores to secondarily contract in light and to expand in darkness. The results obtained with the larvae of Amblystoma tigrinum are thus in complete agreement with those earlier obtained with the larvae of A. punctatum and A. opacum, and do not agree with those of Babak. SUMMARY 1. The reactions of the melanophores of the larvae of Ambly- stoma tigrinum are exactly like those earlier obtained with the larvae of A. punctatum and A. opacum. In light the melano- phores expand (figs. 1 and 2) and in darkness they contract (figs. 3 and 4), in both seeing and eyeless larvae. The melan- ophores of seeing larvae that have been kept for some time (four days or more) in bright diffuse light over an indifferent bottom are, however, partly contracted ({ to + expansion) (fig. 5) while the melanophores of seeing larvae that have been kept for some time in darkness (more than five days) are expanded (2 to 7 expansion) (fig. 6), thus showing, under long continued illumination and darkness, what has been called a secondary reaction. REACTIONS OF MELANOPHORES 205 BIBLIOGRAPHY Basak, E. 1910 Zur chromatischen Hautfunktion der Amphibien. Arch. f. d. ges. Physiol., Bd. 1381, S. 87-118. 1913 Uber den Einfluss des Lichtes auf die Vermehrung der Haut- chromatophoren. Arch. f. d. ges. Physiol., Bd. 149, 8. 462-470. von Friscu, K. 1911 Beitrige zur Physiologie der Pigmentzellen in der Fisch- haut. Arch. f. d. ges. Physiol., Bd. 138, S. 319-387. Laurens, H. 1914 The reactions of normal and eyeless Amphibian larvae to light. Jour. Exp. Zodl., vol. 16, pp. 195-210. 1915 The reactions of the melanophores of Amblystoma larvae. Jour. Exp. Zodl., vol. 18, pp. 577-638. 1916 The reactions of the melanophores of Amblystoma larvae— The supposed influence of the pineal organ. Jour. Exp. Zodl., vol. 20, pp. 237-261. Prernirzscu, F. 1913 Zur Analyse der Rassenmerkmale der Axolotl. I. Die Pigmentierung junger Larven. Arch. f. mikroskop. Anat., Bd. 82, 8. 148-205. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, No. 1 . a eS Vi “ponies pti of ON : : -.. = J 7 a ~ x = - ¢ , | on ates, Ss ar 2; Anyi NA 4%: : " 7 > eed a ae birt er a rho Bp 0 Cy, a fe ’ 7 ol - Me . As 5 “ ~. is Pi Ve alyig s Stee ty oe aw aye 8 a; aera ret Pere ay “ae ae i 7” o ie ee be ; a Based ‘ve, f A ae i ~ ‘ = ; : ahi } ak bist i 1 Aahed) Ee ij free, ey WHE Se sie a Yee if areal: iueehy i 4 a 2+ 65k ake. ei ae mer ey Hn Bist we ; : ae 3 . ps OAL i NRE Rca mals Brigiar || Gab oe of ae via eta ale lat aa os a wh et bs : ‘~ i ’ i% a Aen ‘ j : 7 ity - ss } oz ae ik is ey EVIDENCES ASSOCIATING PINEAL GLAND FUNCTION WITH -ALTERATIONS IN PIGMENTATION CAREY PRATT McCORD anp FLOYD P. ALLEN Detroit, Michigan SEVEN FIGURES INTRODUCTION In the spring of 1915 we undertook to establish the influence of pineal gland substances upon growth and differentiation proc- esses in tadpoles. The tadpoles employed were hatched in the laboratory and immediately placed upon a diet of pineal gland. On the tenth day of larval life it was readily observable that in the pineal fed groups the coloration was uniformly lighter than in the control, muscle-fed groups. This alteration was at first attributed to some unknown difference in environmental conditions—light, background, ete., as the color of these or- ganisms was known to vary considerably in response to such stimuli. These changes when first noticed were trivial in de- gree, but as development progressed the alterations in pigmen- tation became correspondingly greater. Thirty minutes after feeding pineal tissue, the tadpoles which prior to the feeding had been uniformly dark, became so translucent that all the larger viscera were plainly visible through the dorsal body wall (figs. 1 and 2). This translucency appeared with such regu- larity and so punctually after pineal feedings and was so mark- edly absent in the control groups that the phenomenon was made the subject of special study. Out from this work have come many acceptable evidences of a pineal gland influence upon pigmentation, upon the phases of colloidal state, and upon the vegetative nervous system. The following report is the record of the unfolding of this further work. 207 208 CAREY P. McCORD AND FLOYD P. ALLEN Figs. 1 and 2. Drawing of the same tadpole just: prior to (fig. 1) and forty- five minutes after (fig. 2) pineal feeding. PINEAL GLAND FUNCTION 209 NATURE OF PIGMENTATION The color phenomena observable in many animal life forms are due to the absorption or reflection of light rays by chemical substances in the integument of the animal. These materials are usually found as granules of pigment lying in specialized cells, the chromatophores. Chromatophores are divisible into several types, but of these only two are found in frog skin. First, the melanophores, lying in the skin, peritoneum, etc., contain granules of dark-brown or black melanin and are often contractile. Second, the xanthophores, which are found only in the adult skin, contain granules of light yellow xanthin and are never contractile. The relationships between pigmentation and environment are perfectly obvious but little understood. The color changes of the common tree toad (Hyla arborea) have been accounted for in various ways. One writer holds that the pale condition is the result of the stimulative effect of light upon the chromato- phores and that the dark phase is due to the absence of that stimulant (1). Another observer (2) claims that light alone has very little effect, but that changes of temperature control the coloration. The problem is complicated by the fact that the same individual may not react in the same way under exactly similar conditions. Certain it is, however, that animals do respond to environmental changes by alterations in their color- ation. It has been shown by numerous experimenters that these changes are to a great extent, under the control of the eyes. A change of environmental light is caught by the eye and the resulting stimulus transmitted to the chromatophores by the central nervous system. That this is the usual method of procedure is indicated by the atypical reactions of blinded individuals. The mechanism of these changes is entirely dependent upon the contraction or expansion of the melanophores. Both xan- thophores and melanophores enter into the color effects but the former play a passive role and always present the same appear- ance. The melanophores of frog tadpoles are of two distinct 210 CAREY P. McCORD AND FLOYD P. ELLEN types. The simpler and less conspicuous form is limited to the epidermis. It consists of a cell-body with two or more simple processes (Hp. M., figs. 5 and 6). They may lie singly in the epidermis or, in such abundance as to form a definite reticulum. These melanophores are not contractile (3). The second type of melanophore is found in greatest abund- ance in the sub-epidermal connective tissue. During late metamorphosis these cells migrate to the corium. In the ex- panded condition the sub-epidermal melanophores present a very typical ‘mossy’ appearance. The cells lie so closely ap- proximated that they form a nearly continuous sheet (Sub. M., fig. 6). There is a lighter, central space in each cell probably representing the nucleus. In the contracted condition (Sub. M., fig. 7) the melanophores appear as irregular dots in which no structure is visible. It has been fairly well established that the contractile melano- phores are innervated by motor fibres proceeding along both sympathetic and spinal nerve paths (4). Hooker has shown physiologically that the reactions of the melanophores of the frog are synchronized by the action of the central nervous sys- tem. By histological methods, Ballowitz (5) has demonstrated motor nerve endings in melanophores of bony fishes. Laurens (6) working with Amblystoma larvae has shown that the melano- phores may contract as the result of direct stimulation. Often, however, this primary reaction is overcome by an opposite, secondary reaction initiated by the central nervous system. There are three principal theories to account for the mechanics of melanophore contraction. Ballowitz (1) claims that the contraction is an intra-cellular migration of the pigment gran- ules within fixed cells. The protoplasm of a chromatophore is filled with numerous, extremely fine, radially arranged, anasto- mosing canals within which the pigment is forced back and forth by the alternate contraction and relaxation of the protoplasmic canal-walls. Spaeth (7) believes that the chromatophores of fishes are fixed stellate cells, within which the pigment granules, carried in a rather fluid cytoplasm, stream into and out of the processes PINEAL GLAND FUNCTION Dit during expansion and contraction. He explains this migration as a strictly colloidal phenomenon, the contracted and expanded conditions representing respectively the aggregate and disperse phases of a colloidal suspension. This is perhaps the most widely accepted and tenable view. Hooker (8) has advanced a third explanation. He believes that the melanophores of frog larvae le in preformed spaces and that the cells expand and contract as a whole within the spaces which enclose them. The acts of expansion and contrac- tion, according to this theory, are brought about by pseudopodia, the pigment granules being carried in the cell cytoplasm. On this premise the pigment cells are to be considered amoeboid. These expansions and contractions are commonly brought about by changes in the intensity of light or heat, but many other agencies will cause a specific reaction. Spaeth (7) has made a detailed study of the reactions produced by a great variety of stimuli upon the melanophores of Fundulus. He notes that the reactions are in every way comparable to those obtained by the same agents upon smooth muscle. He raises the very pertinent question as to whether melanophores may not be considered as modified smooth muscle cells. In 1910 Babak (9) noted the reversal of the normal reaction to light when Axolotl larvae were blinded. In diffuse lght the melanophores of normal seeing larvae contract. After painting the eyes with an opaque substance the melanophores expand in light. In the same way the melanophores of normal larvae expand in darkness, while those of blinded larvae con- tract. Fuchs (10) explained the phenomenon as due to the intervention of the parietal organ (the pineal gland of higher organisms). He reached this conclusion from a consideration of the phylogeny of this organ. The embryology of the parietal organ in some of the lower reptiles indicates very clearly that this body is a remnant of a third eye. Fuchs assumed that it had retained some of its controlling power over the melanophores. In the normal larvae its influence is completely over-shadowed by the superior power of the functioning eyes. In the blinded 212 CAREY P. McCORD AND FLOYD P. ALLEN larvae the parietal organ again assumes control. Laurens has completely disproven this hypothesis (11). It is noteworthy from the present study that although the pineal gland does not exert a controlling influence upon pig- mentation comparable to that arising from environmental stim- ulation of the retina, nevertheless it contains an active substance capable of directly inducing pigmentation changes irrespective of environmental conditions. MATERIAL AND METHODS Eggs of the species Rana pipiens, Rana cantabrigiensis and Bufo Americana were collected in the vicinity of Detroit, and hatched in the laboratory. Immediately after hatching and before the oral orifice had opened they were grouped in trays into colonies of 200 and food placed in the trays. The food was weighed, each colony receiving the same amount triweekly. All foods were taken voraciously by the tadpoles. By means of a water dropping and disposal system the tadpoles were at all times in fresh, aerated tap water. Moreover the trays were frequently shifted to average environmental conditions of light and temperature. In the observations on pigmentation we used some 12,000 tadpoles. The food consisted of desiccated glandular material and fresh plant food. Of the glands we tested the effect of pineal (adult and preadult) thyroid, parathyroid, and suprarenal. Brain tissue and beef muscle were used as controls. Different lots of tadpoles were fed upon Spirogyra, bread crumbs and hemp seed as a further check. A single lot of tadpoles was fed on desiccated retinae from beef eyes as a particular experiment. Of these tissues the pineal gland alone produced the phenomenon we have called the pineal-pigment cycle. EXPERIMENTAL EVIDENCE Effect of whole pineal tissue on pigmentation Certain endocrinous tissues are known to alter pigmentation in tadpoles. This was noted by Gudernatsch (12), in his feed- PINEAL GLAND FUNCTION 2 ing experiments on these animals. Concerning the pigment altering tissues he states: After five weeks feeding, those (Tadpoles) fed on adrenal cortex be- came much lighter than those fed on adrenal medulla or any other food. This difference in color became more evident as the experiment pro- ceeded, until the cortex-fed tadpoles had an extremely light, greenish yellow tint. The spleen and thymus fed tadpoles became extremely dark during the course of the feedings. Those fed on liver developed a dark greenish color, those on ovary a yellowish color. The type of pigment variation observed by Gudernatsch is obviously distinct from that observed by us. In his animals the pigment alteration was slow in appearing and persistent. In ours the change appeared early in life, occurred sharply in relation to feeding, was cyclic and transient. After the tenth day of larval life pigment changes were always evident after every feeding of the pineal tissues and the animals continued to react until their forelegs protruded. Sufficient blanching of the bodies occurred within thirty minutes after pineal feedings to differentiate these colonies from their con- trols. A maximum condition of translucency was attained in about forty-five minutes, and three to six hours later restoration to the original color was complete. The difference was first noticeable in the region about the eyes due to the absence of larger viscera. It can be demonstrated, however, that the reaction occurs simultaneously over the whole body. At the height of the reaction the integument was so transparent that. the brain, the olfactory tracts, the kidneys, the beating heart and the intestines were all clearly visible through the dorsal body wall. Figures 1 and 2 are drawings of a single tadpole just prior to and forty-five minutes after feeding pineal material. The darker portions in 2 are due to the denser viscera, the pigment conditions being the same over the entire animal. Photography fails to give a true picture of this phenomenon but since actual photographs are more valuable as exact evi- dence than drawings figures 3 and 4 are here included. These are respectively photographs of the same group of tadpoles 214 CAREY P. McCORD AND FLOYD P. ALLEN just prior to and thirty minutes after feeding 5 mgm. fresh pineal gland. A true evaluation of the relative pigmentation may be had by comparison of the tadpoles of figure 3 with the periphery of those in figure 4. The dark color in the center of the bodies in figure 4 is due to the opacity of the denser vis- cera and intestinal contents and not to a difference in the pig- mentation of the skin. —- Ff ) i | SA 9% @ 3 Figs. 3 and 4 Photographs made by reflected light. Fig. 3 Normal tadpoles—just prior to pineal feeding. Fig. 4 Same tadpoles 30 minutes after feeding acetone extract of pineal gland. The darker portions of these tadpoles are due to denser viscera—heart, gills, intestinal contents, etc. The degree of translucency is identical in all parts of the skin. If a portion of the skin from a light and from a dark tadpole be dissected loose and examined under a microscope the reason for the difference in shade will be readily apparent, (figs. 6 and 7). The two types of melanophores are present. In the nor- mal (dark) piece of skin the sub-epidermal melanophores (fig. 6, Sub. M.) are expanded to such an extent that they form an opaque sheet in which there are left a few scattered openings. In the PINEAL GLAND FUNCTION yA ES) light piece of skin these melanophores are contracted to rough spheres of pigment (fig. 7, Sub. M.). The epidermal melano- phores exhibit an unchanged appearance in both drawings. A sagittal section of normal skin (fig. 5) shows the relation be- tween the two types of melanophores and the various layers of the integument. These described alterations in pigmentation are invariably induced in tadpoles upon the administration of pineal materials, be they the fresh minced glands, simple desiccation preparations, or simple aqueous extracts. In an effort to associate these changes with certain constituents of the pineal gland, various fractions of the pineal were prepared and employed and are now about to be described. 2 2 EE 3O° Sub.M. Fig. 5 Sagittal section of integument taken from the eye region of a normal tadpole. Section illustrates the two types of melanophores and their position relative to the tissue layers. ABBREVIATIONS Ep., epidermis Ep.M., epidermal melanophores Tnt., integument, including epidermis Sub.M., sub-epidermal melanophores and sub-epidermal tissue B.M., basement membrane Sub., sub-epidermal connective tissue Effect of pineal fractions on pigmentation In the preparation of these split materials the fresh glands were either ground up and immediately extracted or desiccated and subsequently extracted. From the results of a wide varia- tion in fractionation methods, chief interest centers around the acetone and alcohol extractives and their residues. In the case of acetone the process was carried out in a Soxhlet appara- tus. On freeing the extractives from acetone there resulted 216 CAREY P. McCORD AND FLOYD P. ALLEN a brownish-black fatty mass with an odor suggestive of crude fish oil. Portions of these extractives and the residue were preserved intact for experimentation. Other portions of both were reextracted with alcohol. Likewise fresh pineal material | Ep. id Fig. 6 Surface view of integument from normal tadpole. On the left side of the drawing the sub-epidermal connective tissue has been torn away, leav- ing the epidermis alone. Thus on the right side both types of melanophores are visible, on the left the epidermal type alone. The pigment granules in the sub- epidermal melanophores are evenly distributed throughout the cytoplasm, illus- trating the disperse phase. PINEAL GLAND FUNCTION el Ep.M. Fig. 7 Similar view of integument from tadpole fixed 30 minutes after feed- ing acetone extract of pineal gland. In this case the pigment granules in the subepidermal melanophores are collected into a mass in the centre of the cell— the aggregate phase. The material was fixed in Bouin’s fluid, dehydrated, cleared in xylol and mounted without staining in balsam. The drawings were made with the aid of an Abbe eamera lucida, with Leitz objective 6 and ocular 4, giving an approx1- mate magnification of 590 diameters, but have been reduced 3 for publication. All material was taken from the region between the eyes. 218 CAREY P. McCORD AND FLOYD P. ALLEN was extracted with alcohol and the residue and extractives respectively extracted subsequently with acetone. These several preparations were tested as to their influence upon pigmentation on several hundred tadpoles from the same hatchings. At once it was apparent that the pigment altering principle was com- pletely dissolved in acetone. The typical pigment cycle was induced by this extract while the residue and all acetone ex- tracts of muscle tissues induced no pigment changes. ‘The resi- due from acetone extraction was, however, capable of inducing the growth stimulating action that McCord has described for the pineal gland, while the acetone extracts which were exquisitely active in inducing pigment alterations were only slightly active in stimulating rapid growth. The inference is that at least two separate distinct principles exist in the pineal, the one producing the pigment phenomena, the other stimulating rapid growth. In the case of the alcoholic extraction the active sub- stances were not readily soluble, for the alcoholic extractives, the alcoholic residue, and the acetone reextractives, all yielded positive pigment results. The acetone extractives yielded quite readily to aqueous emulsifying and this form proved to be the most convenient mode of employing this material. Quantitative relations in time and amount of contraction In the feeding of pineal materials to the tadpoles the time interval necessary to establish maximum contraction of the pigment cells increased as the concentration decreased. Tad- poles placed in a 1: 500 pineal emulsion were noticeably lighter in five minutes and required but thirty minutes to arrive at maximum translucency. In higher dilutions the maximum translucency was attained only after a longer interval and in very high dilutions producing only qualitative changes, the maximum was not attained. The dilution of 1:100,000 was the highest that produced a macroscopically discernible qualita- tive action. These reasonably constant quantitative relations have afforded us a means for the evaluing of the strength of our several preparations and may on extended study prove to be PINEAL GLAND FUNCTION 219 a trustworthy method for the standardization of pineal products for at the present time no method exists for testing the strength of such preparations. The quantitative relations between concentration and time will be evident from the following table. DILUTION OF ACETONE EXTRACT MAXIMUM REACTION ATTAINED IN minutes 1: 500 30 1: 1000 45 1: 2000 60 1: 5000 105 1: 10,000 Qualitative change but maximum not attained. 1: 100,000 Maximum not attained. The maximum reaction was determined by comparison with a standard consisting of several tadpoles which had been placed in a 1:500 pineal emulsion thirty minutes prior to the beginning of the experiment. The translucency thus obtained was found to be the greatest possible. It served as the criterion for com- parison as to the degree of depigmentation induced by pineal preparations of unknown activity. In the practical standardiza- tion of pineal preparations, the end reaction of greatest feasibility was the comparative time intervals necessary to attain to maxi- mum translucency. Such a method is obviously open to the criticism that frog larvae are only obtainable for a short period in the year. With Bufo Americana pigment changes were found to be too trivial to be of value in standardization. Rana pipiens were exquisitely responsive and amirably suited for standardization purposes except in that only during the spring months are they obtainable. We are now experimenting with certain amphibian larval forms that may be obtained through- out the year. We have determined that the growth stimulating principle in the pineal is distinct from the principle concerned in pigment changes and this on further investigation may militate against this proposed means of standardization. 220 CAREY P. McCORD AND FLOYD P. ALLEN Effect of pineal gland upon melanophores of other forms! Pineal gland extracts have no demonstrable effect on either type of chromatophore of Loligo (squid) or upon the melano- phores of adult Fundulus. The extracts distinct contraction of the melanophores as may be noted in accompanying table. ONE WEEK-OLD FUNDULI IN BOILED SEA-WATER Time 9.15 9.38 9.46 9.59 10.31 11.51 11.55 1.55 3.01 however determine of young Fundulus No. 1 ONE-WEEK-OLD EMULSION OF ACETONE EX- TRACT OF BEEF MUSCLE IN No. 2 FUNDULI IN SEA-WATER Time Complete expansion | 9.15 of melanophores Complete expansion of melanophores Complete expansion of melanophores Complete expansion of melanophores Complete expansion of melanophores Complete expansion of melanophores All transferred fresh sea water p-m. Complete ex-| 1.56 pansion p-m. Complete pansion 9.40 9.47 10.00 10.32 11.52 to ex- Complete expan- sion Complete expan- sion Complete expan- sion Complete expan- sion Complete expan- sion Complete expan- sion Complete expan- sion Complete expan- sion No. 3 ONE-WEEK-OLD FUNDULI IN EMULSION OF ACETONE EX- TRACT OF PINEAL GLANDS IN SEA-WATER Time 9.15 Complete ex- pansion 9.37 Beginning con- traction 9.45 Contraction in- creasing 9.58 Contraction al- most complete 10.30 Compiete con- traction 11.50 Complete con- traction 1.54 Partial expan- sion 3.00 Complete expan- sion of some of the melano- phores, espe- cially in tail; partial expan- sion of others Mode of absorption of the pigmentation altering principle Several experiments were tried with the object of showing whether the pineal tissue must be ingested or not in order to produce the reaction. 1 Experiment conducted by A. Noble at Woods Hole, Mass. PINEAL GLAND FUNCTION 221 1. Pineal emulsion on anesthetized tadpoles. Several tadpoles of equal depth of pigmentation were completely anesthetized with of ether after which half were placed in an emulsion of acetone extract of beef pineal, the others in a like emulsion of muscle tissue. Five minutes later those in the pineal emulsion were preceptibly lighter, later acquiring a marked translucency. The latter remained unchanged. The tadpoles recovered from the anesthetic and gradually regained their original appearance. 2. Hypodermatic injection of pineal emulsion. Several tadpoles of equal depth of pigmentation were divided into two groups. One received 0.01 cc. of an emulsion (1-500) of acetone extract of pineal gland, injected hypodermatically into the peritoneal cavity; the other received the same amount of normal saline solution injected in a similar way. Shortly after injection the pineal treated animals became lighter, eventually reacting to the maximum degree of translucency. The other tadpoles remained practically unchanged in appearance. As a further control a third group was immersed in the pineal emulsion for the length of time consumed in making the injection and the tadpoles were then washed in tap water. They remained un- changed. This control showed that the effect produced was due to the injected pineal material and not to any accidental absorption through the skin. 3. Effect of pineal emulsion on eviscerated tadpoles. A number of tadpoles were completely eviscerated. These tadpoles live and swim about as freely as their fellows. Following this procedure part of the animals were removed to an emulsion of the acetone extractive of beef pineal gland and shortly passed through the same pigment changes as normal tadpoles placed in this emul- sion at the same time. The eviscerated and normal tadpoles remaining in tap water did not change in color. These observations prove conclusively that the principle involved is directly absorbable through the gills or skin. The effect is produced without the intervention of the processes of digestion. There is no indication, however, as to whether the principle acts directly upon the melanophores, or indirectly through the medium of the central nervous system. 222 CAREY P. McCORD AND FLOYD P. ALLEN Effect of pineal extract upon unstriated muscle Aqueous extracts of fresh pineal glands were tested accord- ing to the method of Dale and Laidlaw (13) upon isolated strips of guinea-pig uterus. The extract produced a typical though feeble contraction of the uterine muscle. Three cc. of a 20 per cent pineal extract was roughly equivalent in activity to 0.004 ec. of a 20 per cent pituitary extract. Thus pineal extracts stimulate certain smooth muscle cells as well as pigment cells to contraction. This similarity of action goes far to con- firm Spaeth’s hypothesis that the melanophore is a type of smooth muscle cell. COMMENT Many acceptable evidences associate the pineal gland with an earlier optical function. In the reptilian stage of evolution this parietal eye probably attained to its highest development. In the embryos of certain lizards (Lacerta agilis) the typical eye structure is still evident, but in no form living at the present time does the pineal gland retain an ocular function, of high order (14). The color changes in forms are obviously in adjustment to environmental conditions. The eye is the essential controll- ing factor in this adjustment. When in blinded animals cer- tain definite changes in pigmentation still occur, on theoretical ground it is tenable to assume that the pineal body retains suf- ficient ocular mechanism to exert its influence upon the pigment cells. This is the hypothesis suggested by Fuchs (10). Laurens has established experimentally that such an activity on the part of the pineal is highly improbable (11). Accepting the contentions of Laurens, it is the gist of our work that while the pineal does not act in the role of its ancient ocular function, it contains within itself an active principle capable of inducing pigment changes independent of and wholly apart from environ- mental conditions. The pineal pigment changes dominate and appear despite environmental conditions tending toward the PINEAL GLAND FUNCTION 225 opposite phase. The salient observations that we have recorded in detail on the foregoing pages are: 1. Up to near tenth day of larval life in tadpoles, pigmenta- tion is not influenced by pineal feeding. Evidences relative to this are not precise, but suggest that this is due to incomplete development of the nervous mechanism involved. 2. Beginning at this time and continuing until near the ter- mination of metamorphosis, the addition of traces as small as 1 part acetone extract in 100,000 parts water determine distinct cyclic pigment changes peculiar to these preparations. Prior to feeding, both controls and experimental animals are uniformly dark colored. Shortly after feeding the pineal fed groups begin to lose color until within thirty minutes, all macroscopic pig- ment is lost so that all the larger viscera are clearly visible (figs. 1 and 2). The condition is transient and the cycle is complete with full restoration of color within from three to six hours, unless further pineal food is added to the trays. As metamor- phosis is completed the pigment is no longer altered by pineal materials, due to rearrangements of chromatophore types and sites in the adult animal. 3. The response in pigment change is quantitative. A method is described for the standardization of pineal preparations. 4. The pineal substance responsible for the pigment changes is wholly extracted by acetone. The residue after acetone ex- traction is an inert substance as to pigment influence. How- ever this residue has an influence on growth and differentiation. The inference is that the gland contains more than one active substance. 5. The reactions produced by pineal extracts add some evi- dence to Spaeth’s contention that the melanophores are modi- fied smooth muscle cells. The similarity of contraction of certain smooth muscle organs under the influence of pineal extracts and the contraction of melanophores is in keeping with Spaeth’s hypothesis. The very nature of this pineal-pigment cycle affords an ex- cellent method of approach to the mechanics of melanophore function and from this the larger problems of colloidal state. 224 CAREY P. McCORD AND FLOYD P. ALLEN LITERATURE CITED (1) BatLtowiTz, E. 1914 Pfliiger’s Archiv, Bd. 157, 165. (2) Harerpr, C. W. 1912 Jour. Animal Behav., vol. 2, 51. (3) Hooker, Davenport 1914 Science, N. S., vol. 39, 473. (4) Hooker, D. 1912 Zeit. F. allg. Physiol., Bd. 14, 93. (5) Battowirz, E. 1893 Zeit. wiss. Zool., Bd. 56, 673. (6) Laurens, Henry 1915 Jour. Exp. Zodl., vol. 18, 577. (7) SparTH, R. A. 1916 Jour. Exp. Zodl., vol. 20, 193. (8) Hooxrer, D. 1914 Am. Jour. Anat., vol. 16, 237. (9) Bapax, E. 1910 Pfliiger’s Archiv, Bd. 131, 87. (10) Fucus, R. F. 1915 Winterstein’s Handb. d. vergl. Physiol., Bd. 3, Teil 42, 1189. (11) Laurens, H. 1916 Jour. Exp. Zodl., vol. 20, 237. (12) GupERNATscH 1914 Am. Jour. Anat., vol. 15, 481. (13) Daur and Larpuaw 1912 Jour. Pharm. and Exp. Ther., vol. 4, 75. (14) Przrson, Human Anatomy, 4th ed., vol. 2, 1124. STUDIES ON SEX IN THE HERMAPHRODITE MOLLUSK CREPIDULA PLANA Il. INFLUENCE OF ENVIRONMENT ON SEX HARLEY N. GOULD Department of Biology, Princeton University CONTENTS IU AOC CHAOT acta ooo ERE EPI aEe ceolnicastooic.o.5 &13.0.0.0 0 coho ene Chee 225 Degeneration of the testis.in male Crepidulas......05.6.2.2....0.025600¢00- 226 Male development in neuter individuals... .¢ccenages ck onic ss Des aeee ces 229 Inniwencexotlarcersonysmallernmalesan.-cap eerie eieiie cree iinet 238 Reversibility of temale differentiation... 8s .cecemeeene ss dees tt cee cee e nee 240 Naturevotrstimulws) toomiale developmentm: ance cere seen salen eee 242 Cenera considerations and Comparisonsan oases aie oe oda 247 SSHUODTD ATEN ei Secccg 3 ig SUIS cht neRe Re RR aoe ote nic ele Cao od co boca time ee eon oa ae 248 INTRODUCTION In a former paper (Gould, 717) the writer has followed out the sexual cycle of Crepidula plana and has shown that the sexual life of the adult may be divided into (A) the male phase, (B) the transitional phase and (C) the female phase. During the transi- tion period the testis degenerates and eventually all the primor- dial male cells in the gonad disappear; while the primordial female cells, present from the beginning, multiply, and form the ovary. It was also pointed out that there was great irregularity in the development of the male phase. A number of specimens of the same size and apparently of the same age, taken at the same time of year, may show widely different sexual states. One may be a fully developed male; one may be a partially developed male; one may exhibit evidence of having been a male though the male characters are being lost; and one may furnish no sug- gestion that any male characters have ever developed. The analysis of this phenomenon was reserved for the present paper; 225 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, No. 2 sguLy, 1917 220 HARLEY N. GOULD and while the analysis is by no means complete, a number of facts have been disclosed which have to do with the influence of environment on sexual development. Light was first thrown on the question by the observation that those animals which we may call ‘sexually inactive’ or ‘neuter,’ though of a size equal to the males, were most frequently found in the ‘new’ hermit crab shells (1.e., Gastropod shells only re- cently acquired by the hermit), in which there were only a few small Crepidulas, and no large females. Of 216 specimens taken from such situations, there were 11 males (5 per cent). The other 205 (95 per cent) were either immature males, degenerate males or sexually inactive. This could be determined in the live animals by the amount of development or the non-development, of the external sex organs, the penis and seminal groove. The majority of all Crepidulas found in the ‘new’ shells had the thin, round, smooth shell which is characteristically formed on an unobstructed surface. In the large colonies, or in fact in any shell where one or more large female Crepidulas were present with the smaller ones, the proportions were strikingly different. Material of this kind was more abundant and 1066 small individuals were examined, of which 662 (62.1 per cent) were adult males, 404 (87.9 per cent) were either immature males, degenerate males or sexually inactive. DEGENERATION OF THE TESTIS An accident disclosed another interesting fact. About 50 males had been removed from the colonies in the hermit crab shells, and placed by themselves in a dish under running salt water. Whey were neglected for several weeks and when subse- quently examined seemed to be perfectly normal except that the penis had in every case disappeared, leaving only a small brown hump behind the right tentacle. Sections of the gonad showed that it had shrunken to small strands which ran here and there in the visceral sac. This chance observation coupled itself with the fact that males are far more numerous in large Crepi- dula colonies than in ‘new’ hermit shells having only a few small INFLUENCE OF ENVIRONMENT ON SEX PAT inhabitants. It seemed advisable to try a controlled experi- ment of removing the males from the colonies, and of examining their gonads after various periods of time. Experiment 1 (tables 1 and 2). Carried on in salt water aquaria of Princeton University vivarium. The smaller Crepidulas were re- moved from shells inhabited by hermit crabs and also containing colo- nies of C. plana, and the males were selected from them. The only Crepidulas left in each hermit shell were the two or three large females. Some of the males were then placed on and around the females to serve as controls (table 2) while the rest were allowed to attach them- selves in other shells from which all the Crepidulas had been removed TABLE 1 Specimens removed from large colonies containing female, found to be males, and transferred, two or three together, to hermit shells in which were no other Crepidulas Z i) 5 2 mn PENIS GONAD SEMINAL VESICLE 61} ? | 10} Long Shrunken to small strands; |} Full of sperm spermatogonia; sperm 62] ? | 10} Long Shrunken to small strands; | Full of sperm spermatids; sperm; dis- torted nuclei 63 | ? | 10} Short Small; spermatogonia; sper- | Full of sperm matids; sperm 67 |15 | 13) Short Small strands; inactive Thick wall; empty 68 | 7 | 13} Short Small strands; few sperma- | Thick wall; few sperm togonia and sperm 69 | 7 | 13} Stump | Small; spermatogonia only | Not included in sections 73 12 | 18) Short Inactive; type A and B cells | Small; full of sperm in wall; few sperm 74 |12 | 18) Short Has considerable lumen, | Full of sperm; unusually empty except for a few large number of apyrenes spermatogonia and sperm 75 |12 | 18} Stump | Empty; inactive Reduced to indifferent goni- duct 79 | 9 | 34) Stump | Small; inactive Thick wall; empty 80) 8 | 34) None Small strands; inactive Thick wall; empty 81| 8 | 34| Stump | Small strands; inactive Reduced in size; a few sperm $2 |10 | 34) None Preparation for oogonial | Not included in sections division. 83 | 72| 34; Stump | Small strands; inactive Not included in sections 228 HARLEY N. GOULD TABLE 2 Specimens removed from large colonies containing females, found to be males, and replaced in hermit shells in which were from one to four large female Crepidulas Z i] z 3 wm PENIS GONAD SEMINAL VESICLE 64] ?] 10) Long Normal testis Full of sperm. 65} ?| 10) Long Normal testis Full of sperm 66| ?| 10} Long All stages of spermatogene- | Very large and full of sis except spermatocytes sperm 70 | 13) 13} Long All stages of spermatogene- | Full of sperm sis but testis small : 71 | 11] 13} Long Good size; normal testis Very large; full of sperm 72] 9] 13) Long Good size; normal testis Full of sperm 76 | 11} 18) Long Normal testis Full of sperm 77 | 11] 18} Long Normal testis; very active | Very large; full of sperm 78| 9] 18} Long All stages of spermatogene- | Full of sperm sis, but testis small 84 | 16} 34) Long All stages of spermatogene- | Full of sperm sis, but testis small 85 | 18} 34| Long All stages of spermatogene- | Full of sperm sis, but testis small 5 86 | 15) 34) Long All stages of spermatogene- | Full of sperm sis, but testis small; mi- nority of spermatocytes 87 | 9] 34| Long Normal testis Full of sperm 88 | 7| 34| Long Normal testis Full of sperm 89| 6) 34; Long Normal testis Very large; full of sperm 90 | 5} 34) Long Normal testis Very large; full of sperm (females as well as males). Several males were put in each hermit shell as was the case with the controls. The two sets of hermits were then shut up in two crates and sunk in the aquarium. After ten days three Crepidulas were taken from each lot and sec- tioned. Specimens 61, 62 and 63 of table 1 were taken from the her- mit shells without female Crepidulas. In two of the three the testis was completely and in the third, partly degenerated. In the controls with females (specimens 64, 65 and 66 of table 2) two were normal males, the third showed no spermatocytes. After thirteen days three more were taken from each lot, and showed a still more marked differ- ence (specimens 67, 68, 69; controls, 70, 71, 72). The external genitalia, as well as the testis, degenerates in the segregated males. Still other samples were taken after eighteen days, and at the end of thirty-four days all those left in each crate which had been males originally, were fixed and sectioned. As the tables show, the males which had been separated from the females suffered regressive changes in the testis INFLUENCE OF ENVIRONMENT ON SEX 229 and the accessory male organs.. Some of the controls showed partial regressive changes but the majority remained perfectly normal. The significance of the words at head of columns in the tables is as follows: ‘Specimen’, identification number; ‘length’, length of shell of specimen in millimeters; ‘days’, number of days duration of experiment; ‘Penis’, ‘Gonad’, ‘Seminal vesicle’, condition of those organs in the prepared specimens. Experiment 2 (Tables 3 and 4). Carried on in floating live-cars of the Marine Biological Laboratory, Woods Hole. This is similar to the preceding experiment except that it was performed under more nearly normal conditions. It shows the same degeneration of organs in the segregated males. Here and there among the controls (table 4) we find a specimen in which the activity of the testis is reduced, or there is slight degeneration; this is not unexpected, since a few degenerate males are found in normal colonies. The results of experiments 1 and 2 strengthened the suspicion that the large female Crepidulas of the colony exercised some influence upon the small male members of the same colony. The preservation of the male character seems to depend on the presence of the larger animals in the same neighborhood with the males. Another experiment confirmed this still further. Experiment 3. All the large females were removed from two colo- nies leaving the smaller animals which had been clustered about them untouched. It was assumed, though it could not be made certain, that the usual majority of males was present. The shells occupied by the colonies and inhabited by the hermit crabs were replaced, after removal of the females, in the aquarium and left for fifteen days. At the end of that time all the smaller Crepidulas were fixed and sectioned. Nine out of the eleven specimens showed that they had been func- tional males, but that the testes had degenerated to various degrees. The presence of the seminal vesicle with sperm in it proved that the testis had recently been active, even though the testes of some indi- viduals were reduced to the neuter condition. Two of the eleven specimens showed that they had not been males when the experiment was begun; for the seminal vesicle was not developed. MALE DEVELOPMENT IN NEUTER INDIVIDUALS It is possible to show, in Crepidula plana, not only that the preservation of the male phase depends upon the proximity of larger individuals, but that ‘sexually inactive’ or ‘neuter’ speci- mens will rapidly develop male characteristics when brought into 230 SPECIMEN 115 116 117 118 134 135 10 DAYS 14 14 14 14 19 19 19 19 19 19 19 PENIS Long Long Half usual length Long Half usual length Long Very short Long Long Long Half usual length = usual length Half usual length Short Long Long 4 usual length HARLEY N. GOULD TABLE 3. Males treated as in table GONAD Small strands; distorted nuclei Small strands; tids; sperm Small strands; inactive sperma- Normal testis Few spermatogonia; sper- matids; sperm Allstages spermatogenesis, but testis small; some follicles nearly empty Very much reduced; some spermatids and sperm; a few resting oocytes All stages spermatogene- sis but much reduced in size Small strands; degenerat- ing cells; some follicles empty Small; interior nearly empty; some spermato- gonia and sperm Oocytes in early synapsis Very small; inactive Small; sperm and coagu- lated matter Small strands; few sperm All stages of spermato- genesis; reduced size All stages of spermato- genesis; only few sper- matocytes; reduced size Spermatogonia, sperma- tids and sperm; reduced size SEMINAL VESICLE Thick wall; full of sperm Full of sperm Full of sperm, mostly apyrenes Full of sperm Full of sperm, largely apyrenes Not included in sections Full of sperm Full of sperm Full of sperm Contains sperm and co- agulated matter Thick wall; few apyrene sperm Small; thick wall Full of sperm few Small; thick wall; sperm Full of sperm Small; full of sperm Full of sperm INFLUENCE OF ENVIRONMENT ON SEX Dot TABLE 38—Continued a ic] 5 é n PENIS GONAD SEMINAL VESICLE Blea Wea n = = 136 | 94} 19) Short Small; lumen empty ex- | Small; full of sperm cept for few sperm 137 | 9 | 19} Short A few spermatogonia; | Full of sperm spermatids; sperm 138 | 8 | 19) Stump Spermatogonia; sperma- | Small; few sperm tids; sperm 139 | 7 | 19) Short Spermatogonia; sperma- | Small; few sperm tids; sperm 150 |17 | 33) Short Small; inactive Not included in sections 151 |14 | 33) Very short | Small; degenerating sper- | Thick wall; some parts matogonia full of sperm, some empty 152 |13 | 33} 7 usual All stages spermatogene- | Full of sperm : length sis, but reduced size 153 |12 | 33) Stump Spermatogonia; few sperm | Thick wall; few sperm 154 |11 | 33) None Small; empty; distorted | Shrunken to nearly nuclei straight tube; occa- sional sperm 155 |11 | 33) Stump Shrunken to solid strands; | Shrunken to nearly distorted nuclei straight tube; occa- sional sperm 156 |11 | 33) Stump Small; nearly empty; oc- | Small; thick wall; empty casional sperm; dis- torted nuclei a colony containing individuals larger than themselves (e.g., the large females). Experiment 4. Carried on in float-cars, Woods Hole, June to August, 1915. The writer collected as many hermit shells as possible where there were no female Crepidulas but only a few small specimens in each hermit shell, all about of the same size. The majority of these had thin, flat smooth shells. They were examined with a lens and the few which showed any development of accessory male organs were discarded. The rest were divided into two lots. Those of one lot (table 5) were allowed to attach themselves inside hermit shells where one or several large females were already attached. Those of the other lot were returned, several together, to hermit shells where there were no other Crepidulas whatever (table 6). It was subsequently found that the sexual condition of an individual is somewhat influenced by the presence of another of only slightly larger size; the experiment 232 120 121 122 12 124 125 126 127 128 140 141 I42 143 I44 145 146 147 lds 149 157 158 159 160 161 162 1638 LENGTH PRNIS Long Long Long Long Long Long Long Long Long Long Long Long Short Short Long Long Long Long Long Long Half usual length Long Long Long Long Long Long HARLEY N. GOULD TABLE 4 Males treated as in table 2 QONAD Normal testis Normal testis Normal testis Normal testis Normal testis. Very large Normal testis Normal testis Normal testis Normal testis. Very large Normal testis Reduced in size; all stages of spermatogenesis Normal testis Warly stages of spermato- genesis but no late stages Spermatogonia; sperm Normal testis; very large Normal testis Normal testis Normal testis. Normal testis Normal testis. Very large Spermatogonia; sperm Large Normal testis All stages spermatogene- sis, but reduced in size Normal testis; small Normal testis Normal testis Normal testis SIMINAL VESICLE Very large but nearly empty; probably after copulation; this condi- tion seldom seen Small; full of sperm Very large; full of sperm Very large; full of sperm Small; many apyrenes Full of sperm ull of sperm Full of sperm Small; full of sperm Full of sperm Full of sperm Full of sperm ew sperm, largely apy- rene Small; full of sperm Large; full of sperm Full of sperm Full of sperm Large; full of sperm Pull of sperm Full of sperm Small; thick wall; some sperm Full of sperm lull of sperm Mull of sperm Very large; full of sperm Full of sperm Full of sperm would have been more striking, therefore, if in the controls each speci- men had been completely isolated. The difference of behavior of the two groups is plainly to be seen, however, in tables 5 and 6, made up of observations on the sectioned specimens at the end of the experiment, which lasted thirty-four days. INFLUENCE OF ENVIRONMENT ON SEX TABLE 5 233 Neuter Crepidulas transferred to neighborhood of large females. Sectioned after thirty-four days. Specimens from each hermit’s shell listed together. W herever sufficiently marked, increase in length of shell (‘growth’) and amount of develop- ment of penis, during course of experiment, have been recorded. Summary of results: with adult testis, 18; testis to spermatids, 6; testis to spermatocytes, 2; with spermatogonia only, 1; sexually inactive, none. Total, 27 & | 5 at a a > = oO = PENIS GONAD GONIDUCT Bl e\e a 4 o Hermit 1 175 |13 Stump Young testis to sperma- | Not included in sections tids; small 176 |13 Stump Young testis to sperma- | Seminal vesicle in process tids of formation 177 |123| 1 | None Few spermatogonia, sper- | Undifferentiated matocytes and sperma- tids Hermit 2 178 |10 | 33} Long Spermatogonia; spermato- | Not included in sections cytes; spermatids; small 179 | 93 Long Adult testis Vesicle, full of sperm 180 | 9 | 13} Short Adult testis, large Vesicle, full of sperm 181 | 9 Very short | Adult testis; very large Vesicle, full of sperm 182) 7 | 13) Long Adult testis; small Small vesicle, full of sperm Hermit 3 183 }11 | 23; None Spermatogonia; sperma- | Not included in sections tids Hermit 4 184 |10 | 2 | Long Adult testis, large Vesicle, full of sperm 185 | 9 Long Adult testis Vesicle, full of sperm 186 | 8 Long Adult testis Vesicle, full of sperm 187 | 8 Long Adult testis Vesicle, full of sperm 188 | 73| 2 | Stump Adult testis Vesicle, full of sperm 189 | 73 Stump Spermatogonia and sper- | Empty tube, somewhat matocytes, very active convoluted 190| 7 | 1 | Long Adult testis Vesicle, full of sperm 234 HARLEY N. GOULD TABLE 5—Continued a rc a 5 PENIS GONAD GONIDUCT 3 z | 0 le 191 | 7 None Spermatogonia; sperma- | Small tube, somewhat tocytes convoluted LO ZT ie) la lsomeg: Adult testis Seminal vesicle, full of sperm 193 | 6 | 14] Long Adult testis, large Seminal vesicle, full of sperm Hermit 5 194} 8 | 2 | Stump Adult testis, small Small convoluted tube 195 | 7 | 13} Long Adult testis, large Seminal vesicle, full of sperm 196 | 7 None Spermatogonia Smali tube, straight 197 | 7 Long Adult testis, large Seminal vesicle, full of sperm 198 | 7 Half usual | Young testis to sperma- | Small convoluted tube length tids LOO ON eles loner Adult testis, large Seminal vesicle, full of sperm 200 | 6 Long Adult testis, large Seminal vesicle, full of ; sperm 201 | 6 Long Adult testis, large Seminal vesicle, full. of sperm The above tables show that the great majority of neuter ani- mals become males when associated with large females; that in the absence of large females, only a very few neuter animals develop male characters, and these few are always found in proximity to somewhat larger individuals. It is to show this fact that the specimens from each hermit shell are listed together in table 6. In no case does the largest individual in a colony ever become a male. The stimulus from the larger to the smaller individuals is quantitative. A number of the latter in table 6 have gone through the first stages of male development, but the change is not often complete. The number of sexual products formed is small, motosis rare; the specimens of table 5, on the other hand, nearly all show full male development, both in primary and sec- INFLUENCE OF ENVIRONMENT ON SEX TABLE 6 235 Neuter Crepidulas returned to hermit’s shells similar to those which they formerly occupied, i.e., without large female individuals. days. Specimens from each colony listed together. Sectioned after thirty-four Summary of results: with adult testis, 3; testis to spermatids, 2; testis to spermatocytes, 1; with spermato- gonia only, 11; sexually inactive, 5; with oogonia, 2. Total 24 See lias = 5 5 PENIS GONAD GONIDUCT Hermit 1 202 |16 | 4 | None Many primordial egg cells | Undifferentiated 203 |15 | 3 | None Many primordial egg cells | Undifferentiated 204 |13 | 2 | None Small; few spermatogonia | Undifferentiated 205 {13 | 2 | None Spermatogonia, degener- | Undifferentiated ating 206 |12 | 4 | None Spermatogonia, degener- | Proximal part somewhat ating wider and - slightly twisted 207 |123 None Spermatogonia Undifferentiated 208 |103 Short Small adult testis Small seminal vesicle with a few sperm Hermit 2 209 |15 None Inactive Undifferentiated. 210 |13 None Inactive Undifferentiated (trans- parent cells) 211 j11 None Few spermatogonia Undifferentiated 212 |11 None Spermatogonia, degener- | Not included in sections ating ae 213 |103 Stump Spermatogonia Proximal end somewhat widened and convo- luted 214 | 9 Stump Adult testis; small Small seminal vesicle; sperm 215| 8 Short Adult testis Seminal vesicle; small; sperm Hermit 3 216 | 9 | 2 | None Inactive Slightly widened proxi- mally 217 | 9 | 2 | None Inactive Undifferentiated 236 HARLEY N. GOULD TABLE 6—Continued 5 BS PENIS GONAD GONIDUCT Ss | ee |S Bea leaalac Hermit 4 218 |15 | 6 | None Few spermatogonia Undifferentiated 219 |13 | 4 | None Few spermatogonia Undifferentiated 220 |10 | 2 | None Young testis to spermatids | Not included in sections 221 |10 | 2 | None Inactive Undifferentiated 222 | 93| 2 | None Spermatogonia and sper- | Undifferentiated matocytes PPS, \| Oey None Spermatogonia Not included in sections 224 | 9 None Spermatogonia and sper- | Slightly widened and matids twisted proximally ; empty 225! 8 | 14) None Spermatogonia Undifferentiated ondary characters, great numbers of sperm and active spermato- genesis. The results of all experiments indicate that the neuter animal, living in isolation and having reached a considerable size, requires only a very slight stimulus to start the develop- ment of male characters, but a greater one to complete it. It is a question whether the production of a limited number of spermatogonia is a sure indication of a stimulus from the out- side; certain specimens which had each been kept isolated in a small vial produced a few spermatogonia; but development never went any farther. Experiment 4 was repeated and samples were taken from the colonies at various times. Space is not available to submit the results in tabular form. In general, the neuters quickly took on male characters when placed in colonies with large females; while the neuters kept apart rarely did. Nine days after hav- ing been transferred to the neighborhood of large females, the formerly neuter specimens had testes developed as far as sperma- tids; adult males were found in sixteen days. The specimens had been marked by notches in the shells to obviate any possible mistake in identity. When left for long periods, the small Crepidulas living near large females become more and more divergent from those living INFLUENCE OF ENVIRONMENT ON SEX OAT in the absence of large females, both in size and in sexual con- dition. This is indicated briefly in tables 7, 8, 9, and 10. The experiments on neuter Crepidulas indicate that the ‘sex- ually inactive’ animals are so because male development cannot take place in the absence of a certain stimulus which proceeds TABLE 7 Neuter Crepidulas transferred to neighborhood of large females; sectioned after sixty-seven days Zz Seats 5 B GROWTH PENIS GONAD GONIDUCT 242 1133} None | Long Adult testis Seminal vesicle, full of sperm 243 |10 | None | Long Adult testis Seminal vesicle, full of sperm TABLE 8 Neuter specimens returned to hermit shells free from other Crepidulas, sectioned after sixty-seven days a mo | = = a | & ms : z PENIS GONAD GONIDUCT 244 |23 | 11) None Early growth period of | Not included in sections oocytes 245 |29 | 20} None Ova with yolk Not included in sections TABLE 9 Neuter Crepidulas transferred to neighborhood of large females; sectioned after seventy-five days Bola le = BS ra o = PENIS GONAD GONIDUCT Senza ay a | & n 4 io} 246 |153] 22} Long Adult testis Seminal vesicle, full of sperm 247 |14 | 23} Long Adult testis Seminal vesicle, full of sperm 248 |103] 2 | Long Adult testis Seminal vesicle, full of sperm 238 HARLEY N. GOULD TABLE 10 Neuter specimen returned to hermit shells free from other Crepidulas; sectioned after seventy-five days! 4 -_ 3, mye iS 5 | PENIS GONAD GONIDUCT sl paul A [| n 4 So 249 | 26) 11| None Ova with yolk; (larvae | Not included in sections in mantle cavity) 250 | 19} 5) Long Adult testis Seminal vesicle, sfull of sperm 251 | 26] 15} None Early growth period of | Not included in sections oocytes 252 | 25| 11; None Synaptic stages of oocytes | Transitional 1 Specimen 250 was found in the same hermit shell with 249 and attached di- rectly behind the latter. The male development in 250 was undoubtedly due to the proximity of the larger animal; 251 and 252 were each alone in a hermit shell. from larger to smaller individuals. The stimulus may act upon animals of quite advanced size. Its effect is seen in a very short time. If male development is to go on uninterruptedly the stimulus must be constantly supplied. Whenever it is removed male development ceases and the male organs degenerate. Individuals which are free from the male-producing stimulus grow much more rapidly than those which remain in the male phase; and the attainment of the large size is correlated with female development (tables 8 and 10). (For other factors influencing growth, see former paper.) INFLUENCE OF LARGER ON SMALLER MALES In the experiments so far recorded, only large female Crepidu- las were used for the purpose of causing the male condition to appear or preserving it, in the smaller animals. It is important to know whether the quality of inciting and sustaining male de- velopment is limited to animals in the female phase. The re- sults of experiment 4, table 6, indicated that larger Crepidulas, though not females, might influence smaller ones. The follow- ing tests were made to determine whether the presence of very large males would sustain the male condition in smaller specimens. INFLUENCE OF ENVIRONMENT ON SEX 239 Experiment 5. (Carried on in float cars, Woods Hole.) The largest males which could be found (18 to 22 mm.) were removed from their colonies and transferred to hermit shells in which there were no other Crepidulas. Much smaller males were transferred to the same hermit shells and allowed to attach themselves near the larger. It was of course realized that the male organs of the large specimens would soon degenerate after being transferred but in the meantime their effect on the smaller males could be observed, and their effect while they were in the transition period. Several of the small males were fixed and sectioned from time to time, and the observations on them may be briefly tabulated as follows: A. Fourteen days after transferring; 6 normal males, 4 more or less degenerate. B. Fifteen days after transferring; 2 normal males, 2 degenerate. C. Twenty-two days after transferring; 1 normal male, 4 more or less degenerate. D. Thirty-one days after transferring; 2 normal males, 2 more or less degenerate, 2 with evidence of regeneration after degeneration. EK. Thirty-six days after transferring; 1 normal male, 2 degenerate. F. Thirty-nine days after transferring; 1 normal male, 2 with evi- dence of regeneration of testis. A fourth specimen in this lot, not one with which the experiment was started, had secured an attachment to the hermit shell probably after being dislodged from another; it shows evidence of recent male development, undoubtedly caused by its new association with the large (formerly male) specimen on which it was found mounted. G. Forty-eight days after transferring; 1 normal male, 1 male with evidence of regeneration of testis. H. Seventy-four days after transferring; 1 normal male, 1 male with evidence of regeneration of testis. I. Seventy-eight days after transferring; 5 normal males, 1 immature male. Examination of the material from experiment 5 brought out several interesting things: Fewer small males degenerated than in experiments where they were completely removed from larger animals; but more than when they were left near large females. At the end of thirty-one days a hitherto unobserved phenome- non is discovered. Two specimens from D show very clearly that after having undergone degeneration the testis has devel- oped anew. ‘The accessory male organs do not yet show regen- eration; the penis has disappeared leaving only a small stump and the seminal vesicle has the thick-walled condition assumed when the sperm are being absorbed (see former paper). There * 240 HARLEY N. GOULD are adult sperm in the vesicle. The testes of these specimens however, plainly exhibit great activity, having all stages of spermatogenesis to spermatocytes in one case, spermatids in the other. Similar conditions are seen in two specimens of F. By this time the large animals about which the smaller are clus- tered are no longer males. Sections through the gonads of sev- eral showed that there was no longer a testis, but no sign of femaleness had yet appeared. Eventually, the large specimens gradually assumed the female condition, growing rapidly as they did so, and the smaller individuals clustered about and upon them were then nearly all found to be in the active male condition (HandI). All the small males which had gone through a period of degeneration subsequently experienced regeneration and finally show no sign of the former cessation of activity. It is possible that some males have not degenerated at all, since every lot taken showed at least one individual with normal testis. Summing up the facts gained in this experiment: 1) Large males will have an effect on the smaller ones similar to the effect of large females on males, but not so marked. 2) A large ani- mal with a degenerate testis will also give a stimulus to a smaller individual. 3) An immature female, which was formerly a male, will likewise give a stimulus. 4) After the degeneration of the testis, a specimen may subsequently regenerate it again, if it receives a stimulus from a larger individual. 5) The largest individual in such an artificial colony as has been described never shows regeneration of the testis following degeneration; there is no indication that a smaller animal can affect a larger. Some attempt has been made to find whether a male freshly removed from a normal colony will cause male development in a smaller neuter; the evidence is incomplete, but suggests that it may do so. REVERSIBILITY OF FEMALE DIFFERENTIATION Although an adult or nearly adult female can apparently not again assume the male condition, it is clearly shown that the partial development of the female organs may pause in its early INFLUENCE OF ENVIRONMENT ON SEX 241 stages and give place to the male phase. Occasional instances found in nature indicate this, and in a few cases the superpo- sition of male on female development has been accomplished experimentally. Two such cases are described in table 11. TABLE 11 Superposition of male on female development. Specimen 313: collected as neuter; placed close to two considerably larger immature females for eight days. Speci- men 314: collected as neuter; placed between two large females in fingerbowl for nineteen days a <3] S 3 eal GONAD GONIDUCT az 3 313 | 12 | 3 mm. distant | Various synaptic and early | Proximal part from a 20 growth stages of oocytes becoming con- mm. specimen. around periphery of go- voluted Other large nad; also, rapid sperma- specimens also togonial multiplication near 314 | 13° | Close to and be- | Various synaptic stages of | Proximal part tween two oocytes; but frequent slightly en- large females division of primordial larged and con- in fingerbowl male cells voluted The above accounts for the occasional presence of oocytes in the male gonad of C. plana. It is due to spermatogenesis in- terrupting the first stages of female differentiation. In such an event the oocytes do not persist very long, but if male develop- ment continues, degenerate. Undoubtedly in C. plana the separation of the two sexual phases is an adaptation so fastened upon the species that sperm and eggs cannot develop under the same conditions in the gonad. The attempt was made several times to cause nearly adult females to return to the male state. All results go to show that after the oocytes are well on in the growth period no more male development is possible. Careful examination of ovaries was made after the effort to incite spermatogenesis, in order to find whether there were at least any mitoses of primordial male cells; and not only were such mitoses absent, but primordial THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, No. 2 242 HARLEY N. GOULD male cells were also lacking. If they completely disappear, as seems to be the case, the inability of females to re-assume the male phase is explained. NATURE OF THE STIMULUS As soon as it became evident that there was actually a stimu- lus passing from the larger to the smaller individuals of C. plana, affecting the sexual development of the latter, experiments were undertaken to discover the nature of that stimulus. Unfortu- nately up to this time all experiments have given negative re- sults. Yet since they have served to limit the field of inquiry and furnish additional proof of the presence of the unique influ- ence, it is thought worth while to include them in this report. Other more precise tests are planned for the future. Copulation When it was first found that males would degenerate on being removed from the vicinity of the females it was suggested that the sex gland atrophied through loss of functional activity; but the development of male organs in neuter animals which had not previously the power of copulation disposed of this possibility. Motility : Males are more motile than females (Conklin, ’98) or than sexually inactive individuals. Is it possible that frequent movement of small animals in large colonies has anything to do with development of male organs? To this it may be answered that motility is not necessarily a property of males; a consider- able number of males have been found whose shells indicated by the conformation to the underlying surface that they had not moved at all during nearly the whole period of their lives; and one neuter animal remained constantly in the same spot for several weeks while undergoing male development under ex- perimental conditions. INFLUENCE OF ENVIRONMENT ON SEX 243 Food In view of the work which has from time to time purported to show that nutrition has an influence on sex, an experiment was performed to show whether a difference in amount of food ma- terial might be responsible for the difference of sexual develop- ment of small Crepidulas. The experiment is based on the assumption that the Crepidulas take the same sort of food as the hermit crab, and that they do in fact live on the fragments thrown free in the water about the hermit’s crab’s head as he tears up food with his chelae and jaws. Experiment 8. Carried on in the laboratory aquarium, Woods Hole. Table 12. Small neuter individuals were marked by a notch in the front edge of the shell and transferred to hermit shells from which all but the large females had been removed. All the hydroids were first scraped from the hermit shells in order to remove all possible sources of food about the Crepidulas. The new colonies were then placed in a glass aquarium about | foot in diameter and the same in depth, into which ran sea water which had first been passed through a Berkefeld filter. After periods specified in the table, the animals were fixed and sectioned. The position in the colony with reference to the females at the end of the experiment is noted in the table. The hermit crabs though unfed remained active and apparently unaffected. Table 13. Other specimens from the same lot as those used above were notched and transferred to hermit shells from which all but the large female Crepidulas had been removed, as had been done with the others, and the new colonies were put in a similar glass aquarium. Sea water which had not been filtered was allowed to run slowly into the jar, and the hermit crabs were fed every day with crushed spider crabs, small fish, etc., large quantities of which they devoured. Specimens from the colonies were fixed and sectioned at the same times as from the starved colonies. It will be observed that the individuals which show least amount of male development or none (specimens 362, 363, 368) had settled themselves after attachment, at some distance from the females. The foregoing indicates that male development is not a mat- ter of food or the lack of it; for development takes place equally well whether the neuters have plenty of food or none at all, provided only they are near the larger animals. A similar experiment, not tabulated here, in which males were used instead of neuters, indicated clearly that starving or feed- 244 HARLEY N. GOULD TABLE 12 Neuters transferred to neighborhood of large females; starved. fixed after fourteen days; 363 and 364 after eighteen days Specimen 357 to 362 SPECIMEN oo or ~J 358 359 360 361 364 : iets oe ey PENIS GONAD - 9 | Close behind fe- | Long Adult testis; male very active 8 | Mounted on fe- | Long Very active male’s shell young testis to almost adult sperm 7+ | Mounted on fe- | Long Young adult male’s shell testis, very active 7 | Close behind fe- | Long Young testis male to spermatids; small; very active 7 | Close behind fe- | Long Young testis male to spermatids; small; active 7 | 10 mm. distant | Stub Inactive from female 11 | 10 mm. distant | None Inactive from female 9 | Mounted on fe- | Long Adult testis; male’s shell very active SEMINAL VESICLE Small, full of sperm; apy- renes in distal end Small; very few sperm, not quite adult Small; very few sperm Small; very few sperm Small; empty Undifferentiated Very small; slightly convo- luted Very large; full of sperm ing would not cause degeneration of male organs if large females were near them in the hermit shell. Stimulating secretion? Several experiments have been made to determine whether the females or any of the large Crepidulas are constantly giving off a substance into the sea water, which may be taken up by the smaller individuals and passing to the sex glands, affect their development. We may already discount the possibility of an ovarian secretion; for it has been seen that the stimulus to male Neuters transferred to neighborhood of females; fed. INFLUENCE OF ENVIRONMENT ON SEX TABLE 13 245 Specimen 865 to 370 fixed after fourteen days; 371 and 372 after eighteen days a 2 : Pie PENIS GONAD SEMINAL VESICLE ele 365 10 | Mounted on fe- | Long Testis to sper- | Small; few sperm male’s shell matids; very in distal part, active majority apy- rene 366 10 |3 mm. to one | Half Young testis | Small; empty side of female usual to spermatids; length| very active 367 9 | Mounted on fe- | Short Young testis to | Small; just be- male’s shell spermatocytes ginning to of the 2nd or- twist der; very ac- tive 368 9 | 10 mm. behind } Stump | Very small;sper-| Nearly straight female matogonia tube; small 369 7 | Mounted on fe- | Short Young testis | Very small; male’s shell to spermatids; empty very active 370 7 | Mounted on fe- | Short Young testis | Small; few sperm male’s shell to spermatids; in distal part, very active majority apy- rene 371 Le mm). distant | Short Adult testis; | Very small; few from female small; not very sperm active 372 7 | Mounted on fe- | Stump | Spermatogonial | Small, thick male’s shell multiplication wall, occasional sperm development may come from an animal which is not yet an adult female, but has its gonad in a very rudimentary or inactive condition. So far all experiments to discover the presence of a stimu- lating secretion have given only negative results. They will be described here. The writer proposes to institute more critical tests along the same line. 246 HARLEY N. GOULD Experiment 9. A number of females were taken from colonies and placed in a small fingerbowl of sea water. Three males freshly re- moved from colonies were placed in another fingerbowl and the water in which the females were was poured into the fingerbowl containing the males. Every day the water standing on the males was poured out and replaced by water in which the females had been; the latter were each time covered with fresh sea water. After sixteen days the specimens which had been males were fixed and sectioned; all showed very advanced degeneration of the male organs. Experiment 10. This experiment was repeated in a slightly different way as follows: A sufficient number of large females were taken from colonies to cover the entire inner surface of an 8-inch evaporating dish. A very small stream of water was allowed to run slowly into this dish from a salt water tap. Small glass tubes then led the water from the evaporating dish full of females to two fingerbowls, one of which con- tained small neuter animals and the other males. Care was taken to keep the small individuals in each fingerbowl separated from each other by some distance. This was left running for a month, and at frequent intervals the small animals in each fingerbowl were examined. At no time did any of the neuter animals begin to develop a penis. The penes of the males gradually atrophied and disappeared, and did not reappear. The numbers were considerable (20-25 specimens in each fingerbowl), and the result was so uniform that it was not con- sidered worth while to fix and section the specimens. Now if a secre- tion were being given off in the water, one might suppose that this experiment would prove its existence; for each of the small individuals would receive secretion from many more females than is the case in the usual colonies. There were 20-25 large females in the evaporating dish, whereas few hermit shells contain more than two or three. The dishes were shallow and whatever substance was present would not be very much diluted, although it might not be in quite so concen- trated form as it would be close to a female Crepidula in the recesses of a hermit’s crab’s shell. In so far as it went, the experiment failed to establish the fact of a stimulating secretion. It might be yet con- ceived that a secretion is given off by the females which is effective only at the moment of liberation, or in the nascent condition. This remains to be investigated. Experiment 11. The bodies of females were ground in a mortar and the extract was added to a fingerbowl of sea water containing neuters. Change of sea water in the fingerbowl was made twice a day, extract of freshly killed female being added at each change. No sign of male development appeared in the neuters. The experiment of course lacks conclusiveness in one respect; the extract was made from the whole body of the large animal, because it is not known what part of the body may give rise to the hypothetical secretion; and it has been shown that for many secretions formed by animals there are also formed antagonistic or inhibitory substances (e.g., anti-fertilizin of F. R. Lillie). INFLUENCE OF ENVIRONMENT ON SEX 247 In view of the negative results of the above mentioned experi- ments, the writer does not wish to do more than suggest the possibility of a chemical stimulus, until the poimt has been further investigated. GENERAL CONSIDERATIONS It must be understood that the peculiar condition which has been found in Crepidula plana, the dependence of the male phase on conditions of the environment, has been developed within the species. So far ds the writer is aware no similar phenomenon has been described in any other Mollusc. Small specimens of the nearly related Crepidula convexa and Crepi- dula fornicata have been kept in aquaria by the writer, in the absence of larger specimens of the same species, under the same conditions as the segregated individuals of C. plana; yet the treatment did not interefere with the development and main- tenance of the male phase. Many specimens were fixed and sectioned which had been found living in nature where no larger ones were in the vicinity; those of the ‘male sizes’ had fully developed, active testes. Apparently, then, the peculiarity of C. plana is a special and not a generic one. It is true, how- ever, that there is great variation in the activity of spermato- genesis In C. convexa and C. fornicata. Occasionally specimens are found (not necessarily segregated) where the gonad is very nearly inactive. It would be interesting to learn whether the European and South American species of Crepidula show any sexual behavior similar to that of C. plana. Outside the Molluscs there is one striking instance of sexual behavior somewhat similar to that which the writer has found. Baltzer (10) in investigating the development of the marine worm Bonellia viridis, has found that if the free-swimming larva attaches itself to a female of the same species, and develops there in a parasitic manner, it becomes a male; but that if it develops solitarily, it becomes a female. The case of Bonellia is clearer than that of Crepidula plana, in that there is an actual contact between the animal which gives the stimulus to male development and the animal which receives 248 HARLEY N. GOULD it; and in that a substance can be demonstrated passing from one to the other. There is not the least suggestion of ‘para- sitism’ in the case of Crepidula plana. An instance of external conditions modifying a male gonad toward the female condition, though not at all as in Crepidula plana, is seen in certain Crustacea. Potts (06) and G. Smith (10) have found in the cases of the hermit crab (Hupagurus) and the spider crab (Inachus) respectively, that the presence of parasites attached to the body causes a degeneration of the testis in the male, and the subsequent appearance of what the authors believe to be ova, in the gonad. Parasitic infection in the female does not result in a modification toward the male condition. Smith believes that the parasitic castration brings out a ‘latent hermaphroditism’ in the male but not in the female. The male would then be the heterogametic sex, the female homogametic. The secondary sex characters are also modified from the male toward the female condition in all degrees. On account of the loose correlation between the changes in the secondary sex char- acters and those in the gonad, Potts believes that the former are not directly consequent upon the latter, but that “both are attributable to some change in the general metabolism.” SUMMARY In the protandric hermaphrodite Crepidula plana the develop- ment of the male phase is dependent upon the presence of a larger individual, not necessarily a female, of the same species. It is evident that some stimulus passes from the larger to the smaller individual. The greater the difference in size between the animal giving the stimulus and the animal receiving it, the more certain and complete is the male development of the smaller. A small stimulus will imitiate male development, but a greater one is necessary to complete and maintain it. When a male becomes removed from the neighborhood of the larger animal, the male organs degenerate, a condition of sexual inactivity ensues, later replaced by female development. If a larval C. plana settles and grows during the first part of its life where no larger individuals are present, the male phase INFLUENCE OF ENVIRONMENT ON SEX 249°: probably never occurs; but if at any time up to the female stage the small individual comes within the sphere of influence of a larger one, it will immediately develop male organs, attaining the male condition in about two weeks. Whether or not the male phase is realized, the female phase is eventually developed. The degeneration of the male organs does not prevent a second or third male development if the small Crepidula comes within the sphere of influence of a larger one after the degeneration. Partial degeneration may be halted and male activity resumed. During the male phase the growth of the body is retarded; after degeneration of the testis and during the sexually inactive condition, or in neuter animals which have never developed the male condition, growth is rapid. The first steps of female development may be interrupted and replaced by male development, under experimental conditions. In this case the oocytes degenerate and the activity of the pri- mordial female cells is suspended; while the primordial male cells multiply and undergo maturation. After the oocytes are ad- vanced in the growth period, male development is no longer possible. The nature of the stimulus to male development in C. plana is at the present time not certainly known. The following state- ments may be made in regard to it: 1. The stimulus depends upon the presence of the actual body of a large Crepidula plana; for if the large specimens be re- moved, leaving all the other conditions of the colony unchanged (even the shells of the large specimens still in their former positions) the stimulus is no longer given. 2. The movement of the smaller individuals, from whatever cause, does not furnish the stimulus; for some males are developed while in a fixed position. 3. Male development does not depend upon the amount of food received; for starved neuter specimens develop a testis as quickly as well-fed ones, when in the presence of large females; and they do not develop any more quickly. 4. No experiment has so far demonstrated the existence of a stimulating secretion; this possibility has not been thoroughly tested. 250 HARLEY N. GOULD 5. The stimulus does not depend upon the presence of the her- mit crab with which the Crepidulas are associated; it will have its effect even if the home of the colony is a fingerbowl. Whatever the manner of working of this peculiar adaptation, there is no doubt of its great advantage to the species; for it provides that the male members of every colony of Crepidula plana shall quickly develop into females as soon as there is no longer a larger female which requires fertilization; and also that there shall be adult males in the colony ready to function as soon as any individual has reached the adult female phase. BIBLIOGRAPHY Batrzer, F. 1914 Die Bestimmung des Geschlechts nebst einer Analyse des Geschlechtsdimorphismus bei Bonellia. Mitt. aus der Zool. Sta. zu Neapel, Bd. 22, No. 1, pp. 1-44. Conxuin, E. G. 1898 Environmental and sexual dimorphism in Crepidula. Proc. Acad. Nat. Sci., Philadelphia, 1898, pp. 435-444. Gouup, H. N. 1917 Studies on sex in the hermaphrodite Molluse Crepidula plana. I. History of the sexual cycle. Jour. Exp. Zodél., vol. 23, no. l. Ports, F. A. 1906 The modification of the sexual characters of the hermit crab caused by the parasite Peltogaster. Quart. Mic. Jour., N. &., vol. 50, p. 599. Smitu, G. 1910 Studies in the experimental analysis of sex. Quart. Jour. Mie. Sci., N. S., vol. 54, pp. 577-604. REACTIONS OF THE WHIP-TAIL SCORPION TO LIGHT BRADLEY M. PATTEN From the Laboratory of Histology and Embryology, School of Medicine, Western Reserve University FOUR FIGURES CONTENTS Statementuotthesproblen: \ 2 :..c eer Ser Poe ci ele -2) «lonerapenenotolie. cists 251 Generalucharacteristics: and behavionsaa-cen ceeds sete emits oi oi. le tether tetera 252 ENTS AERO AILISIA. toate av ne RR Ae PRE AERE SoSe ORE 0000 ORO eg EI 255 - MerSsunementsnoLmneactiOns GO) Lio bitnee serene intemal teers a) slekarsie er iclaverale 256 PTS CUSSION EP RC ict side cu ee Oe ten ea ate te te te es eyo) Sie ever ote leteiatieses = 267 SRUGUTU ODT AY Se ctor dtc, Gtk a ae A EERIE 0 acct Se nicl eee eure Gc) C88 ap aR IIR 274 TUAH ESR LUE OHUG |A 5 Os Os ee ps OR So RL RU ORS AS bic 0n 0 IPAM RRO NEE CLONES 275 STATEMENT OF THE PROBLEM During the past summer I was fortunate in having an oppor- tunity to work with a number of specimens of the whip-tail scorpion (Mastigoproctus giganteus, Lucas). The fact that this -Thelyphonid has median eyes which are well separated anatom- ically from the lateral eyes, made it appear a promising subject for experiments to determine the relative effectiveness of the two types of eyes. Since there have been no extensive observations published concerning the effects of light on any of the Thelyphonids, the present paper has been devoted to presenting data on the reac- tions of normal animals. No attempt was made to treat ex- haustively all phases of their behavior under the influence of light. The object was rather to obtain such reaction measure- ments as would best serve as a basis of comparison for subse- quent work directed toward determining the relative effective- ness of the various parts of their photoreceptive mechanism. 251 252, BRADLEY M. PATTEN GENERAL CHARACTERISTICS AND BEHAVIOR Fabre has said of the Languedocian Scorpion, that he is an ‘“ancommunicative insect, occult in his manners and unpleasant to deal with, so much so that his history, apart from the find- ings of anatomy, is reduced to little or nothing.” Of the whip- tail scorpions this is even more true. The general structure of the group is well covered in an extensive paper on the Pedi- palpi by Borner (04). There is, however, little detailed infor- mation concerning the structure of the eyes. Parker’s paper (87) on the eyes of scorpions deals with a species so far removed from Mastigoproctus giganteus, that it would be unsafe to as- sume the eye structure was similar in the two cases; the ana- _tomical position of the eyes is certainly quite different. As far as I was able to ascertain, there are in the literature only a few casual and fragmentary references to the behavior of this or closely allied forms. The limited distribution and our conse- quent unfamiliarity with the Thelyphonids, together with the very meagre information available as to their habits, makes desirable a brief description of certain points in their anatomy and general behavior. The sketch reproduced in figure 1 shows sufficiently the gen- eral appearance of Mastigoproctus giganteus. Of the four pairs of legs, only the posterior three pairs are used in walking. The - anterior legs are modified and serve as antenna-like feelers. They are long, slender, and flexible... Whenever the animal is moving or is about to move, the anterior legs are constantly waving about feeling out the path ahead. The feelers, as I shall call the anterior pair of legs, were found to be sensitive to touch, to heat, to chemicals, and to moisture; there was no indi- cation that they were photosensitive. The delicacy of their responsiveness may be well demonstrated by breathing on them when they are at rest. Even this slight stimulus will send the feelers into restless activity. The scorpion is, moreover, able to follow up or to avoid stimuli received by the ‘feelers.’ I have seen an animal which was aimlessly wandering about the table chance to get the tip of one of its feelers into a watch REACTIONS OF WHIP-TAIL SCORPION TO LIGHT Doe glass of water; it immediately swung about, thrust its other feeler into the water, then climbed half into the dish and began greedily to scoop water into its mouth with its chelicerae. Sub- stituting a dish with very dilute hydrochloric acid for the water, a clear-cut avoidance reaction was obtained the instant the feelers came in contact with the acid. —--- -- - ---- Centimeters. Fig. 1 Mastigoproctus giganteus Lucas: 1, chelicerae; 2, pedipalps; 3, modi- fied anterior legs which serve as feelers; 4, 5, 6, walking legs; me, median eye; le, lateral eyes. The big pedipalps are surprisingly powerful and can inflict a considerable flesh wound. 280. 282 THE SPLEEN DURING HIBERNATION 1 PLATE 1 FRANK C. MANN AND DELLA PRIPS PLATE 2 EXPLANATION OF FIGURES 3 Photomicrograph of spleen of Spermophile 117, a male captured in the spring of 1915. It had been torpid for thirty-five days. Had access to food. Killed January 10, 1916, by bleeding. At this time it weighed 165 grams and the rectal temperature was 14°C. 4 Photomicrograph of spleen of Spermophile 266, a female captured in June, 1914. It hibernated when kept in the cold during the winter of 1914-1915. Placed in hibernating room and food withdrawn the latter part of September, 1915. Killed January 27, 1916, after hibernating about one hundred and twenty-five days. Rectal temperature at time of death 17°C. Daily observations were not made. The spleen is practically normal. 284: THE SPLEEN DURING HIBERNATION PLATE 2 FRANK C. MANN AND DELLA DRIPS STUDIES ON A RACE OF PARAMOECIUM POSSESSING EXTRA CONTRACTILE VACUOLES I. AN ACCOUNT OF THE MORPHOLOGY, PHYSIOLOGY, GENETICS AND CYTOLOGY OF THIS NEW RACE ROBERT T. HANCE Zoblogical Laboratory, University of Pennsylvania THREE PLATES AND TWELVE CHARTS CONTENTS MearlnGrodieuuoniee eas: 5 dint Skee pk Woes 5 «sa: ty SS oe 5 oD Aer hele 288 eee cine tere tec 5 ons? ASM eet .) y 2oe ana ee 288 ALGTBT FAVA TIN LNO MO ee rey res sess spss eS ae oho Sas «sR ne ey « ae 291 aw General Gescription cee. 2 oss. 2 SE oa) he ceennees 291 baelhenvactioles: 5.0). o. s4seeiici ss 2s. 0-3. elo ome ho oe 292 IV. Observations on the behavior of the vacuoles........................ 294 a. Percentages—range of vacuole number........................-- 294 b. Change of number during the life time of the individual........ 297 e; ‘Numberiof vacuoles imolispring)....:. 5328 cee) oo case 298 d. Effect of various conditions on vacuole number................. 299 Vee te ENy SIO LOD 82 cease soe Dace tea 5. s.1st ee be SE eee SOS eG CHEE. ya AERA TIE Gta cooks « CMe Ae Re | Ai ta -1 n e 314 a. Inheritance of vacuoles for four generations.................... 314 beiiticerrotuselection. 10.0. «++... 58 see ee rin ee a ee 316 c. The immediate and temporary effect of location of the vacuoles in the parent form on inheritance by offspring................ 320 dey inereiect Omconjugation...... .. see Foe. eee eee ee oo. 320 eavauiempts,toveross the two racesvapeseeee.). toes ackloe. Sloe kke 323 ETT VG LO myers ctr Cs coos, ««: .ck e Se eie e o. vu.4y BOB VAG IE DOT SGI Sma G Oe eee cis oot \. an ae 324 2 Gra Ot oI Oya 0) Tk eects A, ere OO 2 os Se CO 327 287 Le) (oe) 6) ROBERT T. HANCE I. INTRODUCTION While examining a number of paramoecia taken from a lab- oratory culture in the early part of January, 1915, one individual was seen to have three instead of the usual two contractile vacuoles. Subsequent examination of the stock from the origi- nal culture showed that a large percentage of the animals pos- sessed the extra organs. Several animals with three vacuoles were therefore isolated and pure lines started from them. It soon became evident that the potentiality for one supernumerary vacuole was not only inherited but that the offspring might possess even more than one extra contractile organ. A prelimi- nary account of the behavior of these organs in heredity was published some months ago (2). The discovery of such a marked variety of a form which has served as a basis for so much valuable study opens an interest- ing field for investigation. Parts of this report must necessarily be of the most preliminary sort and various portions will be completed from time to time and published separately. The finding of the new race of paramoecium affords opportunity for comparison with the common race and for a study of the physio- logical activities of the two varieties. The following points will be considered more or less completely in the following paper: 1. The extra vacuole gives a definite character in the Protozoa whose inheritance can be traced. The existence of two races differing as far as is known only in this one character gives an opportunity of crossing the two races by conjugation and of determining whether inheritance in protozoa is Mendelian as it is in the metazoans. 2. A comparison of the two races may be helpful in the analysis of the function of the contractile vacuole. 3. Some light may be thrown on the process of conjugation through a study of the behavior of both races. | II. TECHNIQUE A separate pipette was used for each culture to guard against cross contamination and was hung in a special holder attached PARAMOECIUM POSSESSING EXTRA VACUOLES 289 to the jar when not in use (3). In following the offspring of a single individual for several generations each of the daughter cells was isolated in a watch glass and as fast as the individuals divided a record was made of the number of vacuoles in each and they were again separated. The syracuse watch glasses that were used were sterilized in boiling water to which a little clean paraffin had been added. ‘This deposited an invisible coat- ing on the glass which was, however, sufficient to prevent the drop of hay infusion from spreading over the surface as 1t would on perfectly clean glass. On the paraffined surface the fluid rounds up and presents the minimum surface for evaporation (3). It was found that the most satisfactory pipette for this work had a short tip of almost hairlike fineness. The method of making these has been described (3). It is rather difficult to find a method of slowing down the animals for the purpose of making accurate observations with- out killing them. ‘The first method used was to place an indi- vidual in a small drop of water and over this a cover glass was lowered with the aid of a fine pair of forceps. The cover had a drop of hardened balsam at two corners which had been filed down to the proper thickness and which prevented it from crushing the animal. The excess fluid was drawn off with a piece of filter paper. To recover the animal the slide was tilted over a watch glass and by means of a stream of hay infusion the cover and whatever was under it were flushed into the crystal. This method was very laborious and slow, and the chances of losing the paramoecium great. The following method has proved to be the better: An individual is placed on a slide in a fairly large drop of the medium. The liquid is drawn off with a pipette under the dissecting microscope, the animal being kept as nearly as possible in the center of the drop. Finally, enough of the fluid is removed so that the adhesion of the sur- face film to the slide exerts sufficient pull to hold the animal quiet. In the center of the drop the pressure is least and it sel- dom causes a paramoecium to burst. Toward the edges, however, the chances are very great that the animal will succumb almost instantly to the greater pressure. In such. a preparation the 290 ROBERT T. HANCE pressure at the center tends automatically to lessen as the sur- face tension slowly draws the liquid from its close adhesion to the slide and rounds it up. This takes a sufficient length of time, however, to allow accurate observations to be made under the compound microscope. Occasionally the pressure is relieved too quickly and the paramoecium is given enough fluid to move about in, which makes observation with the high power impos- sible and the processs has to be repeated. As soon as the ex- amination has been made, a drop of fresh hay infusion is added. To drop this liquid immediately on top of the animal after it has been under pressure may frequently crush it and the safer way is to allow the fresh fluid to flow from the side into the drop containing the animal. The paramoecium may then be picked up in a pipette and placed in its respective watch glass. When examining paramoecia in pure lines a separate pipette is used for each line. Under the 16 mm. lens when the animal is compressed, the vacuoles stand out with diagrammatic clearness appearing as so many holes punched through the cell and as a rule are to be found close to one side and almost invariably lying in a straight line. This latter characteristic was one of the criteria for differentiat- ing between the contractile vacuoles and food vacuoles when these were so numerous as partly to obscure the former. The comparative lack of refraction of the pulsating organs was an- other basis of distinction. When, however, the refraction of the food vacuoles was almost identical with that of the contractile vacuoles under the low power, the 4 mm. lens was swung into place and the contraction of the supposed contractile vacuoles was watched for. It has been observed that the vacuoles in animals raised in watch glasses are more difficult to see than they are in animals living in larger amounts of medium. In the watch glasses the food vacuoles have a lack of refraction which gives them an appearance very much like the contractile vacuoles whereas the food vacuoles in animals raised in the large culture jars frequently appear very nearly black. Cytological technique. For measuring and drawing the whole paramoecium Worcester’s fluid was used (10 per cént formalin -PARAMOECIUM POSSESSING EXTRA VACUOLES 291 saturated with mercuric chloride). Animals fixed in this way can be immediately cleared in glycerine which brings out many of the internal parts very well, and the vacuoles, if they happen to be expanded at the time of fixation, are visible. For whole mounts and sectioning, paramoecia were fixed in Schaudinn’s, Gilson’s, Flemming’s and Worcester’s solutions. As com- paratively little time has been devoted to cytological studies I am not prepared at present to offer any criticism of the fixatives. To Dr. M. H. Jacobs I am indebted for his constant interest, advice and criticism during the progress of the work recorded here and I also feel under great obligation to the other mem- bers of the Zoological Department of the University of Penn- sylvania whose many kind suggestions have been of great assistance to me. Ill. MORPHOLOGY a. General description In general form, the animals of this race are apparently identical with the common slipper-shaped Paramoecium cauda- tum. The newly discovered individuals average rather larger than any of the representatives of the two-vacuoled race I have found about Philadelphia. Chart 1 shows a curve plotted to illustrate the range in size. The peak of the curve is at 233 pu and is very nearly midway between the two extremes. On this same chart are plotted the ends of two curves formed by animals taken about Philadelphia. The range of one race is from 153 u to 207 » and in the other from 106 u to 173 uw. In the collection of slides belonging to the Department I have found paramoecia somewhat larger than the new race ranging from 197 yu to 325 u. The extremes of all the races studied by Jennings were 50 » and SO2 [- In certain cultures where the individuals were unusually fav- -orable for study a band slightly darker than the surrounding protoplasm could be seen across the center of the animal in the region where the constriction appears at the time of division (figs. 4 and 6). It is interesting to note that in these cultures 292 ROBERT T. HANCE Chart 1 On the axis of abscissas are marked the various sizes in » while on the axis of ordinates are placed the percentages. The heavy solid line represents the — multivacuoled race of paramoecium while the light broken line indicates the larger sizes of a small two-vacuoled race and the light unbroken line is based on figures obtained from fixed preparations of two-vacuoled animals in the Laboratory collection. the percentage of animals with three and four vacuoles was very high. The consideration of this phenomenon will be left for future work. b. The vacuoles 1. Location. So exactly are the contractile vacuoles located that a single straight line parallel to the outline of the animal might be drawn passing through the center of each. In a very few cases I have found one or two vacuoles slightly displaced from the usual straight row condition (fig. 9). In the majority of individuals the extra vacuoles are located in the posterior part of the animals as can be seen from the drawings. In a few PARAMOECIUM POSSESSING EXTRA VACUOLES 293 instances, however, one extra vacuole was found in the anterior end (fig. 3), but I have never found more than two vacuoles located in the foremost half of the body. It is a curious fact that, rare as this condition is in the normal medium of hay infusion, in cultures to which a little sea salt has been added paramoecia with two vacuoles in the anterior end have been found to be much more numerous. As yet no plausible explanation for the increase of this rare arrangement has been found—if, indeed, it is not merely a coincidence.. There can be no doubt that the contractile vacuoles are very definitely located organs and fixed in position for a given indi- vidual. When an animal is swimming the vacuoles can be seen to turn with the cell and always maintain the same position in relation to each other. As far as I have been able to determine, both the radiating canals and what appears to be the excurrent pore or tube aid in holding the vacuoles in position and particu- larly the latter. That there is a connection with the outer wall of the animal can be seen when a paramoecium bursts directly opposite to a fully expanded vacuole, when the vacuole, drawn by the outflowing protoplasm, can be seen straining at some retaining fastening. 2. The vacuoles. Under normal conditions the vacuoles in this race measure on the average about 10 » in diameter with a capacity of 500 cubic micra. There may be some variation in this size due to the age of the vacuole, i.e., the length of time since it had appeared, but I think that it is safe to say that the diameter of fully formed vacuoles is very nearly the same. There can be no question that these vacuoles originate separately as they generally lie at some distance from each other and no cases have been seen when a vacuole appeared as though it had been separated by division from another vacuole. The average size of the vacuoles in the common race which I have observed is 11 » with a cubic content of 664 cubic micra. In my fixed preparation the vacuoles are surrounded by what appears to be a definite morphological membrane (fig. 16). When animals have been kept at a temperature a few degrees above freezing for a few hours the vacuoles expand and do not contract and it is THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 2 294 ROBERT T. HANCE interesting to note that the anterior vacuole becomes from three to four times the diameter of those in the posterior end. This, in the cases studied, was not true for the common two-vacuoled race, neither vacuole, exceeding the other in size. IV. OBSERVATIONS ON THE BEHAVIOR OF THE VACUOLES To sum up briefly our knowledge of contractile vacuoles: The contractile vacuole is a pulsating organ surrounded by a membrane of physiological importance, belonging to the ecto- plasm. These vacuoles form an osmotic system and the evi- dence at hand shows them to function in part as excretory and respiratory organs. ‘a. Percentage of variation in contractile vacuole number After the pure lines that had been started had multiplied con- siderably examination was made of a number of individuals in each line. Apparently these high numbers of vacuoles were not in every case passed on to the offspring for in all of the cultures, whether started with three or four vacuoled paramoecia, there were considerable numbers of two vacuoled forms. But on the contrary in the pure lines started with three vacuoled forms there were found individuals possessing four contractile organs. Ob- servations made on one culture in the early days of the work showed a range of variation in vacuole number in the following percentages. per cent Mworvacuoles. ) 2 20.5 em Single 20 em 21 algal 1) of 27.5) em Single ¢ 26.5 cm 22) hol 1] o 24.5 em Singlet Gi? 2B tcl 1} & 18 cm. Single Sf 17.5 em 24 | 22 21 cm. each Single; nar- row con- nection Pha) || 11.2 cm. each | ? Injured 26 1 Lot 125 5%em Single ¢ 12.25 cm MATERNAL OVARIES Ovaries absent Ovaries absent Both present. Corpus luteum in each Ovaries absent One missing, other had 1 corpus luteum Ovaries absent Ovaries absent One ovary absent. Other con- tains corpus luteum Ovaries absent Both ovaries present. Corpus luteum in each One ovary absent. Other has 1 corpus luteum Both ovaries present. Corpus luteum in each One ovary absent. Corpus lu- teum in other One ovary absent. Other has 1 corpus luteum ACTION OF SEX HORMONES IN FOETAL LIFE 387 SEX NUMBER SIZE CHORION MATERNAL OVARIES (8/2) Die |e 42.5 em. each | Not examined | One ovary absent. Other has 1 corpus luteum DS M74 17 cm. each Single One ovary absent. Other has 1 corpus luteum Oa 2 15 cm. each Single Both ovaries present. Corpus luteum in each 30) 2 23 cm Single One ovary absent. No corpus 22 em luteum in other 31 2 12 cm. each Single Both ovaries present. Corpus luteum in each oy) |i a o 18 cm. Single Both ovaries present. Corpus S 16.75 em. luteum in each 33 «| 2 10.5 cm. each | Single Both ovaries present. Corpus luteum in each o4 2 25 em. (about)| Single ‘Both ovaries present. Corpus luteum in each So 12 13 cm. each Single Both ovaries present. Corpus luteum in each 06 | 1 o 18 cm. Single Both ovaries present. Corpus So 17.5 cm. luteum in each Sia mee o 16 cm Single Both ovaries present. Corpus C152 5)em luteum in each 38 | J o 23.5 cm Single Both ovaries present. Corpus G 22.5 em luteum in each oH) |) 2 18 cm. each Single Both ovaries present. Corpus luteum in each AON | Lia o 10.4 em Two separate | Both ovaries present. Corpus ? 10.2 cm chorions luteum in each Shel o 22.7 em. Single Both ovaries present. Corpus ¢ 21.8 cm luteum in each 388 | FRANK R. LILLIE SEX 5/Q21¢ NUMBER 42—Isele | Nljote 5 43 2 44—|se]e 45 2 46 2 47 1 48 2 49 PL & 50 1 51 2 52 2 53 2 54 2 55 , 56 Dy 57 1 Niote 5 SIZE CHORION MATERNAL OVARIES See 10.75 em. Single Not observed 34 em. Single Both ovaries present. Corpus 31 (25) cm: luteum in each 23 cm. (about)| Almost sepa- | Both ovaries present. Corpus rate luteum in each 6 22.75 Single Both ovaries present. Corpus @ 22.25 em. luteum in each 20 cm. (about)| Single One ovary absent. No corpus 1.5 em. each o' 21 em. ¢ 21 cm. 5 em. each 18 em. 19.5 em. 19 cm. 20.5 em. 21 cm. 13.75 cm. 12.5 em. o 19.25 cm. ¢ 18 cm. Separate, but overlapping Single Single Single Single Single Single Two chorions separate Single luteum in other Both ovaries present. Corpus luteum in each Both ovaries present. Corpus luteum in each Both ovaries missing One ovary absent. Single’ cor- pus luteum in other Both ovaries absent Both ovaries present. Corpus luteum in each Both ovaries absent Both ovaries absent Both ovaries absent ACTION OF SEX HORMONES IN FOETAL LIFE 389 1 Case 7 was received in my absence, and the entire uterus was placed in formalin; preservation of its contents was bad, and condition of chorion must be recorded as doubtful. 2 Case 13 uterus injured by butcher; chorion cut in two. 3 Case 16 uterus injured by butcher; chorion cut in two. 4 Case 22 uterus injured by butcher; chorion cut in two. > Cases 42 and 44 are not included because they were selected heterosexual pairs taken after birth. Ill. THE TIME OF-FUSION OF THE TWIN CHORIONS AND THE DE- VELOPMENT OF THE VASCULAR ANASTOMOSES BETWEEN THE TWINS In order to form an estimate of the probable time of fusion of the twin chorions it is necessary to present a few data concern- ing the development of the usual single chorion. F igure | shows the non-pregnant uterus of the cow partly dissected. It will be noted that the horns of the uterus open by constricted apertures into the small body. The blastodermie vesicle forms in the horn of the uterus on the same side as the ovary from which the ovum was derived, as I have observed in numerous cases. It grows out into a long strand-like sac extending both distally and centrally. The embryonic area forms near the.center in the sheep (Bonnet) and presumably also in the cow. The growth of the strand-like vesicle in length is extraordinarily rapid, and it soon enters the body of the uterus centrally, and penetrates into the opposite horn. By the time that the embryo is 10 mm. long the vesicle has extended completely through the body of the uterus and far into the other horn (two cases observed); the embryo is thus excentrically placed in the very long vesicle. The allantois forms later than the blastodermic vesicle; it grows from the embryo both centrally and distally, and ultimately completely fills the blastodermic vesicle and occludes its cavity. In the case of an embryo of 19 mm. length the allantois had passed from the horn of the uterus containing the embryo well into the body of the uterus. In another case of an embryo of 21 mm. length the allantois had extended through the entire horn of the uterus opposite to that containing the embryo. I have one case of a twin pregnancy in the cow in which the embryos were only 15 mm. long (no. 49). Unfortunately the THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 2 390 FRANK R. LILLIE collector did not recognize the case as a twin pregnancy until after the uterus had been opened and one foetus removed, thus cutting the membranes; the other foetus was also removed in the same way, and when the uterus and specimens reached me for examination it was necessary to reconstruct the original condition from the parts. This was, however,. successfully done, as the central end of the one chorion was found still in place in the body of the uterus and extending into both horns. In this one the chorion with contained allantois had passed the body of the uterus. In the other the end of the chorion had been drawn out of the uterus with the foetus, but measurement showed that it also with the allantois contained had passed the body of the uterus. The two chorions were thus not fused at this stage, but they overlapped and were in closest apposition in the body of the uterus. ‘The conditions precedent to fusion were thus fully established at this early stage long before sexual differen- tiation begins. Comparison of these twins with the stages of 19 and 21 mm. described above indicates some variation in the degree of devel- opment of the chorion relative to the length of the embryo. But these stages demonstrate the possibility of fusion of twin chorions a considerable time before the stage of beginning sex- differentiation, which I estimate at about 25 mm. Vascular anastomosis between the twins is possible as soon as the allantoes from the two sides meet, or even earlier, because after the allan- tois has once fused to the chorion the blood-vessels tend to spread out more or less in the chorion beyond the area of fusion. Owing to the extreme difficulty of obtaining early stages of twin pregnancies in cows the next earliest stage that we have (no. 51) is a case of male twins 5 em. long in which there is no evidence of the place of fusion of the twin chorions, and there is a perfect vascular anastomosis between the two sides. Fusion is already perfect and any overlapping parts have entirely dis- appeared. The next case is a two-sexed pair (no. 19) in which the male foetus was 80 mm. and the female 75 mm. long. The twin chorion was single with a broad connection provided with cotyledons between the two halves; no evidence of the place of ACTION OF SEX HORMONES IN FOETAL LIFE 391 fusion of the twin chorions remained. The urinogenital system of the female, described in section 4, was already definitely of the sterile free-martin type. The inference is, therefore, that, fusion had taken place some time previously in order to account for the completeness of the fusion and the transformation of the reproductive system of the female. In the case of twin pregnancies in cattle, therefore, the two vesicles starting in opposite horns of the uterus will meet in the body of the uterus before the 10 mm. stage; the allantoes of the two vesicles will not however meet until about the 15 mm. stage, and the opportunity for vascular anastomosis therefore dates from this time. Bonnet (89) describes a very early twin pregnancy of the sheep, which confirms in the strongest way my conclusion con- cerning the early time of fusion of twin chorions in ungulates. His description is of so much interest that I quote it entire, he describes a pair of sheep twins 6 mm. long, secured 18 days and 6 hours after copulation, deren serdsen Hiillen an den sich berithrenden Enden auf eine Strecke von 6 cm. in elnander eingestiilpt und verklebt, aber noch nicht ver- wachsen waren. Sie liessen sich vielmehr noch leicht auseinander- losen. Beide Eier maassen zusammen vom freien Ende des einen bis zum freien Ende des anderen 35 em. und waren in maximo 1.5 em. weit. Die Kirze der Eier ist eine im Vergleiche zur Linge einzelner Kier in diesem Stadium auffallende; sie betrug bei einem 15, bei dem anderen 17 em. Wahrsecheinlich behindern sich die bald eimander mit den Spitzen beriihrenden Eier einigermaassen in der sonst normalen Langenentwicklung. It will be noted that in this case the ova met at their apices and invaginated one another, and that the stage of such union was only 6mm. ‘The sheep’s uterus is of precisely the same type as the cow; fusion follows the union of the ova in the sheep as in the cow; but vascular anastomosis does not occur in the sheep, as I describe in detail later on, and for this reason the female of two-sexed twins remains unaffected in the sheep. We have already referred frequently to the vascular anasto- moses between twin foetuses of the cow, and it is now time to describe the matter fully. The working hypothesis with which 392 FRANK R. LILLIE the investigation began was that the free-martin and its twin were monozygotic, and it was not until after 27 cases had been _ examined that I was convinced that they were dizygotic. The real explanation of the phenomenon then for the first time be- came evident. No vascular injections were therefore made during the first part of the investigation, and the evidence for vascular anastomosis among these rests upon incidental obser- vations, the significance of which was not realized at the time. Relatively few of the twin ova received thereafter were in a fit state for complete injections. Of the 28 cases involved injections were made only in seven cases; two of which will be described in detail below. But in 21 of the 28 cases vascular anastomosis could be satisfactorily demonstrated either in the uninjected chorions or in injections. Some of the uninjected cases were just as demonstrative as though they had been injected. In four cases of the remaining eight there was no anastomosis; one too young (no. 49); the second was a case of normal male twins in which the connection between the two chorions was merely a narrow band-like connection (no. 46); the third was a case of male twins with entirely separate chorions (no. 56); the fourth was another case of completely separate chorions (no. 40) of the greatest theoretical interest because one foetus was male and the other a normal female. Finally there were three cases with inadequate records. Eliminating these three we have 25 cases, in 21 of which, including the three possible twin combinations, vascular anastomosis could be demonstrated and 4 in which it was absent (nos. 40, 46, 49, and 56). This is not, however, the only evidence that more or less com- plete vascular anastomosis between the pairs is the rule in cattle twins.. I can distinctly remember the continuity of the thickened chorionic band that carries the main arteries as the rule in the first 27 cases, and this was recorded in certain cases In my notes. There cannot be the least doubt that in bovine twins fusion of the chorions usually occurs and is followed by anastomosis of the blood vessels of the two sides, and that intermixture of the blood of the two foetuses results. Nor can it be doubted that ACTION OF SEX HORMONES IN FOETAL LIFE 393 rarely this does not occur either because the chorions fail to fuse (cases 40 and 56) or because a slender connection is not vascu- larized (case 46). The significance of the exceptions is very great. The nature and extent of the vascular connections may now be illustrated by a detailed study of two cases. 1. Case no, 33 Males, 10.5 em. long (figs. 2 and 3). The entire arterial system of both chorions was injected from one umbilical artery of one partner; the mass easily passed the constriction between the two halves of the chorion, and penetrated even into the umbili- cal arteries of the other; every cotyledon was injected on both sides. The venous system was also injected from one of the umbilical veins of the same specimen; the injection mass also passed the constriction far into the chorion of the opposite side, but the blood present in the veins prevented as complete an injection of the veins as of the arteries. The two umbilical arteries of each foetus have a cross connec- tion at the distal end of the umbilical cord, so that an injection from one artery outwards flows both centrally and distally. The two veins lack such an anastomosis. The arterial anastomosis (fig. 3). The main artery from the right of the drawing divides in three branches 1, 2, and 3 as it approaches the center. Branch 3 need not be farther considered as it does not anastomose with the opposite side. Branch 1 can be followed directly through into communication with the arterial system of the other side, branch 2 has a strong anastomosis with the through trunk /-/ (at 1-2), but branches for the most part within its own venous territory. The side branches of the through trunk /-/ are of considerable interest, inasmuch as some are oriented in the direction of the blood flow from the right, and others from the left. Thus following the trunk from the right the first branches that we meet are directed against the blood stream coming from this direction (1a); immediately after pass- ing the anastomosis (/—2) we meet a branch /b, directed with the blood flow from the right; the next two branches, /c and /d, are directed similarly, but the large branch /e immediately beyond has the reverse orientation. If we suppose the blood 394 FRANK R.- LILLIE flow to come from the left /e:is directed with the current, 1d, ic, and 1b against it, etc. The orientation of these branches seems to indicate an alternation in direction of flow in the main trunk, as is to be expected with a beating heart at each end of it. It is significant that this is the only place in the arterial system of the membranes where reversal of orientation of branches is found in the course of a single trunk. The venous anastomoses. ‘The venous anastomoses are two in number (4 and 64 in figure 3). The larger one 4 was on the opposite side from which the drawing was made, and is there- fore represented as a broken line; 5 comes from the same main venous stem. There is no reversal of orientation of side branches. The circulation. It is obvious that any arterial blood that is pumped by either foetus into the capillary system situated be- yond about the line A—B will be taken up by the venous system to the other foetus. There must therefore be a constant inter- change of blood between the two foetuses, which, considering the size of the arterial intercommunications, must be very con- siderable. The venous anastomoses are not significant for the intermingling of the two circulations. The direction of flow along the main arterial trunk (/-/) will depend on the blood pressure on the two sides. If for any reason an excess of blood is received by one of the two foetuses, this will have a tendency to raise the blood pressure on that side and thus to equalize the distribution. There is of course the possibility that the beat may alternate on the two sides, but nothing is known of this, and the effect of such an arrangement would not be easily deduced. 2. Case no. 47. o& 22.75 em. and $22.25 em. (fig. 4). Ar- teries injected yellow; veins blue. The injection was made first into an umbilical artery and vein of the male. The arterial injection flowed regularly into the opposite chorion and through to the free-martin; the venous injection also flowed into the op- posite chorion, but not so freely. The injection was then com- pleted from an umbilical artery and vein of the free-martin in order to fill the vessels on this side more completely. 9) ACTION OF SEX HORMONES IN FOETAL LIFE 395 The arterial anastomosis is a single strong vessel, the relation- ships of which are shown clearly in the figure and require no further description. The stage is much more advanced than the preceding case, and the cotyledons are much more developed. Most of the arterial branches are distributed directly to the coty- ledons. The venous anastomosis is much less viable than the arterial; macroscopically it consists exclusively of a connection between the two veins of one cotyledon (2, fig. 4) one of which returns to the male side and the other to the side of the free- martin. This is the only cotyledon that appears to be connected with the umbilical veins of both sides; therefore any other venous anastomisis must be through the capillary circulation of the extra-cotyledonary chorion if it exists. The circulation in this case must be according to the same principles as in the preceding: whenever the arterial pressure is higher on one side than the other blood must be distributed from the side of higher pressure to that of the lower pressure; it will thus reach the veins and the foetus of the opposite side; varia- tions in pressure on the two sides must constantly occur, if there is any difference in the time of occurrence of systole and diastole of the twin hearts. The blood of the twins must therefore in- termingle intimately, and internal secretions of either must reach the other. These cases adequately illustrate the time and nature of the vascular anastomosis; we may therefore turn to the question of duration of the intermingling of the blood during foetal life. We have seen that the vascular anastomosis probably begins at the stage of about 19-20 mm. The two cases we have consid- ered in detail indicate a strengthening of the arterial anastomo- sis, and a weakening of the venous anastomosis after a certain stage as development proceeds. This is to be expected because the arterial flow is stronger and toward the center, whereas the venous flow is slower and away from the center. The circula- tion itself tends therefore to strengthen any primitive arterial connection, and to diminish relatively any venous connection. Moreover as development proceeds the cotyledons increase in size, and the intercotyledonary circulation in the chorion becomes 396 FRANK R. LILLIE correspondingly reduced in a relative sense with the result that the prominent arteries and veins become exclusively cotyledon- ary with the single exception of the artery connecting the two sides; and any intercotyledonary venous connections become insignificant. It is an important question whether this condition persists throughout foetal life, even though completely sterilizing effects on the female reproductive system are produced by the stage of 7.5 em., as we shall see in more detail in another section. The question therefore relates to possible influences on later stages of the female reproductive organs, and on the somatic characters of both twins. The latest stage that I have examined with reference to this question was a pair of female twins, 35.3 em. and 31.25 em. in length respectively. The arterial connection was even stronger than in earlier stages proportional to the more advanced stage of development. There is no reason to suppose that the connection is interrupted until birth, but the actual observations have not been made. Thus the available records indicate a growth of the arterial anastomosis throughout foetal life and a consequent duration of action of the male hormones up to the time of birth. ‘The possi- bility exists that in certain cases the connection may be inter- rupted at different stages of development; but so far no such cases have appeared. In any event the decisive effects on the reproductive system of the female are determined very carly and they are presumably irreversible in their character. Triplets occur rarely in cattle, and cases of even more young at a birth are on record. Unfortunately records of their breeding history appear to be very rare. The only one that I have been able to discover is given by Pearl (12). In this case there were two females and one male. The females were kept until they were about three years old, but they never came in heat. They were then killed, and ‘‘The man that dressed them said that they never would have bred. Neither uterus nor tubes were recog- nized, but the vagina apparently ended at its anterior end as a blind sac.’’? Both were apparently sterile free-martins. The male was put in service and got good calves. We have here, ACTION OF SEX HORMONES IN FOETAL LIFE 397 therefore, in all probability a case in which the circulations of the three individuals anastomosed, and in which the male sterilized both females. IV. THE HORMONE THEORY OF THE FREE-MARTIN We may now proceed to a consideration of the argument for the hormone theory. In our previous considerations we have dwelt upon the separate zygotic origin of the free-martin, and the foetal vascular connections; it is obvious that these conditions suggest a hormone theory; but, before such a theory could be regarded as demonstrated an explanation of the existence of fertile free- martins would need to be offered, and the hmitation of the phe- nomenon of sterility of the free-martin to cattle as a common oecurrence would have to be explained; the possibility of the existence of sex hormones at such an early period of the foetal life would also need to be demonstrated, and reason for limitation of the sterilizing effect to the female is needed. We shall consider first the fertile free-martin; three cases of a normal female twin to a male have been found in my 24 cases of bovine two-sexed foetal twins. These are readily explained a priort on the hormone hypothesis on the supposition that they represent cases in which anastomosis of the foetal blood-vessels did not occur. It is important to notice that such cases are ex- ceedingly crucial, for if we should find a case of two-sexed bovine twins in which foetal vascular anastomosis was absent, and in which the female was nevertheless a sterile free-martin, the hormone theory would have to be abandoned. The first two cases of fertile free-martins were nos. 8 and 9 of my series (figs. 7 and 8); they were collected before the hormone theory was formed and the records are incomplete. In my note- book I had merely recorded that the connection between the two chorions of each pair was narrow; it was probably not vascular, but this cannot be certainly known, and these cases must be left out of consideration. Fortunately the third case, no 40, is a veritable experimentum crucis. In this case organic connection of the two chorions was entirely lacking. The central ends of the two chorions merely overlapped in the body of the uterus, and fell 398 FRANK R. LILLIE apart when removed; injection of the chorion of the male showed its circulation to be entirely closed. Dissection of the female showed its reproductive system to be perfectly normal (fig. 6); sections of the gonad showed it to be an ovary (Chapin, 717); each maternal ovary had a corpus luteum in it. Even though this case stands alone, it is obvious that it fulfills all the con- ditions of a radical experiment; so that we can say that foetal vascular anastomosis of two-sexed twins involves the sterile condition of the female, and absence of such anastomosis its fertile condition. The sheep and other normally uniparous ruminants should furnish another test of the theory; for though twin births are fairly common in sheep the female of two-sexed pairs is usually normal. This is a matter of common experience among breeders, and is strikingly demonstrated by Prof. Alexander Graham Bell’s well-known experiments (Bell 712) on the production of a multi- nippled race of sheep; 36 per cent of the lambs born on Professor Bell’s farm were twins; and in 1912, 60 per cent of the lambs born from three year old ewes were twins; the records show that the twin ewes are used commonly for breeding purpose, which would not be the case if any considerable percentage were sterile. The fact that there is no reference in this very careful series of experiments to sterility of ewes from two-sexed twins would also show that such a phenomenon must be at least very uncommon. On the other hand Bateson states that it sometimes occurs among sheep; though, on what authority, I do not know. In response to a letter of inquiry Wm. John G. Davidson who has had charge of the breeding operations at Dr. Bell’s estate for a great many years writes: I may say that in all my experience in sheep breeding I have yet to find a case where lambs born twin to males have turned out. sterile. In fact when lambs are born twin male and female if they have the desired qualifications required in the flock both lambs would be retained in the flock and I have not had the slightest trouble with either male or female being unfruitful. I know there is nothing in the free-martin theory in sheep breeding. It was therefore very interesting to examine twin pregnancies of sheep with reference to the relations of their membranes. I ACTION OF SEX HORMONES IN FOETAL LIFE 399 found in the four cases, that I examined, that the twins were dizygotic (in one case both corpora lutea were in one ovary) and that the membranes were fused in the body of the uterus as in cattle. But when injections were made, as was done in all four cases, it was found that the circulation of each individual was entirely closed; the injection mass could not be forced from one side to the other, either through the arteries, or through the veins. Figure 5 gives a faithful representation of one case; it will be observed that the arteries and veins of each side end in a central neutral zone that they do not cross; this zone is no doubt occupied by capillaries, and it is possible that these anastomose from the two sides, though it is uncertain. The other cases were similar, though in one of them a single centrally placed cotyledon received an artery from each side; each artery was accompanied by its own strong vein returning to the same side, which indi- _cated that there was little, if any, intermixture of blood in the cotyledon; the starch injection masses, yellow on one side and red on the other were not forced through. In the sheep we have, then, all the necessary conditions for the production of sterile free-martins except the actual vascular anastomosis. If the vascular anastomosis should also occur exceptionally, such a condition should be accompanied by ster- ility of the female in the case of heterosexual pairs. This lends probability to the assertion that this condition actually occurs occasionally in sheep. The hormone theory thus gives a satisfactory explanation of the occurrence of occasional fertile free-martins in cattle as well as of the usual condition of sterility of the free-martin; and it fits the case of the sheep equally well. As regards other rumi- nants we have unfortunately almost no information. But I have been much interested to find that the famous discoverer of the circulation of the blood, William Harvey, in his “‘ Pzer- citationes de Generatione Animalium” 1651 has some statements on the subject of twin pregnancies in ruminants: thus in Ex. 69, p. 487 (Sydenham Society edition, translated by Willis), he says of the deer, ‘‘if the conception be double, one in either horn (of the uterus), each sends its umbilical vessels to its own 400 FRANK R. LILLIE horn alone; the embryo in the right horn deriving nourishment from the right part of the conception, that in the left from the left portion of the same.’’ He made similar observations on the sheep, goat, and “other bisulcated animals” and notes that ‘in the dog, rabbit, hog, and other animals that produce a consider- able number of young at a litter, the thing is otherwise. In these each foetus has two humors, these being severally surrounded with their proper membranes.”’ So far as I know there are no other published observations on the foetal membranes of twins in ungulates from Harvey’s time to the present with the exception of Bonnet’s single case already referred to. Harvey’s observa- tions show that fusion of chorions is wide spread in twin preg- nancies in ungulates; but he states definitely that in the deer the umbilical vessels of each foetus are distributed to its own side only, in which it resembles the sheep. A more careful examina- tion of the female of two-sexed twin pairs in these animals would be of interest in order to determine the possible sporadic occur- rence of sterility. The theory requires that if the same condition of common circulation of the foetal blood were to occur in other mammals as in twins of cattle the sterile free-martin condition should occur there also. Now in multiparous mammals such conditions cer- tainly do not occur commonly; for, if they did, the very numerous researches on their embryology would have brought them to light. In the pig one can find occasional, but rare, fusions of adjacent chorions, but I have never found any vascular connection. XK "Youve “uD COT ‘9.9 6e ‘ou aso fuotjooful oyqnop {Moo JO opOISAA OTUOTLOYO WIM], Z% “SI GG Are + *sepIs OM] oY} ULOIT S[PSS9A-poOoTq JO SISOUIOJSvUB OU ST O94} ING “pasny OAVY SopIsoa OTUOLIOYD VY], ‘yoRo UT WN] sndaoo & py SOIVAO [RUTOJVUT oY, “Yovo “WO ET 6 6 “AoP Ud dy} paBMo} uorzoolur sfqnog “f X “daeys Jo spoIsed oTuOTIOYO WIM], ¢ ‘Siq (‘uo1ydrtosap TOY JANI 10] FEE “d 4x0} 99g) “aTVU YFIM “JO (8}B0} JO JUOTMOSUVIIG o[BULeZ 9}0U ‘ULJABU-90If JO SIAOJIPO ‘fF {pouedo sovs oTJOTUUIE “¢g ‘SOpIS YJOG YIM WOT}I9TUO) SNOUdA Y}IM UOpaTAyoo ‘g {Yun} YSNoyy [VMopIw “TP “TK “UW EZ" “Wd C)°Z © ‘Lp ‘ou osvo fuorpoofur a_qnop :MO0d jo 9OISeA OTUOTIOYD UM], P “SI 428 FRANK R. LILLIE Fig. 6 Case 40. Reproductive organs of fertile free-martin; 10.2 cm. long. *. In this case there was no fusion between the two chorions, and the repro- ductive system is normal. See meus 10 for male twin. 1, ovary; 2, remains of Wolffian body; 3, Miillerian duct; 4, umbilical artery; 5, vagina; 6, urino- -genital sinus; 7, clitoris; 8, neck of allantois; 9, ureter. 429 HORMONES IN FOETAL LIFE ACTION OF SEX. Reproductive organs of fertile free-martin 20 em. long. Fig. 7 Case 9. < 4+. The chorion of the twins was single with a narrow connecting part be- tween the two halves devoid of cotyledons for a space of about three inches. 1, ovary; No record of vascular connections, which were presumably lacking. 4, ureter; 5, vagina; 6, urinogenital 2, left horn of uterus; 3, round ligament; 4, sinus; 7, clitoris; 8, umbilical artery; 9, neck of allantois; 10, fallopian tube. 430 FRANK R. LILLIE Fig. 8 Case 8. Reproductive organs of fertile free-martin 23.3 em. long. * 4, The chorion of the twins was single with a narrow connection between the two halves. Presumably no vascular anastomosis. The male was 26.5 cm. long, nearly 80 per cent heavier than the female, and its skin was unpigmented, whereas the female was darkly pigmented. 1, ovary; 2, left horn of uterus; 3, round liga- ment; 4, vagina; 9, clitoris; 6, neck of allantois. ACTION OF SEX HORMONES IN FOETAL LIFE 431 Fig. 9 Urinogenital system of normal female from twin females, pair 5. 17 em. long. Collected February 11, 1915. Chorion was single constricted be- tween the two foetuses. Figure B shows part of the right side x 5 with the horn of the uterus lifted to show the insertion of the round ligament. Contrast the round ligament of the female with the gubernaculum of the male (fig. 11) to which it corresponds exactly in position. The horns of the uterus begin to show the spiral coil; body of the uterus small; vagina distended; the urinogenital sinus much shorter than in male (fig. 11). A. X44. 1, ovary; 2, round ligament of uterus; 3, vagina; 4, rectum; 4, urinogenital sinus; 6, clitoris; 7, fallopian tube; 8, right horn of uterus; 9, allantois. B. xX. Part of same specimen with horn of uterus raised to show inser- tion of the round ligament. 1, ureter; 2, ovary; 3, right horn of uterus; 4, round ligament; 5, umbilical artery; 6, rectum; 7, body of uterus. 432 FRANK R. LILLIE Fig. 10 Normal male 10.4 em. long, from two-sexed pair 40. > 5. Note that the gubernaculum is well formed on the left side, but not yet on the right. 7, kid- ney; 2, testis; 3, Wolffian ducts; 4, inguinal fold; 4, left gubernaculum; 6, scrotal sacs; 7, teats; 8, penial tube; 9, urinogenital sinus; 10, umbilical arteries; 17, umbilical veins; 72, umbilical cord; 13, anus. ACTION OF SEX HORMONES IN FOETAL LIFE 433 Fig. 11 Normal male 15.8 em. long. X 4. From two-sexed pair 6 collected March 4, 1915. Chorion was single, constricted between the two foetuses. The long gubernacula have grown into the groin but have not yet entered the scrotal sacs. The testes are still in the body cavity, though close to the entrance to the saccus vaginalis. The left testis, posterior in position to the right, corre- sponding to the more posterior location of the left kidney. The seminal vesicles are well formed with distal buds. The urinogenital system of the free-martin twin is shown in figure 16. 1, spermatic artery; 2, testis; 3, Wolffian duct (vas deferens); 4, vesiculae seminales; 5, gubernaculum; 6, urinogenital sinus; 7, root of penis; 8, rectum; 9, umbilical artery; 10, ureter; 1/1, allantois. 434 FRANK R. LILLIE Fig. 12 Normal male 26 cm. long; from twin one-sexed male pair 3; collected January 28, 1915. X43. Single chorion, constricted between the two foetuses. This figure shows the entire male urinogenital system; the gubernacula have en- teredthe scrotal sacs. The testes are drawn into the vaginal sacs. The disposi- tion of the teats for the normal male should be noted. /, rectum; 2, vasa deferentia; 3, epididymis; 4, testis; 4, urinogenital sinus; 6. eubernaculum withdrawn from scrotal sac; 7, cut end of umbilical cord; 8, prepuce; 9, wall of penial sheath; 10, penis; 11, vesiculae seminales; 12, teats; 13, allantois; 14, gubernaculum in scrotal sac; 15, retractor muscle of penis. ACTION OF SEX HORMONES IN FOETAL LIFE 435 Fig. 13 Urinogenital system of sterile free-martin 7.5 em. long. XX 4; case 19. Specimen fixed in Flemming’s fluid; not so fully dissected as the following cases, as it was preserved for microscopical study. (Other drawings from the same specimen are figures 4 and 5, Chapin 717.) 1, gonad; 2, Wolffian body; 3, rectum; 4, genital duct; 5, allantois; 6, umbilical artery; 7, clitoris. 436 FRANK R. LILLIE Fig. 14 Sterile free-martin 13.1 em.long. > %. From two-sexed pair 17. Ceol- lected October 9, 1915. Gubernacula are not developed; ef. male of earlier stage (fig. 10). 1, gonad; 2, Wolffian duct; 3, inguinal fold; 4, umbilical artery; 5, ureter; 6, urinogenital sinus; 7, clitoris; 8, allantois. ACTION OF SEX HORMONES IN FOETAL LIFE 437 Fig. 15 Sterile free-martin 15.5.em. long. % 3. From two-sexed pair 37. Collected January 25, 1916. Gubernacula are developed as typically as in a male (figs. 10 and 11).. Gonads small. . Ducts also appear as in male. 1, gonad; 2, gubernaculum; 3, urinogenital sinus; 4, clitoris; 5, Wolffian ducts; 6, umbilical artery; 7, allantois. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO-2 438 FRANK R. LILLIE Fig. 16 Sterile free-martin 6, 16.8 em. long. X 3. (Cf. fig. 11 for male twin.) Gubernacula are somewhat smaller than in the male (fig. 11). Small gonads (cf. figs. 9 for normal size, and fig. 7 of Chapin for histological structure). The urinogenital sinus is intermediate in length between male and female (fig. 9); no seminal vesicles. 1, kidney; 2, gonad; 3, rectum; 4, Wolffian duct; 4, gubernaculum; 6, urinogenital sinus; 7, clitoris; 8, umbilical artery; 9, allantois. ACTION OF SEX HORMONES IN FOETAL LIFE 439 Fig. 17 Sterile free-martin 16.75 em. long. X 4. From two-sexed pair 32. Similar in all essential respects to figure 16 except that gonads are slightly larger, and possible rudiments of Miillerian ducts occur in the broad ligament. 1, kidney; 2, gonad; 3, rudiments of horns of uterus; 4, gubernaculum; 5, Wolffian ducts; 6, clitoris. 440 FRANK R. LILLIE iS Fig. 18 Part of urinogenital system of sterile free-martin 17.5 cm. long. x }. From two-sexed pair 23. The gonads are unusually rudimentary. 1, gonad; 2, urinogenital fold with ducts; difficult to interpret; 3, umbilical artery; 4, gubernaculum; 5, ureter; 6, allantois turned back. Fig. 19 Urinogenital system of sterile free-martin 17.5 em. long; X #, From two-sexed twin pair 36. The gonads are exceedingly rudimentary in this speci- men; the gubernacula are present, but not as well developed as in figure 18. 1, kidney; 2, gonad; 3, rectum; 4, gubernaculum; 5, urinogenital sinus; 6, clitoris; 7, Wolffian ducts. ACTION OF SEX HORMONES IN FOETAL LIFE 441 20B 20A Figs. 20A and 20B Urinogenital system of sterile free-martin 21.5 cm. long. From two-sexed pair 2. 20A. X +4. On the right side the gubernaculum, instead of growing into the body wall has evaginated into the body cavity and lies partly in the utero- rectal recess. Both Wolffian and Miillerian ducts appear to be present; latter very rudimentary. Gonads very small. 1, gonad; 2, gubernaculum, left side; 3, umbilical artery; 4, Wolffian duct; 5, ureter; 6, Miillerian duct; 7, urinogenital sinus; 8, gubernaculum of right side evaginated into body cavity; 9, allantois. 20B. xX 3. Part of 20A with the right gubernaculum withdrawn from the utero-rectal recess and turned over to expose the gonad and Wolffian duct. 1, gonad; 2, Wolffian duct; 3, Miillerian duct; 4, gubernaculum. 442 FRANK R. LILLIE Fig. 21 Urinogenital system of sterile free-martin 21.8 em. long. 4. From two-sexed pair 41. Exceedingly small gonads drawn close to entrance of the saccus vaginalis. 1, gonad; 2, Wolffian duct; 3, gubernaculum; 4, urinogenita sinus; 9, clitoris; 6, allantois. Fig. 22A Urinogenital system of sterile free-martin 22.5 cm. long. X #4. From two-sexed par 4. The right gubernaculum is evaginated into the body cavity. 22B. Part of 22A. X $ with the right gubernaculum turned over to show the gonad and Wolffian duct. 1, kidney; 2, gonad; 3, Wolffian ducts; 4, gubernacu- lum; 5, allantois; 6, clitoris; 7, umbilical artery; 8, ureter. 444 FRANK R. LILLIE Fig. 23. Urinogenital system of sterile free-martin 22.5em. long. x {. From two-sexed pair 38. The typical features recur here; the gubernaculum of the right side has grown only partly into the body wall; compare the left side. 1, gonad; 2, Wolffian duct; 3, gubernaculum; 4, urinogenital sinus. ACTION OF SEX HORMONES IN FOETAL LIFE 445 Fig. 24 Urinogenital system of sterile free-martin about 24 em. long. X 5. From two-sexed pair 22. The typical features recur here again; the right guber- naculum lies in the body-cavity and is relatively undeveloped; compare the left side. Parts of the cornua uteri seem to be developed in this case. 1, gonad; 2, Miillerian ducts (cornua uteri); 3, Wolffian duct; 4, gubernaculum; 4, rudi- ment of corpus uteri, cervix and vagina. 446 FRANK R. LILLIE 25A ACTION OF SEX HORMONES IN FOETAL LIFE 447 25B Fig. 25A Urinogenital system of sterile free-martin 26.5 em. long. ™X 4. From two-sexed pair 21. In this case the gonad of the left side has entered the saccus vaginalis; compare figure 12 for normal condition of the male. The right gubernaculum has grown into the body-cavity. Observe the female disposition of the teats and development of the glandular tissue of the mammary gland, and compare male (fig. 12). Fig. 25B Part of 25A further dissected to show. the left saccus vaginalis containing the gonad on the left side; on the right side the gubernaculum is turned over. Designations for 25A and 25B. 1, ovarian artery; 2, remains of urinogenital ridge; 3, entrance to saccus vaginalis; 4, gubernaculum; 5, Wolffian duct; 6, teats; 7, mammary gland tissue; 8, clitoris; 9, gonad. Fig. 26 Urinogenital system of sterile free-martin 27 cm.long. X +. From two-sexed pair 14. Although this case 's beyond the stage in which the testes of the male normally enter the saccus vaginalis, the rudimentary ovaries are here in the body cavity. In this case there appear to be rudiments of the cornua uteri. 1, rectum; 2, gonad; 3, cornua uteri; 4, urinogenital sinus; 4, umbilical artery. Fig. 27. Urinogenital system of sterile free-martin 28 em. long. X 4. From two-sexed pair 12. In this case both gubernacula have grown into the body ‘cavity; the right gubernaculum lies in the utero-rectal recess of the body-cavity. In figure B the gubernacula are rearranged, the right one being drawn out of the recess, and the genital cord is cut across and turned forward. Rudiments of both Miillerian and Wolffian ducts present. 7, gonads; 2, gubernaculum; 3, sex-ducts; 4, cornua uteri; 5, corpus uteri, cervix and vagina; 6, Wolffian ducts. 448 Fig. 28 Reproductive organs of a seven weeks old free-martin. X ys. Born twin to a male, case 44. The dissection shows a dorsal view. Description in text. 5; Fig. A 1, left saccus vaginalis containing gonad; 2, right saccus vaginalis containing gonad; 3, Vas deferens (Wolffian duct); 4, broad hgament; 5, ureter; 6, bladder; 7, seminal vesicles; 8, urogenital sinus; 9, vulva; 10, clitoris. Figs. Band C Left and right sacci vaginales opened. 1, testis; 2, epididymis. Fig. 29 Reproductive organs of a free-martin described by Numan (’43) from his plate XI. Description in the text (p. 413). The description of this plate was missing. The explanation of the letters is therefore my own inter- pretation. a, epididymis with testis above; b, Saccus vaginalis; c.c, cut wall of saccus vaginalis; d.e.f, spermatic artery, vein and nerve (?); g, Vasa defer- entia (Wolffian ducts); h, bladder; 7, broad ligament; k.l/, ligaments of bladder; (2); m, ureters; n, seminal vesicles; 0, entrance of vasa deferentia into the urino- genital sinus; p, urinogenital sinus; g, prostate; 7, penis; s, retractor muscles of penis; ¢, ?; uw, external opening of urinogenital sinus (urethra) beneath the glans penis; v.w, accessory openings in the urethra; 2, vulva; 7, anus; 8, perineum. 451 ons... as See (EE: A MICROSCOPIC STUDY OF THE REPRODUCTIVE SYSTEM OF FOETAL FREE-MARTINS CATHARINE LINES CHAPIN Hull Zoélogical Laboratory, University of Chicago SIXTEEN FIGURES The following study of one phase of the free-martin problem was suggested to me by Prof. F. R. Lillie and has been pursued under his direction. To him my thanks are due for his kindly advice and constructive. criticism. The foetal free-martins used in this investigation are those described by Professor Lilie in the preceding paper (this Jour- nal, p. 371 to 452). Some of these specimens were preserved in toto in 5 per cent formalin. Cther specimens were dissected as soon as possible after being brought to the laboratory and parts to be used for histological study were preserved in Zenker’s fluid or in strong Flemming solution. The gonads and related organs, including in most cases the Wolffian body and the Wolf- fian and Miillerian ducts, of the foetal free-martins were sec- tioned. These organs were studied also in normal males and females of approximately the same size as the free-martins. Two series of records of specimens were kept; one, of the series of twins described in the preceding paper and one, of the series of normal embryos collected to study in comparison with the free-martins found in the twin series. Individuals of the twin series, which includes twins of both the one sexed and the two sexed types are designated by the letter T and their serial num- ber; individuals of the normal series are designated by N and their serial number. Histological preparations were made from some of the normal males and females for comparison with the free-martin. There is no indication in the early stages studied that, at a given degree of development, there is any marked dif- ference in size between a single embryo and a twin. Thus a 453 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 2 454 CATHARINE LINES CHAPIN 20 em. free-martin may justly be compared with a 20 cm. normal embryo of either sex, as well as with its twin, the normal male. In the description of the reproductive glands and related or- gans which follows, most of the terms are employed in their usual sense; Wolffian body, epididymis, epodphoron, Wolffian or mesonephrotic duct and Miillerian duct, germinal epithelium and others. A few terms, which have not been used in their ordinary sense may best be explained at this point. Rete is used to de- seribe the network of tubules of the rete testis, the rete ovarl which for the most part degenerates, and the modified rete of the _ free-martin which persists. The term sex cords is used to des- ignate the proliferation of the germinal epithelium in the two sexes. In the male, there is one set of sex cords, the seminif- erous tubules. In the female there are two sets of sex cords; first, the medullary cords which degenerate during foetal life and second, the cords of Pfliiger, at the inner ends of which are formed nests of cells including the primordial ova, the primary ovarian follicles. The term albuginea is used to designate the tissues lying just beneath the peritoneum and surrounding the sex cords, in- cluding not only the tunica albuginea, but also the tunica vascu- losa which, in the male embryo, merges into the tunica albuginea. According to Allen! this term is equally applicable to the similar structure found in the adult female, between the cords of Pfluger and the germinal epithelium, but which has not yet developed to any great extent in a 29.5 cm. female (N20). For the sake of clearness this structure is designated definitive albuginea, to distinguish it from the primary albuginea which separates the medullary cords from the cords of Pfliiger. The term interstitial cells is used to refer to those cells de- scribed by R. H. Whitehead? as the cells of the interstitial gland; interstitial material refers to all the material between the seminiferous tubules, including not only the interstitial cells, but also the connective tissue stroma. 1B.M. Allen, Am. Jour. Anat., vol. 3. 2R. H. Whitehead, Am. Jour. Anat., vol. 3. REPRODUCTIVE, SYSTEM OF FREE-MARTINS 455 The following pages comprise detailed descriptions and com- parisons with normal males and females of eight free-martins, Viz) ime, P19) (2) time toe oii. 26, (4) fms. 13, 2) and 4, (5) fm. 12, and (6) f.m. 42. 1. FREE-MARTIN, 7.5 CM. T 19 The smallest free-martin which has been accessible for study, T19, measures 7.5 cm. in total length. Lillie, figure 13. The gonads are very small, being only 2.07 mm. long, whereas a testis of its twin brother is 3.5 mm. long, and an ovary of an 8.5 em. female (N4) measures 3 mm. The gonads of this free- martin are lemon-shaped structures which lie close to the Wolf- fian body, near its anterior end. The area of the central cross section is about one-fourth of that of the testes of normal males measuring 7 em. (N10) and 8 em. (T19) and of the ovary of a normal female, 7.3 cm. long (N8), (figs. 2, 3, and 5). a. Normal conditions at this stage In the normal male T19, twin with free-martin T19, and measuring 8 cm., the sex cords, seminiferous tubules, are dis- tinct, much branched, and separated from each other by con- nective tissue and the cells of the interstitial gland. (Unfortu- nately, the preservation of # T19 was not made with a view to histological study and the central portion of the gonad is indis- tinct.) In o& N10, 7 em. long and preserved in strong Flemming (figs. 1 and 2), the seminiferous tubules make up the larger part of the testis. The outer layer, in both cases (T19 and N10) is the typical albugineal layer of connective tissue with the long axis of the cells parallel to the peritoneum. The rete enters the testis near the anterior end and runs posteriorly, forming the core about which the seminiferous tubules radiate, figures 1 and 2. Most of the germ cells of the seminiferous tubules are still indifferent but a very few are found in the early stages of the growth period.? In a normal female 8.5 em. long (N4), the medulla is relatively large in cross section. The medullary cords have not yet begun 3’ Schoenfeld, Arch. de Biol., T18, 1901-02. 456 CATHARINE LINES CHAPIN fn vnnennn-— PA re 10) REPRODUCTIVE SYSTEM OF FREE-MARTINS 457 to degenerate. In an 8.3 cm. 92 N7 (fig. 3) andina7.3cm. 9 N8, primary follicles are found in the medulla. The inclosed ovum is usually indifferent but may have begun to degenerate. Separating the medullary cords from the outer layer of sex cords, the cords of Pfliiger, is a layer of connective tissue, the primary albuginea. Strands of connective tissue extend out- wards from this layer, toward the periphery of the gland, sepa- rating the cords of Pfliger from each other and forming the stroma of the cortex. This cortex is narrow. The cords of Pfliiger are still in an early stage of development, the cells not yet being arranged in ‘nests’ surrounding growing ova. The germ cells are still in the indifferent stage. No interstitial cells can be distinguished. The germinal epithelium is a single layer of cuboidal epithelial cells. The cords of Pfliiger have not yet been separated from it by the development of the definitive albuginea. The normal ovary has a different shape from that of the testis. The rete, entering near the anterior end, lies close to the mesen- tery which connects the ovary with the Wolffian body. The rete ovarii does not grow far posteriorly at as early a stage as does the rete testis. During the further development of the foetal ovary, the cortex grows more rapidly than the medullary region, extends toward the Wolffian body on each side of the mesentery, and the two sides approximate each other second- arily, making the rete appear to be in the center of a round gland as it is primarily in the testis. Fig. 1 Cross section of testis and Wolffian body of 7 em. & Bos embryo, N10, near anterior end, showing rete entering the testis. 1, glomerulus; 2, secre- tory tubule; 3, collecting tubule; 4, Wolffian duct; 5, Miillerian duct; 6, rete cord, (with lumen); 7, interstitial material; 8, seminiferous tubule; 9, albuginea; 10, superficial epithelium (peritoneum). X 20. Fig. 2 Cross section of testis and Wolffian body at 7 em. <& Bos embryo, N10 near middle of gonad. Designations as in figure 1. X 20. _ Fig. 3. Cross section of ovary and Wolffian body of 8.3 em. ? Bos embryo, N7, near middle of ovary, showing position of rete in 2 as compared with that in o, figure 2. 1, rete; 2, medullary cords; 3, primary albuginea; 4, cords of Pfliiger; 5, germinal epithelium; 6, Bowman’s capsule (Malpighian body); 7, Wolffian tubule; 8, Wolffian duct;.9, Miillerian duct. 42. 458 CATHARINE LINES CHAPIN The Wolffian bodies of a normal 7 cm. & N10, and of a nor- mal 8.3 em. @ N7, are shown in figures 1, 2 and 3. These con- tain the Malpighian bodies and Wolffian tubules of the young embryo. The tubules in all the specimens of this stage which were examined, showed the typical form of wall, low epithelium in collecting portions and tall, columnar epithelium in secretory portions. The tubules are a little less numerous in the 8 em. & T19, but N10 was a better preparation from which to make a drawing. The relations of the Wolffian duct, Wolffian body and rete are still in the indifferent stage. In both @ and 9 the rete connects sex cords with the Malpighian bodies, from which the Wolffian tubules lead into the Wolffian duct. In the normal male T19 and female N8, measuring 8.0 and 7.3 em. respectively, not only the Wolffian, but also the Miil- lerian ducts are present. In the female the Millerian duct, at its anterior end, opens into the body cavity by the ostium abdominale, a funnel-shaped opening lined with the large cili- ated type of epithelial cell of which consists the inner layer of the whole duct. Degeneration of the Miillerian duct in the male starts at an earlier stage than that reached by an 8 cm. male. The ostia abdominalia are present in a 4.8 cm. male N13. In N10, 7 em. long, there is a suggestion of ostia ab- dominalia, but the preservation is poor in the critical region. In the 8 cm. male 19, which is twin with a free-martin, no ostium abdominale is present. Slghtly anterior to the testis there begins a rod of connective tissue which runs posteriorly, parallel to the Wolffian duct. For a short distance the anterior end consists simply of a few concentric layers of connective tissue, but throughout the greater part of its course this structure en- closes the epithelial Millerian duct. The wall of the duct is made up of epithelial cells but these are not ciliated, as they are in the female. In a few sections (not consecutive) the lumen is obliterated, but this may be due to preservation and staining rather than to an abnormality in the duct. (This specimen 4 J. B. MacCallum, Am. Jour. Anat., vol. 1. REPRODUCTIVE SYSTEM OF FREE-MARTINS 459 had not been preserved carefully for microscopic study.) The whole duct is smaller in diameter than the duct of a normal female of the same size. b. Condition in the free-martin The gonad of the free-martin 19, on microscopic examination, is found to consist of a central, rather dense mass of deeply staining cells, surrounded by a layer of cells less dense than the central mass, figures 4 and 5. The outer structure is albuginea, similar to that of the testis, but it is not arranged in definite layers as in the normal ~# T19. In the anterior part, the cen- tral mass shows two divisions. The smaller one, round in cross section, is the rete. This is easily traced from its con- nection with the glomeruli in the Wolffian body, into the gonad which it penetrates almost at the anterior apex, figure 4. A short distance posterior to its entrance into the central mass of the gonad it becomes scarcely distinguishable from the rest of the mass, figure 5. The rete is not differentiated into cords. The remaining portion of the central mass of the gonad partly surrounds the rete. It is an undifferentiated mass of cells, but represents the medullary cords. A very few germ cells are found. These are in Schoenfeld’s growth stages c, d and e. Interstitial cells cannot be distinguished. A single layer of peritoneum covers the whole gonad, as in the testis. Although the gonad is much smaller than those of the normal @ and ¢ studied in comparison with free-martin 19, the Wolffian body is of normal size and structure. The Wolffian tubules and Mal- pighian bodies in 7.5 em. free-martin 19 are as numerous as they are in the 7 cm. &@ N1O and 7.3 cm. 2 N&8, and more numerous than in its twin, the 8 em. #@ T19 and in the 8.3 cm. Q N7. The Wolffian duct of the free-martin is normal. The Miil- lerian duct, on the other hand, is abnormal. At its anterior end it is like the Miillerian duct of the normal male twin 19. The connective tissue rod lies in the normal position of the Miillerian duct, but the epithelial duct which it encloses is discontinuous. The lumen is present only at irregular intervals and even the 460 CATHARINE LINES CHAPIN REPRODUCTIVE SYSTEM OF FREE-MARTINS 461 epithelial cells which form the duct are not present through its entire length. Summary. The gonad of this 7.5 em. free-martin resembles a reduced testis in the presence of albuginea, thin covering of peritoneum, and absence of cords of Pfliiger, and differs from the ovary in the same respects. It is like the ovary in the posi- tion of the rete. The ducts resembles those of the male in that the Wolffian duct is complete and degeneration of the Miillerian duct has commenced. The Wolffian body is like that of normal embryos of the same size. 2. FREE-MARTIN, 12.5 CM. T26 The next size of free-martin which was examined microscopi- cally, T26, measured 12.5 cm. This may be compared with the 12.75 em. male T16, and the 14 em. female N26. a. Normal conditions at this stage The testis of the male is 4.45 mm. long. The seminiferous tubules are already branched, contorted, and very numerous, but have not yet developed lumina. They are separated by a small amount of interstitial material; most of this material is con- nective tissue, but some of the cells resemble interstitial cells as described by B. M. Allen! and R. H. Whitehead.2. Throughout the seminiferous tubules are found primitive sex cells, some of which have started upon the growth period.? A very thin layer of peritoneum covers the surface of the testis. Beneath this lies the albuginea which is wide and compact. Fig. 4 Cross section of Wolffian body of 7.5 em. free-martin T19, at point where rete is pushing out to enter anterior end of gonad. 1, rete; 2, Wolffian tubule; 3, Wolffian duct; 4, Miillerian duct; 5, Glomerulus. X 42. Fig. 5 Cross section of Wolffian body and gonad of 7.5 cm. free-martin embryo, T19 through middle of gonad. 1, rete; 2, sex cord region; 3, albuginea; 4, germinal epithelium; 5, glomerulus; 6, collecting Wolffian tubule; 7, secretory Wolffian tubule; 8, Wolffian duct; 9, rudimentary Miillerian duct. X 42. Fig. 6 Cross section through Wolffian body and middle of gonad of 12.5 em. free-martin, T26. 1, medullary cords; 2, superficial or germinal epithelium; 3, rete; 4, albuginea; 5, degenerating Wolffian tubules; 6, Wolffian tubules; 7, uro- genital fold in which ducts normally lie. X 42. 462 CATHARINE LINES CHAPIN The rete tubules branch and anastomose. The lumina are wide. The epididymis is formed from the anterior end of the Wolffian body, but as yet it is very small, consisting only of a few tubules, the vasa efferentia, which connect the rete with the Wolffian duct. Lateral and posterior to the testis the Wolffian body is degenerating. No glomeruli are to be found. Tubules are fewer than in the Wolffian body of younger males and cells filling their lumina indicate degeneration.' The Wolf- fian duct is fully developed. The Miillerian duct has undergone complete atrophy in the region of the gonad. The entire length of the ducts was not sectioned so the vestiges of Miillerian ducts —uterus masculinus—were not observed. The ovary of the 14 cm. 9 N26 measures 3.4 mm. Com- pared with an 8.3 cm. @ the medulla is small and the cortex large. The cortical region is less dense and the cells stain less deeply. The cords of Pfliiger are separated by connective tissue stroma. No ‘nests’ of cells are as yet formed at the inner ends of the cords of Pfliger. The interstitial material consists mainly of connective tissue but a few large cells are found which may be the interstitial cells of Leydig.1¢? The germ cells which are distinguishable in the cords of Pfliiger are mainly in the indifferent stage In the deeper part of the cortex are found primary odcytes in the early stages of growth.’ The germinal epithelium is a layer of cuboidal cells on the surface of the ovary, continuous with the cords of Pfliger; no defini- tive albuginea has as yet developed. Some of the follicles which were formed in the medullary cords at a much earlier stage, have degenerated in the 14 cm. @ leaving only round clusters of cells, sometimes arranged in concentric layers. Others contain germ cells, but the stage which these ova had reached, could not be determined. - The rete is smaller in cross section than that of the testis and does not extend as far into the ovary. The rete cords are less numerous and less branched than those of the male, but they also have lumina. The rete can be traced into the Wolffian body. Only a few glomeruli persist in the Wolffian body. Those are in the posterior part. There remain none of the large REPRODUCTIVE SYSTEM OF FREE-MARTINS 463 Wolffian tubules which were such a noticeable feature of the Wolffian body of younger females (N8, etc.). Opposite the anterior end of the ovary, a few tubules form the epodphoron, which is the homologue of the epididymis in the male. Through- out the Wolffian body are signs of degeneration such as a large amount of connective tissue, and cells in the lumina of tubules.‘ The Wolffian duct has atrophied, although it is present in a 17 em. female sectioned. The Miillerian duct is complete. b. Conditions in the free-martin . The gonad of the 12.5 em. free-martin is 1.735 mm. long. The area of the central cross section is one-fifth that of the ovary of the 14 em. female and one-eighth the size of the testis of the 12.75 cm. male. The shape of the gonad resembles that of an ovary of an8 cm. @ (fig. 6). The rete enters the gonad at one side, near the anterior end, but runs posteriorly near the mesen- tery. Itis more clearly defined than the rete of the 14 cm. ? N26. The cords are distinct and have lumina. Connective tissue sur- rounds the rete. On the distal side the connective tissue forms the fan-shaped stroma of the medullary region. In the anterior part of the gland, this region is large but the sex cords are with difficulty distinguished from each other. In the posterior part of the gland are found some degenerating medullary cords which look like those of the female. The few germ cells present are in Schoenfeld’s growth stage d. Outside the sex cords and just beneath the peritoneum lies a layer of albuginea, less com- pact than that of the testis of a 12.75 em. #. Both the peri- toneum and the albuginea are strikingly like the male of this stage. There seems to be but one set of sex cords present—the homologue of medullary cords of the @ and seminiferous tubules of the #. There is no structure present to correspond to the cortex of the normal ovary. Neither the Wolfian nor the Miillerian duct is complete. An- terior to the gonad, a groove runs along the genital fold at about the place where Miiller’s duct is normally found. In cross section this groove is suggestive of the ostium abdominale. 464 CATHARINE LINES CHAPIN On a level with the anterior end of the gonad there is a portion of the Miillerian duct, 0.080 mm in length, surrounded by the connective tissue structure which normally encloses it (ef. f-m. 138, page 474). The Wolffian duct begins anterior to the gonad. At intervals, other ducts enter it from the Wolffian body. It is irregular, in some places large and clearly defined, with a wide lumen, at other points small with the lumen indistinct. Summary. The gonad of the 12.5 em. free-martin resembles a testis in the appearance of albuginea and peritoneum, and in the fact that only one set of sex cords is present. In the position of the rete, it resembles an ovary. The Wolffian duct is incom- plete as in the female and the Miillerian duct is degenerating, as in the male. 3. FREE-MARTIN, 16.3 CM. T6 (See fig. 16, Lillie.) Both the free-martin and the normal male twin were studied. The free-martin measured 16.3 em. and the male measured 16.8 cm. The gonad and Wolffian body were sectioned in both embryos; in the free-martin, the ducts posterior to the Wolffian body were also sectioned. a. Normal conditions at this stage The male, as is usual in this type of twins, is normal. The testis is 4.86 mm. long. The rete enters the gonad almost at its extreme anterior end. The seminiferous tubules which radiate from the rete are numerous and more branched than those of younger males (cf. # T16 or T25). They are separated from each other by connective tissue and interstitial cells. Germ cells in the seminiferous tubules are found in the indifferent stage and early stages of the growth period. A wide, compact albuginea is present. The anterior part of the Wolffian body has become the epididymis, the tubules of which are more con- torted than those of the epididymis in smaller males. Degen- eration is going on throughout the rest of the Wolffian body. The Wolffian duct is complete and, through part of its course, lies in the fold of peritoneum overhanging the testis The tubular part of the Millerian duct has atrophied. (Posterior part of ducts not sectioned.) REPRODUCTIVE SYSTEM OF FREE-MARTINS 465 The 17 em. female N23 may also be described for comparison with the 16.3 em. free-martin T6. The ovary measures 4.39 mm. In cross section its area is about half that of the testis of male T6. The medulla still makes up a large part of the ovary. Medullary cords are found containing many primary follicles and surrounded by abundant connective tissue. The cords of Pfliiger are in the same condition in which they were found in the 14 cm. 2 N26, with the exception that at their inner ends some of them have formed ‘nests’ of cells, primary follicles enclosing primitive ova. A few germ cells are found in early growth stages. There is no albuginea formed; cords of Pfliiger are still at- tached to the germinal epithelium. The stroma separating the cords of Pfliiger from each other is wider than that of a 14 cm. 2, but no interstitial cells are distinguishable. The rete ovarii is much smaller in cross section and extends a shorter distance into the gonad than does the rete testis. Lumina are very few but have clearly defined epithelial walls. Degeneration of the Wolffian body has gone farther in the 17 em. ° thanin the 16.8cm. @. The Wolffian body of the former is smaller in cross section than that of the latter. The tubules of the epodphoron remain in the anterior part. In the posterior end are found glomeruli, fewer in number than were found in the 14 cm. stage. The Wolffian duct, although lacking in the 14 em. 2 studied, is present in this 17 em. 2 and complete as far as it was sectioned. It is also present, though small in diameter, in a 20 em. @ studied, showing some irregularity in time of atrophy of vestigeal structures in normal embryos. The Miil- lerian duct is complete. The ostium abdominale is relatively larger than that of smaller females and its walls are becoming convoluted. The uterine horns open into the uterus, which by this stage is large and has a muscular wall. (The uterus was studied by dissection.) b. Conditions in the free-martin The gonad of the free-martin is approximately 2 mm. long, as compared with the 4.86 mm. testis and 4.39 mm. ovary which have been. described for comparison. The area of the cross 466 CATHARINE LINES CHAPIN section through its widest region is one-fourth the area of the central cross section of the aforesaid ovary and one-eighth that of the testis. The medulla of this 16.3 em. free-martin is relatively small, being little wider than the rete (fig. 8). It is about the size of the medulla in the 12.5 em. free-martin T26, previously described. It shows little sign of differentiation, but occa- sional primary follicles are found. In some of these the vacuo- lated condition of the contained germ cell suggests degeneration, in this point, resembling the medullary cords of the female. The rete enters the gonad at the anterior end. It is prominent and well developed. The more anterior rete cords have lumina. The albuginea can hardly be distinguished from the undiffer- entiated sex cord region. Especially is this the case in the an- terior part, where the medullary region is little specialized (fig. 7). The albuginea looks like a compact layer of connective tissue, whereas the medullary region looks like a loose mass of connective tissue. Farther posterior, the medulla has a more dense structure, though still homogeneous and undifferentiated, and the albuginea is quite distinct, a relatively wide and com- pact layer. It should be noted that, as is true of the other free-martins investigated, only one set of sex cords is found. No second set, corresponding to the cords of Pfliiger, the cortex of the ovary, is ever developed. Anterior to the gonad, the Wolffian body of the free-martin seems to have the relations found normally in the male. The Wolffian tubules connect the rete with the Wolffian duct, as in the epididymis. These tubules, however, are smaller than those Fig. 7 Cross section through Wolffian body and anterior end of gonad of 16.3 em. free-martin, T6. 1, degenerating Wolffian tubule; 2, fold of peritoneum overhanging gonad and in which Wolffian duct normally lies; 3, albuginea; 4, rete; 5, sex cord (medullary cord). X 42. Fig. 8 Cross section through Wolffian body and middle of gonad of 16.3 em. free-martin, T6. 1, degenerating Wolffian tubule; 2, Wolffian tubule not yet degenerated; 3, urogenital fold; 4, rete; 5, sex cord region, showing little differ- entiation into cords; 6, albuginea. X 42. Fig. 9 Cross section through epididymis of 24 em. co’ Bos embryo, T4. 1, vas epididymis; 2, rete; 3, vasa efferentia; 4, spermatic artery. X 20. 467 REPRODUCTIVE SYSTEM OF FREE-MARTINS potee os ties, aay st 468 CATHARINE LINES CHAPIN in the normal male and, in some cases, cells are found in the lumen indicating degeneration. The Wolffian body of free- martin T6 is as large in cross section as that of &@ T6, and is larger than the Wolffian body of the @ N23, examined for comparison. Both sets of ducts of the free-martin are irregular. The Wolffian duct is almost entirely atrophied. Anterior to the gonad its relations are normal. In the region near the gonad it appears only at irregular intervals. Posterior to that it is lacking for some distance. It reappears near the caudal end of its normal course where it seems to be regular. As for the Miillerian duct, the tube is entirely absent. The enlarged posterior part of the Miillerian duct is present and seems to be complete. In a nor- mal 14 cm. female, the horns of the uterus are much larger in diameter and unite to form the body of the uterus. In this 16.3 cm. free-martin the uterine horns do unite (of the free-martins examined, this is the only case in which any union of the cornua was found), but only for a distance of 1.905 mm. Anterior to the union, one duct lies ventral to the other. When they sepa- rate, posterior to their junction, they are once more lateral to each other. Summary. The gonad of the 16.3 cm. free-martin resembles that of the male in the absence of cortex, the appearance of the albuginea, and peritoneum, and the large development of rete. It resembles the female gland in the structure of the sex cords and in the position of the rete. The atrophy of the oviduct and retention of the reduced uterine horns is characteristic of the male, whereas their partial union and the degeneration of the Wolffian body suggest the condition of these organs in the female. 4. SEVERAL FREE-MARTINS STUDIED MEASURED 20 CM. OR THEREABOUTS Of these, T13 measured 20 em., T2 measured 21.5 cm. (Lillie, fig. 20) and T4 22.5 cm. (Lillie, figs. 22A and 22B). These embryos were presumably of about the same age, but, as might be expected from the possible variations in the cause of their abnormalities, they differ among themselves, in development of REPRODUCTIVE SYSTEM OF FREE-MARTINS 469 reproductive organs, much more than do normal embryos which vary even more in size. For comparison the normal # and @ of the same size will first be described. a. Conditions in the normal The males sectioned for comparative study are N21, measur- ing 20 em. and T4, the twin of free-martin 4, which measures 24 em. The testis of the 20 em. male, which had not yet be- gun its descent, is 4.042 mm. long. The left testis of the 24 cm. co’ measures 8.67 mm. and had begun its descent into the saccus vaginalis. The right testis in the same specimen had almost reached the distal end of the scrotal sac. Otherwise, the testes of these two specimens are approximately the same. Their condition is shown in figures 9 and 10. The seminiferous tubules are still solid cords, longer and more branched than in testes of younger animals. They contain germ cells, some of which are as yet in the indifferent stage, while others are in the growth stages described by Schoenfeld. In the 20 em. &@ one finds primary spermatocytes in stages a toe. In the 24 em. specimen they are in stages a,e and f. In neither case were any mitoses found. The spaces between the sex cords are filled with a large amount of interstitial material made up chiefly of typical em- bryonic connective tissue, but some of the cells have a wider layer of cytoplasm around the nucleus and are polyhedral in shape. These are young interstitial cells. The interstitial space is wider in T4, the interstitial cells more numerous. The characteristic male albuginea, consisting of layers of connective tissue pressed compactly together, lies just beneath the superficial peritoneum, surrounding the sex cord region. Blood vessels lie in the inner deeper layer (tunica vasculosa). The rete cords can be seen connected with the seminiferous tubules, but distinguished from the latter by their lumina! (fig. 10). The Wolffian body lateral to the gonad is almost completely degenerated. From the anterior part of the Wolffian body is formed the epididymis, much larger in these males than it was THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NG. 2 470 CATHARINE LINES CHAPIN REPRODUCTIVE SYSTEM OF FREE-MARTINS 471 in the 16.75 ecm. male. Numerous, much convoluted vasa efferentia lead from the rete into the coiled vas epididymis which in turn opens into the Wolffian duct. The Millerian duct has long since atrophied. In the case of the females, there were sectioned the left ovary and ducts of a 20 cm. 2° N24 and the left ovary and ducts of a 23 em. 9 N25. A dissection was made of the reproductive sys- tem of a third female, measuring about 20 em. The sectioned ovary of the 20 cm. 9 was 4.440 mm. long. That of the 23 cm. 2 measured 6.830 mm. The condition of the ovaries is almost the same in these two cases. The medulla is relatively larger in the younger specimen. In N25, figure 11, it is composed mainly of fibrous stroma and contains fewer medullary cords than does the medulla of the smaller female. ‘Nests’ of cells, primary follicles, first found in the cortex in the 17 em. 2 N23, are more numerous in each succeeding stage. The individual cords of Pfliiger are surrounded by wide sheaths of stroma, but in this large amount of interstitial material, no interstitial cells have been identified with certainty. Most of the cells are the long slender spindle shaped cells typical of embryonic connec- tive tissue. In the smaller female, most of the germ cells which are found in the growth period are in Schoenfeld’s early stages, though a few have reached as advanced a stage as i. In the 23 cm. @ they are found in these same stages, the majority of Fig. 10 Cross section through Wolffian body and middle of testis of 24 em. o bos embryo, T4. 1, superficial epithelium; 2, blood vessels in tunica vas- culosa; 3, rete; 4, interstitial material, including connective tissue stroma and interstitial cells; 5, seminiferous tubules; 6, albuginea; 7, degenerating Wolffian tubule; 8, Wolffian duct. 15. Fig. 11 Cross section through Wolffian body and middle of ovary of 23 em. 9 Bos embryo, N25. Note that the rete does not extend as far as the middle of the ovary. 1, tubule of epodphoron; 2, degenerating Wolffian tubule; 3, Millerian duct; 4, primary follicle formed at inner end of cord of Pfliiger; 4, primary albuginea; 6, medullary cords; 7, cords of Pfliiger; 8, germinal epithelium (scarcely distinguishable from cord of Pfliiger) ; 9, beginning of ovarian albuginea. Seo) Fig. 12 Cross section of gonad and Wolffian body of 20 cm. free-martin T13, and entrance of rete into gonad. 1, degenerating Wolffian tubule; 2, rete; 8, sex cord region (no differentiation), 4, rudiment of Miillerian duct; 4, fold ‘of peritoneum overhanging gonad. X 28. 472 CATHARINE LINES CHAPIN them having advanced at least as far as stage e. No mitoses are in progress. The germinal epithelium is thick and scarcely distinguishable from the outer ends of the cords of Pfliger ex- cept that in places, the strands of stroma on either side of a cord of Pfliiger are uniting at their distal ends to form the begin- ning of the definitive albuginea. The rete ovarii, small in cross section compared with the rete testis, extends only a short dis- tance into the ovary. The Wolffian body, in both females, is represented only by the epodphoron and parodphoron. In N25 the Wolffian duct has completely atrophied, but in N24 it still persists, though it is exceedingly small in diameter. The Miillerian duct is large and. lined with ciliated epithelium. The mouth of the ostium abdominale is becoming convoluted. A macroscopic study shows the uterus large, with a thick, muscular wall. b. Condition in the free-martin The three free-martins, 13, 2 and 4 (figs. 12 to 16) resem- ble each other in regard to some structures. All have well developed rete with distinct lumina in the cords. All have distinguishable medullary cords, although in case 4 they are relatively fewer in number and more widely separated by the connective tissue stroma. In every case the albuginea is a wide, compact layer, and the superficial peritoneum is a thin epithelial layer as in the male; the cortex, cords of Pfliiger, is absolutely lacking. Fig. 13 Cross section through middle of gonad and Wolffian body of 20 em. free-martin T13. 1, degenerating Wolffian tubules; 2, urogenital fold; 3, super- ficial epithelium like that of &; 4, rete; 5, medullary cord connecting with rete (as seminiferous tubule connects with rete in ); 6, medullary cord; 7, albuginea. X 20. Fig. 14 Cross section through middle of gonad of 21.5 em. free-martin, T2. 1, sex cords resembling medullary cord; 2, rete; 3, sex cords resembling semi- niferous tubules; 4, Wolffian duct in fold of peritoneum overhanging gonad; 4, rudiment of Wolffian body. X 20. Figs. 15 and 16 Cross sections through gonad and Wolffian body of 22.5 em. free-martin, T4. 1, Wolffian tubules (epodphoron) ; 2, rete; 3, sex cord; 4, Wolf- fian duct in fold of peritoneum overhanging gonad; 4, sex cord connected with rete. XX 20. SYSTEM OF FREE-MARTINS 473 REPRODUCTIVE A474 CATHARINE LINES CHAPIN In the 20 em. free-martin T13, the medulla, in cross section, is shaped like the cortex and medulla of a much smaller, normal female, 7.3 em., N8 (fig. 13). In size, the gonad is like the ovary of an 8.3 em. female N7 (fig. 3). Very rarely one finds a germ cell. enclosed in a primary follicle. In case 2, a few of the medullary cords (fig. 14), have a very definite arrangement of cells, like that of the seminiferous tubules. Such an arrange- ment, in a larger number of cords (in an older individual) is doubtless what D. B. Hart® found and illustrated. It was partly because of this that he interpreted the free-martin as an abnor- mal male. Occasional gérm cells are found. In case 4 connec- tions between the rete and the medullary cords can be seen (fig. 16). It should be noted that such connection is not found in the normal female, but that in the male the rete is connected with the seminiferous tubules, which are homologues of the medullary cords. No germ cells were distinguished. The Wolffian body in all three cases has degenerated, with the. exception of the part which forms the epididymis. In case 13 there are several connecting tubules between the rete and an ~ enlargement of the Wolffian duct. The Wolffian duct extends straight anteriorly for some distance. Posterior to the vasa efferentia which are straighter than those of the epididymis of a normal <, the Wolffian duct ends in a posterior enlargement. In the region of the uterus, the Wolffian duct again appears. Here, the duct is not continuous. In places it is represented only by a cord of cells; in other places there is a lumen through this cord. In no place does it have the ciliated lining which is characteristic in the normal ¢@. The tube of the Miillerian duct in T13 is lacking, except for a very small portion, the lumen of which is not more than 0.025 mm. long, figure 12 (cf. f-m. 26, page 464). The anterior part of the uterine horns is present, the middle part is lacking and the most posterior part is present but very small and not like the typical Millerian duct. In case 2, the epididymis consists of several vasa efferentia. The Wolffian duct is complete. The more convoluted part lies in 5D. B. Hart, Proc. Roy. Soc. Edinb., vol. 30, 1909-10. REPRODUCTIVE SYSTEM OF FREE-MARTINS 475 the fold of peritoneum which overhangs the anterior part of the gonad (fig. 14). This position of the Wolffian duct is character- istic of the male. As for the Miillerian ducts, the tube is en- tirely atrophied. The horns of the uterus are present and like those of a normal female, in histological structure, but much smaller in diameter. However, in the preparation studied, they do not unite, although they were sectioned posterior to the point at which they normally unite in the female. In the 22.5 cm. free-martin T4, the epididymis and anterior part of the Wolffian duct resemble those structures in free-martin 2. The convoluted portion of the Wolffian duct (figs. 15 and 16). lies in the fold of peritoneum overhanging the gonad. Poste- rior to the gonad, the Wolffian duct ends in an enlargement. The caudal part of the Wolffian duct is present and, as in case 13, the lumen is discontinuous. The entire tubular portion of the Miil- lerian duct is atrophied. In the location where the anterior part of the horns of the uterus are found, there is a large structure which may be a rudiment of one of them. It has a typical, epi- thelial wall and is about the diameter of the normal uterine horn. It is closed at both ends, having no connection with any other structures. Lateral to the posterior ends of the Wolffian ducts are coiled tubules, one on each side. These are presum- ably the anlage of seminal vesicles which develop in the male from the Miillerian ducts at their posterior end. Summary. The gonads of the free-martins measuring 20 cm., 21.5 em. and 22.5 em. resemble the testis in the entire absence of cortex, the appearance of the albuginea, peritoneum and rete, and the connection of the rete with the Wolffian duct through the Wolffian tubules. They resemble the ovary in the position of the rete and in the structure of the medullary cords, with the excep- tion of the few sex cords in T2 which have the appearance of seminiferous tubules. T2 resembles the male in the condition of both ducts. T13 and T4 resemble the female in the partial atrophy of the Wolffian duct and resemble the male in the atrophy of the oviduct, and non-union of uterine horns. 476 CATHARINE LINES CHAPIN 5. FREE-MARTIN 28 CM. T12 The largest free-martin embryo studied measured 28 em. (Lillie, figs. 27A and 27B). The male (N19) and female (N28) sectioned for comparison measured respectively 31 em. and 29.5 cm. a. Conditions in the normals of this stage These specimens differ so little from the smaller normal em- bryos that they will be described but briefly and illustrations seem unnecessary. In the male, the seminiferous tubules are even more contorted than those of T4, and slightly larger. The nuclei, in general, are arranged along the periphery of the sex cords but no lumina are as yet formed. ‘The interstitial material is about equal in volume to the sex cords and contains many interstitial cells. The germ cells which are found in the growth period are even more numerous in N19 than in T4. Most of them are in the later stages of the growth period (i). Otherwise the structures in the 31 cm. male are like those of the 24 cm. #7. In the female also some advance may be noted. The medulla is made up more largely of connective tissue stroma, and many more of the medullary cords are degenerating. At the inner ends of the cords of Pfliiger are found many ‘nests’ of cells some of which are enclosed by more than one follicular layer. Many germ cells are in the growth period, those in the follicles being more advanced than the others. The germ cells in the follicles are in Schoenfeld’s ‘stage 1 while most of the other germ cells are in stages d, e, etc. No maturation or odgonial mitoses are found. The interstitial, connective tissue stroma is vol- uminous. A few cells in it look like young interstitial cells, but have not been identified with certainty. The albuginea is a little more advanced than it was in the 23 cm. @. The rete extends less than half way through the ovary and has no con- nections with the medullary cords. The Wolffian body, and Wolffian and Miillerian ducts are in the same condition as those of the 23 em. 9 N25. REPRODUCTIVE SYSTEM OF FREE-MARTINS 477 b. Condition in the free-martin The gonad of the 28 em. free-martin T12 measures 3 mm., in comparison with the 8.260 mm. testis of N19 and the 7 mm. ovary of N28. The rete is the most prominent feature of the gonad. It is much longer than the gland, extending posterior to the sex cord region as well as anterior to it. The diameter of the rete is about one-half the diameter of the entire gonad. The rete lies at one side of the gonad, as in the female, sur- rounded on three sides by sex cord region. The rete cords are very numerous and have large lumina. The sex cord region shows no evidence of organization. It seems to be a homogeneous mass of connective tissue, with no differentiation into cords. A few cells are seen which have large, round nuclei resembling those of germ cells (indifferent stage or Schoenfeld’s stage spe. a.) but they do not have the definitely limited cytoplasm char- acteristic of germ cells and probably are not germ cells. No interstitial cells are distinguishable. The thin epithelial covering of the gland and the compact albuginea lying directly beneath it are typical of the male. The Wolffian body is represented only by a few tubules, the epodphoron, ete. The Wolffian duct is absent. The anterior portion of the Miillerian duct, the oviduct, is absent but the horns of the uterus are present. Summary. The 28 cm. free-martin resembles the male in the absence of cortex, condition of the albuginea and superficial epithelium, extent of the rete, atrophy of the oviduct and pres- ence but non-union of the uterine horns. It is like the female in the position of the rete, degeneration of the Wolffian body, and atrophy of the Wolffian duct. 6. FREE-MARTIN TWENTY-ONE DAYS AFTER BIRTH Besides the free-martin embryos studied, the gonad of a free- - martin killed twenty-one days after birth was sectioned (T42). The gonad measured about 13.5 mm. in length and 5 by 3 mm. in cross section at its largest point. Data concerning the size of glands of normal calves is not at hand. 478 . CATHARINE LINES CHAPIN The medullary region in this free-martin is relatively larger than in the other free-martins studied, and more organized. Almost the entire region is differentiated into sex cords, sepa~- rated by connective tissue stroma. Some of these sex cords resemble the seminiferous tubules, as do a few sex cords in free- martin T2; others resemble the medullary cords. Degenerating follicles are found but no germ cells. Interstitial cells have not been identified in the stroma. The superficial peritoneum and albuginea are like those structures in the male. Again there is complete absence of cortex. The rete resembles the rete testis in structure, relative size and distance of penetration into the gonad, but, lying at one side of the sex cord region, near the mesentery, it resembles the rete ovarii in respect to its location. There is a large amount of rete anterior to the gonad in the position which, in the male, is occupied by the epididymis. This resemblance to the epididymis is still more marked because in the region where the vas epididymis leads from the vasa efferentia into the vas deferens, are rudiments of three ducts. These ducts are lined with ciliated epithelium but are closed at both ends and, although the walls of tubules from the rete come in contact with the walls of these ducts, in no place do they actually open into each other. However, this mass of rete, which resembles epididymis in position does not resemble it in structure, being, in that respect like the typical rete testis. The Wolffian body has degenerated, but lateral to the gonad are many tubules. Among other structures are found large blood vessels like those of male T4 (fig. 9). Summary. The free-martin T42 resembles the male in ab- sence of cortex, presence of albuginea, structure of the super- ficial peritoneum, and structure of the rete. Some of the sex cords are like medullary cords; others are like the seminiferous tubules. The position of the rete in the free-martin is that of the rete ovarii. TABULAR SUMMARY OF CONDITIONS OF SEX ORGANS OF BOS EMBRYOS, INCLUDING NORMAL MALES, NORMAL FEMALES AND FREE-MARTIN ~ -' GONAD MULLERIAN DUCT x Germinal 3 Arca cross | Sexcords, lot sot Cords of Pager | Interstitial material Germ,colls epithelium Albuginen Reto Wolffian body. Wolffian duct Tube Horns of uterus_| Body of utorus - Seanty conneative tis- | Indifferent Absent Thin, compact Enters at anterior | Larger than testis in | Complete mesoneph- | Complete, except for | Present Not formed in io Soe ee eae ond of testis, figure | cross section and| roticduct, Nocon-| ostium abdominale zl amr 1; docs not extend | much longer. No | nection with reto far posteriorly degeneration. No formation of opi- didymis as 35 | 6Xfm. | Distinct, branching, More than in N10 ? (Preservation poor) | Absent Compact Extends farther into | Cross section = con- Complete Complete, except for | Present No union of horns 4 no lumen tral cross section ostium abdominals of uterus of testis. Dogonor- ted with glomeruli} ation has begun; of W. b also formation of epididymis a | —|- xem. | Medullary cords, not | Present, short, little | Connective tissue | Degenorating or in-| Wide Cord of | Primary albugi-| Enters anterior end | As large as ovary in | Complete Complete, including | Present Horns united to : yet dogenerating. | differentiated from | stroma only differont in me-| Pfdger still at- | nea presont of ovary. Lies near | cross section. De- ostium abdominale form body of Figure 3 ‘one another or from dulla;indifferentin | tached mesontery, in me-| generation has be- uterus germinal epithelium cortex dulla gun but has not gone far. Glom- eruli fewer than in & or free-martin b: 1azem. o | Medullary region | Absent Absent Present, but vory | Absent! Wido Enters anterior end | Larger than gonad in Irregular, discontinu- | Not sectioned 1/3 8.3 om. | small, Not differ few. Growth stage of gonad, Figure 4, | cross section and ous : 9 entiated into cords. a, or indifferent Position like that | in length. No de- Figure 5 in 9. Cords with | genoration as yot. lumina not formed. | Glomeruli numer- Figure 5 ous @Xfm. | Branching tubules, Thin connective tis- | Many indifferent. Absent ‘Compact Runs almost entire | Much smaller than | Continuous with | Absen Not sectioned No lumina. Cells sue stroma. Some in early testi cross sec-| Wolffian tubules beginning to lie growth stages a-d tion, Degeneration | which form epi- with nuclei near has gono far. | didymis periphery of cords with seminiferous Glomeruli reduced tubules in size, Cells in lumina of tubules, Epididymis formed st anterior end, W. tubules, con, Ss with rete, contorted aa xm, | Medulla relatively | Cortex relatively larg- | Connective tissue | Majority indifferent. | Present Indi+| Primary albugi-| Lumina in cords dis- | Glomeruli still pres- | Atrophied Complete. Lies close | Complete. Large | Formed by union , smaller than in 8.3 er than in 8.3 em, stroma. A veiy In deoper Jayer of | tinguishable| neapresont, Sug-| tinct ent in posterior to ovary in diameter of horns. Wall 9. Cords contain | 9. Cordsdistinctly | few cells ure sug-| cortex, some pri-| from cords of | gestive of defini- part. No large W. thick and mus- primary follicles separated from each gestive of young in- mary spermato- PAlger tive albuginea tubules like those cular other by connective torstitial cells cytes in early of 7.3 cm. 9, NS tissue stroma growth stages. and 8.3 cm. 9 N7. Odgonial mitoses Signs of degenera- f tion-cells in lumina = of tubules 1.735) Veo Anterior part of med- | Absent Absent Preeent; few Absent Presont, narrow | In position of rete | Smaller than W-. b, | Anterior part ends | Absent, except for | Not sectioned wag ulla little differen- ovarii, Rete cords| of 11.2 cm. and| anterior to gonad.| small portion (ho tinted. Medullary 5 connected with | 12.75 cm. co s,| (Posterior part not| mologous with cords found in pos- some medullary | Much degenerated, | sectioned) hydatid morgagin) terior part, Figure 6 cords as with semi- | asin 9 7 niferous tubules in the o. Wide, composed of indiffor- | Absent Wide and com-|Large in diameter. | Bpididymis large, | Complete Atrophied. Lillie, fig- | ? connective tissue primary pact Cords much branch- Posterior part of ure 1 | stroma and some in ed. Possess lumina, | W, b. much de- interstitial cells stages Connect with sem- | generated iniferous tubules ‘Medullary cords pres- | Still attached to | Connective tissue. No | Majority indifferent. | Present Primary albugi:| Present—less exten- | Degeneration has | Present continuous as | Complete. Ostium | Present Present, wall thick ent. Connective | germinal epithe- | _ interstitial cells Some rimary: nea wide sive than in @ gone farther than | far as sectioned large, wall becom- tissue stroma vol-| lium. A few nests odcytes in early in @. Fewer glom- ing convoluted uminous of cells formed at stages eruli than in 14 em, inner ends of cords g Anterior part of me- | Absent. Figures 7 | Absent In primary follicles | Absent Compact, rela- | Relatively large. Lu- | Degeneration has | Present anterior to | Atrophied. ie, fig- | Present; small Horns of uterus Gulla small; poste- | and 8 in medullary cords, tively as wide | mina of cords dis | gone far gonad. Presentin | ure 16 diamoter united for 1.905 rior larger—2 X area Some degenerating as that of ot tinguishable in an- region of horns of mm. cross section of rete, terior part. Posi- uterus, but discon- Fow cords distinct tion of rete ovarii tinuous Figure 7 Seminiferous tubules Primary spermato- | Absent Wide, compact Extends almost to | Degeneration except | Complete Atrophied Not sectioned many and branched cytes in growth posterior end of tes- for large opididy- stages a toe tis. Smaller than in mis, ‘ v4 Seminiferous tubules | Figure 10 ‘As in N2I, or more | Primary spermato- | Absent Wide, compact Large in cross sec- | Epididymis very vol- | Complete Atrophied Not sectioned longer and more voluminous. Area | cytes in growth tion, Tubules much | uminous, W. tu- branched than in in sny section =| stages a to f, No bules composing it, N21, No lumina, area semi mitoses mush —_ contorted. Cells arranged with tubules ous tubules Figure 9, See 26 nuclei near periph- section cm. io, fig~ ery of cords ure 12 14a T4 | Cords degenerating. | Separated by stroma. | Much interstitial ma- | Primary odeytes in | Present Primary albugi-| Smallerthaninmales, | Mostly degenerated, | Present, but very | Largo, Ostium large | Presont Thick walls, mus- Medulla composed | Primary follicles ut but no inter- | growth stiges up nea, Suggestion] Luminalarge. Con- | Tubules of epo- | small in dinmeter cular mainly of connoc- | inner ends of cords; cells to stage i of definitive al-| nected with gonad | ophoron and paro- tive tissue stroma more numerous buginea and with W.b ophoron remain than in 17 om. 9 N23 2.0 | @ | N25) 6.83) 1/4. TA | Few medullary cords | As in 20cm. 9 As in 20cm. 9 Asin 20cm, 9 Prosent Asin 20cm. 9 | Extends on As in 20m. ¢ Atropbied Complete. Ostium | Present Thick walls, mus- = 9 NUM presont, Figure 11 distance large. End of tube cular ovary curves around end of ovary 20.0 | fm. | T13 | 2.175] 1/6c%,T4 | Some distinct cords, | Absent. Figure 1% Tnterstitial cells ab- Absent Wide, compact Much degeneration. | Rudimentary. Ends | Suggestion of hyda- | Reduced No union of horns Some seem to be sont, Connective No glomeri i tid. Figure 12 com- connected rete. tissue stroma pres- Three W. tubules plete with 12.5 om, Medulla, 3 X cross ent connective tissue connecting W, duct f-m. T26 section rete and rete represent epididymis 21.5 | fm.| T2 | 2.85] 1/6c',T4 | Medulla large. Many | Absent. Figure 14 ‘An nbove A few present Absent Wide, compact Large. Many cords. | Epididymis repre | Complete. Position | Absent Reduced. See | No union of horns medullary —_ cords. Lumina distinct, | sented by a few | of W. duct in nor figure 20A (Lil- Somo which re- Not separated from | tubules (moro than | mal go, Figure 14 lie) Cf. figures 7 somble seminiferous rete, as in T13 in T13). Rest of and § (Lillie) for tubules of ot. Wolffian body de normals e generated 22.5 | f-m.| T4 | 2.205] 1/8 ot, TA Figures 15- | Connective None present Absent Wide Connected with me | Epididymis — repre- | Complete region | Absent Represented by | Contorted ducts, = stroma dulls cords, in a| sented by fewer | of gonad and in interstitial cells few cases. Large. | tubules than in| region of uterus. resombling degen- Many cords. Lu-| TI3, Rest of W. Discontinuous be- erating medullary mina distinct body degenerated tweon == aa 31.0 | co | N19) 8.26] 4 fm, | Much contorted. No Interstitial cells nu- | Many in growth | Absent Wide, compact | Many tubules lined | Degenerated, except | Complete Absent Not sectioned 7 lumen. Nuclei of merous i with columnar ¥, cells, near periphery epitholium < 20.5 | @ | .N28/7.0 | 25% fm. | Cords mostly degen- | Many follicles grow- | Stroma. A few Thick Primary presont. | Extends Jess than 4 ‘Atrophied Large Present Prosont erated, Follicles de- | ing may be intera Definitive grow-| way through gland. generating cols most advanced, i; ing Smallor in diame- | ophoron and paro- those in cords, d ter than that of | ophoron ps =A) eo) ee and © Som. ot 8.0 | (-m.| T12] 3.0 1/4 | Medulla not differon- | Absent Not distinguishable | A fow in indifferent | Absent Wide, compact | Prominont. Longer | Ropresonted by a few | Atrophied. Li Atrophied Present No union of horns EM) tinted into sords stage oF growth than medullary re- | tubules ures 27A, stage a? gion, In_ position — | tJ of rete in 9 Killed | ‘Ta2 [13.5 | Average di- | Modulla relatively |” Absent No interstitial cells | None Absent Like o } cross section of | Rete extends into r- a ameter| largo and well dif- gonad. Resembles | gion of epididymis eae esvesemeraalt, rerenuated 9, bilo, @. in structure; | of ci. Rudimon- ( Not examined). ) after tion 4mm. cords, some of which @ in position tary vasa offeren- birth resemble seminifer tio—3 ous tubules. Degen. crating follicles A ay oh HE Peeler, a — 0g anny | ea eae it phe CHET | ee. Gh miei | soludys ‘suoaiictoadll smim base asaaol . ni wedtt bedaaed -egiauit OVE. eK dihw bayserts alivO -dyemg mon isfosse PD on oe amos 7 boiewmdel Se, esloiflo? scrawzixd jabres lo zhao totiel lin02o mu @ Ane. 9.109 vt ay pos? :. eae +r pats | ere eee qb ed pat Dnt germ (au to rere 423 aero “wa, IND UeahtE: REPRODUCTIVE SYSTEM OF FREE-MARTINS 479 SUMMARY AND CONCLUSIONS . The conditions found in foetal free-martins described in the preceding pages, show the effect of the introduction of intersti- tial secretion from the male embryo into the circulation of a female embryo. The possibility of such an introduction of interstitial secretion and its effect upon the secondary sex characters has been fully discussed by Professor Lillie in his paper which pre- cedes this. There follows a summary of the conditions result- ing from the introduction of the interstitial secretion of the ¢# embryo into the @ embryo. The sex cords present in the free-martin are homologous with the medullary cords of the normal female and with the seminif- erous tubules of the male. In some specimens the sex cord region is simply an unorganized, homogeneous mass of cells. In the majority of embryos in which the cords are differentiated from each other, they resemble the medullary cords of the female, but in a few cases some of the cords exhibit an arrangement of cells ike that of the seminiferous tubules. This latter con- dition is found only in free-martins in which the sex cord region is large, compared with that of other free-martins. In no free-martin are sex cords ever formed which correspond to the second set of sex cords of the female, the cords of Pfliger. The latter are characteristic of the female and have no homologue in the male. The proliferation of the cords of Pfliiger from the germinal epithelium marks the beginning of differentiation from the indifferent stage to the female condition. In the male, - apparently all the primitive germ cells are carried down into the gland with the first proliferation of the germinal epithelium, leaving as the surface covering of the testis, only the thin meso- thelial peritoneum from which the cords are soon separated by a compact layer of connective tissue, the albuginea. In the fe- male, relatively few of the primitive germ cells enter the medul- lary cords. The primary albuginea, an irregular layer of loose connective tissue cuts these cords off from the germinal epithe- lium which is still a thick layer composed largely of cuboidal epi- thelial cells, and containing also many primitive germ cells. 480 CATHARINE LINES CHAPIN The cords of Pfliiger are formed by a second proliferation of the germinal epithelium, from which they become separated by the development of the definitive albuginae, Just making its appear- ance in the older normal female embryos studied, 23 em. and 29.5 em. In the free-martin, but one set of sex cords is formed. The primary albuginea becomes a compact structure like the tunica albuginea of the male. Instead of the germinal epithelium of the normal female, the gonad of the free-martin is enclosed by a thin layer of flattened epithelial cells like the superficial peritoneum of the male. - The rete is present in the indifferent stage and in the early stages of both sexes, after differentiation. In the male it con- tinues to grow larger with the testis. In the female, it gradu- ally diminishes until in a 29.5 em. @, it is of comparatively small extent. In the free-martin the rete continues to grow large, as it does in the male, sometimes to the point of becoming even larger in a certain free-martin than in a male of corresponding size. In the youngest embryos studied, 7 and 8 cm., the Wolffian bodies differ in the two sexes chiefly in the larger size and num- ber of Wolffian tubules of the male. The condition in the free- martin is like that in the male. In larger free-martins the Wolffian body is variable. In some, vasa efferentia connect the rete with the Wolffian duct as in the <’, in others, there is no such connections, the Wolffian body having almost entirely degenerated as in the female. In some free-martins the Wolffian duct undergoes complete atrophy, or at least atrophy of the entire anterior part. (Gonad to region of the body of the uterus.) In others it is present an- terior to the gonad and is connected with the rete by vasa effer- entia, as in the male (no such connection is ever established in the female). In some of these free-martins the Wolffian duct ends blindly, anterior to the gonad. In others it ends posterior to the gonad and in a few, is complete. In the latter two con- ditions it lies in a fold of the peritoneum overhanging the gonad just as it does in the male. REPRODUCTIVE SYSTEM OF FREE-MARTINS 481 The Miillerian duct becomes irregular in young free-martin embryos as in young males. In older free-martins the tube has atrophied. The posterior enlargement may persist. This is homologous with the uterus in the normal female and the uterus masculinus or prostatic vesicle in the male. The two Miillerian ducts unite in the female to form the body of the uterus. In the male they do not join. In only one free-martin were they found to unite—T6—and in that case the union was for a distance of less than 2mm. The failure of these ducts to unite to form the body of-the uterus is in accord with Paton’s® finding of non- development of the uterus in castrated female guinea pigs. In two free-martins, 20 cm. T13 and 12.5 cm. T26, a rudiment of the Miillerian duct is found near the anterior end of the gonad. This is probably the homologue of the hydatid of Morgagni, a vestige of the Miillerian duct which persists in that region in the male. From the foregoing facts it is evident that, 7. As a result of the introduction of the interstitial secretion of the male, those organs in the free-martin which are present in the indifferent stage, de- velop toward the male condition (rete, first set sex cords, primary albuginea), and those which develop in the normal female at sex differentiation or later are inhibited from developing (cords of Pfliiger, definitive albuginea, union of Miillerian ducts to form uterus). 2. The high degree of variation found in the organs of the re- productive system in free-martins is indicative of the variability of the time at which the interstitial secretion of the male embryo may first be introduced into the circulation of the female embryo, and the amount which may be introduced; in other words, the variability of the time and degree of anastomosis of the extra embryonal blood vessels of the two embryos. a. The fact that in some free-martins the Wolffian body and Wolffian duct have degenerated more than in a male of corre- sponding size, though not more than in the female, suggests a later introduction of the secretion of the male, or the introduc- 6D. N. Paton, Regulators of Metabolism. 482 CATHARINE LINES CHAPIN tion of a smaller amount, allowing some development toward the female condition. This is also suggested by the partial union of the horns of the uterus in the 16.3 em. free-martin T6. b. The arrangement of cells in the sex cords of the free-martin as they are arranged in the seminiferous tubules of the male and the relatively large size of the sex cord region suggest an early introduction of the male influence, made before the beginning or early in the process of degeneration of medullary cords which normally take place in the female, or the introduction of a sufficiently large amount of male secretion to inhibit female development completely and to cause development toward the male condition. The development of the Miillerian ducts of the 22.5 em. free-martin, T4, into contorted seminal vesicles of small diameter suggests that the male hormone was introduced into the female earlier or in larger amount in this case, than in the 28 cm. free-martin, T12, in which the Miillerian ducts are still straight ducts of large diameter. BIBLIOGRAPHY ALLEN, B. M. 1904 Embryonic development of testis and ovary of mammals. Am. Jour. Anat., vol. 3, p. 89-141. Hart, D. Berry 1909 Reproductive organs of the free-martin. Proc. Roy. Soe. of Edinb., vol. 30, 1909-10. Linuiz, F. R. 1916 The theory of the free-martin. Science, N.S8., vol. 43, pp. 611-613. 1917 The free-martin; a study of the action of sex-hormones in the foetal life of cattle. Jour. Exp. Zo6l., vol. 23, pp. 371-452. MacCauuum, J. B. 1902 Wolffian body in higher mammals. Am. Jour. Anat., vol. 1, pp. 245-260. Paton, D. N. 1913 Regulators of Metabolism. MacMillan & Co. ScHoENFELD, H. 1901 La Spermatogénése chez le taureau. Arch. de Biol. T., 18, pp. 1-64. WHITEHEAD, R. H. 1904 Development of the interstitial cells of Leydig. Am. Jour. Anat., vol. 3, pp. 167-182. The literature on the free-martin, and on the interstitial gland and the effect of its secretion is included in the bibliography of the preceding paper by Prof. F. R. Lillie. AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE BIBOLIGRAPHIC SERVICE AUGUST 6. MICRODISSECTION STUDIES II. THE CELL ASTER: A REVERSIBLE GELATION PHENOMENON! ROBERT CHAMBERS, JR. Cornell University Medical College, New York City ONE PLATE I, INTRODUCTION 1. Historical The periodic appearance and disappearance of the aster in the cell, the very definite structure which it offers to the eye, and its very evident relationship to cell division make it one of the most striking phenomena in cell protoplasm. Any idea which one may advance regarding its structure is necessarily based on a conception of the structure of protoplasm. There can be, therefore, as many interpretations of the astral structure as there are theories of the physical make-up of pro- toplasm. Taking for a basis the reticular theory of protoplasm, the astral rays have been considered fibrous strands whose radiate arrangement is produced by being drawn together at a point in the protoplasmic fibrous network. In accordance with Biitschli’s foam theory of protoplasm the aster has been explained as an arrangement of the protoplasmic alveoli into more or less definite rows radiating from a common center. The experimental work of Morgan (’08, 710), Lillie (09), Conklin (12), and others on the centrifuged eggs has shown conclusively that whatever be the structure of protoplasm the mitotic spindle at least is of such a consistency that it preserves its structure on being driven through the cell substance. The 1 This paper was read before the American Society of Zoologists, New York City, December 27, 1916. It is based on the work done during the summer of 1916. The writer wishes to express his indebtedness to Prof. H. V. Neal, for accommodation and facilities accorded him at the South Harpswell Laboratory. 483 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 3 auGust, 1917 484 ROBERT CHAMBERS, JR. astral radiations often persist about the poles of the spindle being apparently dragged along with them (Spooner 711). F. R. Lillie (09), suggests that the aster about the poles of a dis- located spindle is a new configuration, and due to forces (see Hartog ’05), which are focussed in the centrosomes and influence the cytoplasmic granules in situ. A significant phenomenon is the occasional occurrence of distortion of the astral rays not only in centrifuged but also in normal eggs (Mark ’81, Coe 99, Conklin ’02, and Painter ’16). Investigators generally agree in considering the distortion to be a proof for the existence of more or less extensive movement in cell protoplasm and some conclude that the rays may be firmer in consistency than the surrounding cytoplasm. In a recent paper Heilbrunn (’15) describes in the cytoplasm of sea urchin eggs a gelatinization produced by chemical agents which cause the eggs to undergo parthenogenetic development. While investigating the structures of various marine ova by micro-dissection, the author found it possible to detect changes in the consistency of the protoplasm during aster formation and to demonstrate that during cell division we meet with definite reversible gelation phenomena. 2. Material and method The microdissection method, first introduced for the study of protoplasmic structures by Kite (Kite and Chambers, ’12), affords means of collecting direct evidence of the physical con- sistency of many constituents in the living cell. The apparatus used for holding the dissection needle is the mechanical pipette- holder of Barber. One specimen only; accidentally lost before being identified. THE COLORATION OF REEF FISHES 549 one’s shoulders, and air pumped into it through a hose reaching a boat above continually replaces that which is exhausted and escapes in turn below its rim. A total weight of about 80 pounds is necessary to steady the diver in 15 to 20 feet of water, where excursions may be made up to at least half an hour, and probably much longer, without discomfort. For the prosecution of a research such as the present the hood has all the advantages and none of the objectionable features of a regulation diving suit. With a wax-coated slate and sharp-pointed nail ample records may be kept and transcribed at leisure. Some of the fishes one encounters when diving are so little afraid that they will almost feed from one’s hand. Hence, even minor changes in their coloration may be observed to advantage. This encouraged an attempt at submarine photography which was at least as successful as might reasonably be anticipated when working under novel conditions with untested apparatus. A No. O Graphic camera, taking a picture 22 by 13 inches, was enclosed in a brass box, windows in which permitted the use of the finder, while the necessary manipulation of shutter and film was accomplished by means of a screw and plunger protected by water-tight packing. Since the lens was of uni- versal focus adjustment for distance was unnecessary. Some of the pictures secured are reproduced herewith, and serve to illustrate the striking changes in pattern which the fishes un- dergo, and suggest also how slight ground there is for consid- ering the animals conspicuous. Much remains to be done, but in the light of present experience it is apparent that adequate exposition of the facts should be possible by the photographic method. Regarding color change it seems perfectly safe to go much farther than Townsend (710), who notes that his fishes showed more color phases as their surroundings were more varied, 1.e., that their environment played at least a subordinate part in determining their aspect; farther even than Mast (’14), who states his conviction that adaptive color changes in fishes occur not only in marked degree, but that they are rather widespread. There is, indeed, within the limits of my own experience almost 550 W. H. LONGLEY no evidence that in nature any other factor than the color and shade of the surroundings exercises notable control over those changes in coloration which occur by day, modifications in pattern excepted. Fishes in tanks, it is true, often show color phases which baffle interpretation, but when they are unconfined this is not commonly so. This suggests that in the former case the behavior is not entirely normal, and two sets of observations lend some support to this conclusion. In the first place, the whole behavior of some species seems to be modified by restraint, to which they do not become accustomed in weeks of confinement. It is also known that in some animals processes underlying growth, secretion, etc., are directly affected by comparatively slight disturbances in their environment. The rate of growth of rats, for example, is distinctly modified, if unfamiliar operations are carried on by strangers in or about the creatures’ quarters. That is to say, modified color reactions may be anticipated with reason in fishes in confinement, since they sometimes show its effects plainly in other ways, and since, in addition, in other animals processes apparently no more complex than those con- cerned in adaptive color changes are inhibited by no more evi- dent stimuli, and without more marked departure from normal behavior in other respects. But wholly apart from complications which might originate in disturbance of organic function through confinement, there is a possible source of error in experimentation upon fishes under such conditions. This lies in the fact that the color- complex in which most captive fishes find themselves, only remotely resembles that in which they normally occur, and to which they are able to adjust themselves accurately. But when to match a given background color is wholly impossible, and there is bound to be maladjustment in any case, much greater variation is to be expected in one fish at different times, and in different ones at the same time, than if a stereotyped response well within the creatures’ capacity were demanded. Finally, exhibition tanks such as those in the New York aquarium unquestionably fall under the head of mixed environments, in THE COLORATION OF REEF FISHES 551 which, even under the most favorable conditions, the least intelligible results are obtained. Therefore, when all is con- sidered, the surprising thing is not that the significance of color change should have been imperfectly understood, but that its meaning should have been grasped at all. Except as banded patterns sometimes appear upon fishes as they come to rest, unconfined specimens give one little reason to believe that psychic states, special activities, or uncontrolled internal stimuli determine their color reactions to any appreci- able degree. The following records are rather exceptional. A hogfish (L. maximus) feeding in the gray phase over light colored bottom is noted as flushing momentarily and then returning to and remaining in its previous condition. Similarly a Nassau grouper (E. striatus) lying in a dark phase in the shadow of a large coral head came into the open over clear sand (fig. 3), turned pale as it crossed it (fig. 4), and darkened somewhat (fig. 5) as it commenced to feed upon the half of a crawfish (Panu- lirus argus) which had been provided for it. Such irregularities in behavior seem inconsistent with the impression conveyed in the preceding pages, but they are apparently mere swirls upon the great current of evidence, and move on with the tide they seem to oppose. Flounders adapted to black or white bottom will reverse their coloration completely, while the whole body rests upon the color to which it conforms, if the head is upon the other (Mast, 714). Sparisoma flavescens reacted for me in the same way in a tank with a slate bottom, half of which was covered with white sand. It was observed repeatedly swimming slowly toward the white, where for a moment its passage was impeded’ at the border. Then, with only a part of its head across the line, it assumed in full the color and pattern it regularly showed over the light material before it. The whole system of chromato- phores in the most changeable species seems to be in a state of highly unstable equilibrium. Hence, dominance for even an instant by a dark object in which interest might be centered for the moment might very well induce the exact aberrations which have been noted in the preceding paragraph. Ty W. H. LONGLEY How largely independent of activity, ‘states of mind,’ and internal stimuli the color phases are, may best be illustrated by reference to specific instances. On July 28, 1915, I broke open a large, long-spined sea urchin (Centrechinus) over clear sand in a small bight surrounded by large coral heads, which were at no place within 8 or 10 feet of the bait. A small hogfish about 10 inches long came and circled about many times, for the broken test was lying spines upper- most, so that it could get nothing, although smaller fishes could go under and feed from the inside. Finally it found a detached spine which it picked up and masticated base foremost. It remained consistently in the gray phase throughout the whole period during which it was under observation (fig. 6). I next moved to a dense gorgonian patch 3 or 4 feet in diam- eter, and placed food beneath the brown branches of the clustered colonies. Within a few minutes a larger hogfish, 18 or 20 inches in length, went in under them and displayed an almost uniform. brown color, or, as it moved about, replaced this by a mottled pattern of rich reddish brown and gray (fig. 7). When it was driven out over the open bottom, it turned gray, and swam off in that phase with its mouth full of food. It went under gorgonians nearby and turned brown for the second time and finally swam away low down over mixed bottom in its mottled livery. Coming back after a little, it approached the original station, ate and swam about in brown phases. Then a broken Centrechinus was placed about 10 feet away on bare sandy bottom; the fish was driven out to it with a long-handled dipnet, and assumed its gray phase in the open. The food was moved back and forth a number of times, and color and pattern were changed regularly with each significant change of position, but neither variation in activity, nor any incipient alarm engendered by the repeated seizure and removal of its food seemed to have any effect in modifying the fish’s appearance. The facts re- garding the color changes of the other species mentioned in table 1 are perfectly comparable with these and seemto point to only one rational conclusion. Whether or not a familiar object minus its color might by association induce a color change appropriate to its normal but THE COLORATION OF REEF FISHES es unsuited to its modified condition seemed worth investigating. The turtle grass (Thalassia) provided suitable material for mak- ing the test. The rootstock of the plant is usually deeply im- bedded in the sand. The leaves spring from short offshoots, and for half their length, on the average, are not exposed to the light. Their buried portions are white, therefore by cutting off the green parts and making shallow plantings of the basal seg- ments it was possible to manipulate the materials in a small tank, so that the fishes were exposed to two sets of surroundings, whose essential difference was in color. © In the white grass Iridio bivittatus, Monacanthus hispidus, Siphostoma mackayi and Sparisoma hoplomystax all reacted as they did over bare sand, except that one young Siphostoma was even lighter in color than in that case. To the green they responded as indicated in table 1. Hence color changes in fishes appear to be induced directly by the color of surrounding objects,® and not indirectly through suggestion derived from familiar forms. There is, therefore, nothing here to lend col- lateral support to such hypotheses as that of Steinach (’01), re- jected indeed by Cowdry (11), that the chromatophores in the octopus are controlled by reflexes from the suckers, and that, as a consequence, the texture of the bottom, rather than its color, determines the color changes that occur. At this point one should refer for a moment to an idea one frequently encounters, and which seems in fair way to become an article of faith in the matter of animal coloration. The reasoning upon which it rests is wholly illogical, as the reader will observe. Sometimes it is simply affirmd ex cathedra: ‘Absence of movement is absolutely essential to protectively colored animals.’ (Beddard, ’92, p. 90.) . Sometimes it is stated with some attempt at justification: ‘““No color whatever could make a flying butterfly invisible to its enemies, because the background against which its body shows is continually changing during its 6 In view of what is known of the path of nerve-impulses, the fact that the fishes are able to match different colors proves, of course, that they possess color vision. The point has already been made by Mast (’14), but the demon- stration of adaptive color changes in additional unrelated species emphasizes a conclusion that has been commonly mistrusted. 554 W. H. LONGLEY flight, and, moreover, the movement alone is enough to betray it, even if it is of a dull color.” (Weismann, ’04, p. 74.) ‘‘No ob- server of Nature can have failed to remark how the least move- ment on the part of an animal will betray its whereabouts, even though in color it assimilates very closely to its environ- ment. . . . Thus in order that protective coloration may be of use to its possessor the latter must remain perfectly motionless.” (Dewar and Finn, ’09, p. 200.) The same sentiment is expressed by Werner (’07), Selous (08), Palmer (09), and is quoted from Beddard with approval by Roosevelt (10, p. 493). It reappears in Allen’s (11) review of Roosevelt’s Revealing and Concealing Coloration in Birds and Mammals, yet is diametrically opposed to the just inference from the facts noted in the present section of this paper. It is one of the ‘obvious’ things, the number of which used in constructing theories of coloration is so great, that if all were eliminated, the skeleton remaining would be scarcely recognizable. It is so inconsistent with the fact that an un- usually active fish, such as Iridio bivittatus, which seems never to rest by day, possesses three color phases, which it changes appropriately as it passes from one environment to another, that farther comment is unnecessary. Some of the species whose color changes have been discussed in the preceding pages wear bright colors and bold patterns commonly considered conspicuous. Five are included in and constitute 25 per cent of a list of fishes for which, among other animals, Reighard has felt it desirable, if not imperative, to restate and extend Wallace’s (’67) hypothesis of immunity color- ation. There is some reason for believing that adaptive color changes may yet be demonstrated in other listed species. There- fore one feels safe in saying that the advocates of the color hypotheses which postulate conspicuousness must face the fact, that in an important group of animals, whose colors and patterns rival those of any other group in brillianey and contrast, many which to the casual observer seem among the most conspicuous possess, in addition to countershading, an elaborate mechanism which enables them to reproduce upon their own bodies the dominant hues of the different environments in which they rormally find themselves. Ou Ou or THE COLORATION OF REEF FISHES Correlation of color with habit When it stands alone, the inconspicuousness implied by the occurrence of countershading is insufficient to force the abandon- ment of current explanations of the significance of bright colors; for once grant that there are ‘conspicuous’ animals, and it is incontestable that in a species springing from an inconspicuous line countershading, having outlived its usefulness to the ances- tral type, may persist as a vestigial character. But when coun- tershading and adaptive color change are coupled with one another, as they are in many highly colored species, the pressure brought to bear upon the hypotheses which assume that certain types of coloration must be conspicuous is greatly intensified, for the interpretation naturally placed upon the two phenomena is the same, and is opposed to this conception. The second, more- over, can scarcely be explained as a survival from another age, for its manifestation depends upon the efficient codperation of several of the most highly differentiated organs or organ systems in the body. Yet the facts of adaptive color change are not of general application to bright colored animals. Hence it is difficult to exaggerate the importance of a successful attempt to discover a rational system of distribution of the colors themselves, which the different species wear, and with which countershading and adaptive color change are associated. If the attempt to define such a system should fail, it is still possible that the creatures are as inconspicuous as may be under the conditions in which they live. If, upon the contrary, the effort should be successful, and a consistent relation between color and habit be demonstrated, such that conspicuousness might thereby be supposed to be reduced to a minimum, the uniform suggestion flowing from this fact and from countershad- ing and adaptive color change will constitute as strict proof of the concealing function of color as may be had, short of unimpeach- able feeding experiments. The first attempt to determine whether the reef fishes repeat the colors of their environment, as the Sargassum fauna does, was made by analyzing their patterns and listing the few. shades that stand out at a distance of feet, rather than the great variety 556 We HH. LONGLEY appearing in flecks and vermiculations upon closer scrutiny. The method is crude; allowance for the personal equation of the observer must be large, and the number of species examined was neither great nor thoroughly representative. Moreover, as will appear later, the implicit assumption that all hues that accord with those of the environment must repeat those of the bottom is without foundation. The results obtained are not, however, without interest, and may be stated as follows: Upon 30 species considered, yellow occurred nineteen times; brown and gray, sixteen times each; blue, eight times; red and green, five times each; and black, three times. These facts might seem to warrant the conclusion that, roughly speaking, colors occur upon the fishes in the same proportions as those in which they appear upon the reefs; for the gray of bare sand and dead corals, and the brown of large algae, or the micro- scopic ones living symbiotically in some corals, and the brown gorgonians, are the commonest colors from the shore line to depths where the bottom becomes invisible. Turtle grass is abundant over some parts of the reef flats, and at places upon the reefs its color is supplemented by the vivid green of Zoo- anthus or Halimeda. Yellow heads of Porites astraeoides and yellow gorgonians are common upon the rougher bottoms where fishes most abound, and red and blue are not wholly absent, though forming only an infinitesimal part of the color mass as a whole. But the conservative inference that, at least, no positive evidence appears that different laws prevail in the distribution of color upon animals in the Sargassum and on the reef, is all that is permissible; for it may be shown that some greens, most blues and apparently all true reds are not related at all to the colors upon the bottom, but have another significance. It was noted next that the remarkably large eyes of the squirrel fish, Holocentrus ascensionis, and of Priacanthus cruen- tatus are correlated in each case with red body color. This observation, and the knowledge that the upper limit of the range of certain red, deep-water animals is also the limit within which most of the sun’s rays are absorbed (Murray and Hort, 712, p. THE COLORATION OF REEF FISHES 557 664), led to careful study of the habits of the five species of red fishes which occur in the shallow waters of the inner reefs at Tortugas. The conclusion that these animals belong to a well defined ecological group was soon reached, and is supported by the following facts. The fishes are very rarely seen in the open by day. Pria- canthus cruentatus has not been seen fully exposed, of its own initiative, except at or after twilight. One specimen of Amia sellicauda was observed a few inches from cover, and the squirrel fishes, Holocentrus ascensionis, siccifer and tortugae, are little less retiring in habit, for weeks may pass without one being noted outside the shelter of the coral stacks,’ although the observer may be on the alert to catch them. The five species may be secured in the daytime by blasting with dynamite at almost any station among the stacks in whose crevices they lie hidden. As many as six individuals, represent- ing four of them, have been taken at once, and complete failure rarely followed when using dynamite among the heads. In attempting to visualize what this means regarding the com- parative abundance of hidden and exposed specimens one should bear in mind that the explosive, in the quantity used, is effective up to a distance of only 4 or 5 feet from the point of discharge. But the fishes taken are not uniformly distributed throughout its ‘sphere of influence,’ which lies largely outside the heads among which they lurk. This is apparent from the fact that where the work was done the water averaged little more than 8 feet in depth. In addition, it was always necessary to place the shot within a yard of the outer face of the stack, which in water of the depth mentioned, commonly rises nearly verti- cally toa height of 5 or 6 feet. If this precaution were not taken, the fragments of coral, failing to be thrown out into the open, would fall in a heap from which the specimens could not be re- 7 The coral stacks are masses of heads rising nearly vertically from the bottom in water from eight to fifteen feet deep. They are, in respect to the shelter they afford to fishes of small and medium size, upon a level with a pile of boulders loosely cemented together. 558 W. H. LONGLEY trieved; but, obviously, each condition limits the space in which the fish secured must have lain. Still more vivid impressions of the actual abundance of the red fishes, in spite of their infrequent appearance by day, may be obtained by watching them come out of hiding at dusk. If one goes at that time to a suitable place, and flashes a strong light down into the water, it is possible to demonstrate that they are more in evidence than all other species combined. One may easily have several in sight at once through a glass-bottomed pail. Just before dawn the fishes may be seen again with the same frequency in the same places, as they are about to retire into seclusion. In neither case is there any possibility of undue concentration of individuals being induced by the hight, for the first flash may reveal the situation described. Farther and decisive evidence to the same effect may be obtained by studying their feeding habits, which have not yet received the attention they merit. As matters stand, an ex- amination of the stomachs of nine specimens gives no reason for supposing that they feed at all by day. The facts are stated in detail in table 2. It is worthy of note that the nearest relatives of these red fishes, also red, are large eyed and live at consider- able depths. Red and yellow appearing during the breeding season of fishes which spawn at depths which the sun’s rays of those colors do not attain (Hess, 1913) may have the same significance as the other reds under discussion. These discoveries, if it is proper to use such a term in con- nection with facts which may be common knowledge among fishermen, but do not seem to have been used in any attempt to arrive at an understanding of the function of animal coloration, lead one to oppose the application to the red fishes of hypotheses of warning or immunity coloration, or signal or recognition marks. But it is not really probable that any one will maintain that, natural selection having failed to curb the tendency of color to run riot in this group of animals of the same habit, the same color has been produced independently and accidentally in each case; and this is the essence of the immunity hypothesis. Nor is it more likely that any one can be found who will seriously assert THE COLORATION OF REEF FISHES 559 that through the operation of natural selection, in view of their undemonstrated possession of disagreeable characters, there has been developed in five species of one habit, and without any direct relation to it, a single type of warning coloration which has not reappeared once among many warningly colored species of other habits. That this hypothetically conspicuous color should be that one of all possible shades which first loses its distinctive quality in dim light, in which the animals that bear it habitually live, is an additional difficulty. The five species under discussion represent three families of two widely separated groups, and even the two families of the same group are not closely related (Jordan and Evermann, ’96, p. 1237). Their common color does not, therefore, seem to be due to common descent, unless it is held that they resemble a very ancient ancestral type, from which all related families have di- verged. Even so the question remains, why types of one habit should have been stable, or persistent, while others underwent modification. There are apparently only two possible solutions to the problem: either the environment through some direct action controls the pigmentation of these creatures, or red, under the conditions in which they live, possesses selective value hitherto unrecognized. In any case the correlation of red color with a definite habit renders it highly desirable to have detailed information regarding the habits of fishes in general in order to determine whether this is simply a sporadic instance, or a strik- ing example of conformity to a general law. The knowledge desired is the answer to the question, where and how the species investigated normally spend their time. One must narrow the inquiry, however, so effort has been concen- trated upon the determination of three sets of facts. These seem to be of fundamental importance and may be ascertained with comparatively slight difficulty. When the investigation is completed, one should be able to say when the different species normally feed, where they are usually found by day, and at what level they habitually swim. Nocturnal feeders are little given to diurnal wandering, differ- ing decidedly in this respect from those that feed during daylight. 560 W. H. LONGLEY Hence, if large numbers of a species of such fishes can be defi- nitely located by day, one may have almost complete confidence that an inconsiderable proportion of the whole is in other sur- roundings, searching for food and subject to exposure which might result in adaptation to an environment differing from that in which the great majority lies. The importance of knowing the exact range of the animals studied should be self-evident, for if one undertakes to as- certain whether their colors repeat those of their normal habitat, it would seem to be axiomatic, that the nature of the places over or through which they wander should be accurately determined. But some writers take it for granted that the observation of individuals of any number of species in what we may loosely describe as the same place at the same time, proves that they have the same habit. This uncritical attitude is manifested in extreme form by Dewar and Finn (’09, p. 88-89), who argue that because the moose, Greenland whale, and a farther mis- cellaneous assortment of mammals, including seals, narwhals and musk-oxen, are colored, the prevailing whiteness of the arctic fauna has been greatly exaggerated, and that the common impression that adaptive coloration is dominant in that region is misleading. This means that to these authors the difference in habit and habitat between terrestrial animals ranging south to the latitude of northern Spain, aquatic animals and polar bears, is so inconsiderable, that their difference in color demon- strates its essential lack of correlation with habit and its slight biological significance. It is of importance to know at what level fishes commonly swim, for, as they rise or fall, the proportion of the finny population in whose sight they appear against a background of blank water, or against the variegated bottom or its vertical excrescences, 1s continually changing. As a first step toward the separation of the fish fauna of the Tortugas into ecological groups, the time of feeding has been conclusively determined for a number of species by examination of their stomach contents at different times during the day. The record appears in the following table. A few observations are 561 THE COLORATION OF REEF FISHES ‘Ur’ g 4B Udy} OOM ‘AJCUIO OOM UOAOS YOTYM jo ‘suouttods [euotjippe AjueMy, ‘a10Y} UBY} oLoY SIoys -S¥IS OIOW OSTB 918 Loy T, ‘“Sotveds Sutpedoid oy} ul ueyy pelMva o1our aie osoyy, “Avp Aq oj oJIUYop ynoqe stoyyer ‘ULB QT 7B Way} otoM ‘AjduId O.10M ue} yorum jo ‘suoutroeds [RUOT}Ippe aNoj-AJuOMT, “ABP Aq a10ys Suoje svovjd Ayoor 10 ‘spwey ynoqe Aj[asoyo spooyog “ULE § 4B UDB} 10M ‘jydulo a19M daIG} YOryM Jo ‘sueumtoeds [eUOTyIppe UaAVG ‘snuas oy} jo Sorods 19430 YIM spvoy 9y} JNoqGe [oOoYS (YjSue] UL SeyOUT FT OF 2) OzIS WMIpeuT Jo ssoy} ynq ‘Aep ay} Sulimp joot uedo oy} uo A[SUIS UdeS o1v SUOUITDEdS OSIETT Aep &q soyoyed UBIUOS105 UT IO Spvoy [B109 JNO" S19Y}VS Sotoeds sty], Avp oY} SuLInp suvtU0s10S5 SUOUIG puv SpBoT, [B10 qnoqe [Ooyos sotveds sty} JO STenptArIput jo Ajiol[vul yeoIs oY], Aep Aq spooyog Aep Aq SyovJS [B109 JNOGR pue UT spooyog [ny sea ‘Ure 8 4B Udye} 9UG, ‘up Aq SuoTZe{S poxy JNoqR [OOYOS 0} suees ‘UOUTUIOD JON SMUVNaY N G Adu | 1M TIVNVO AUVLNAWITV 410 NOILIGNOO (aoddvus Avi) cp | 61 | &6 | FIT Snes *N (1e}sBur [OOYIG) z I Ke Bie snpode -N (ysy u0zyny) ¢ OL 61 ST[BUB STUDBUIOI NT (Junds MOT[PX) SE SY SP SIDS SET (quni3 WOUIUIOD)) 98 WS | rotund “Fy (ao10yo L j Z | Sdoyreg) “waaed “yy UINULOJSO.108 UL 9 I I UO[NULIE FT (ysyytod) SNOIUISITA C PT Pit Snue.1}OsSIUy Aqydugy | [Ina Wi deiG"| r= op ent enna aa 1G Na@MVI. TWNVO NAVE saloads TVLOL AUVINGANITY TYLOL 10 NOILIGNOOD Burpaaf fo amy saysif uodn ving 6 WIaVL LONGLEY Winkle. 562 “Ayduro SBA “UB (0'G 7B U9YBY 9UG’ “UOUTUIOD ssoy ST soroeds sty} yey} ydooxe ‘oroy A[ddv owow “gq uodn syusuTUIOD 'Gh'6 pus CEG 7B pus ‘00° pue 00°€ “(Z) 00'E PUF 0N'Z UoEM}0q QO'g Jnoge “UI QO'C }8 poinoes ataM souo Ayduy “CFs puB OS's ‘(Z) 00'S ‘O0'F puv 00's ‘(Z) 00'S PUF 0N'Z U9eMJoq “(Z) 0O'S Jnoqe ‘CFT “ure QZ'ZI 38 usyey 910M SuoUTTOeds |[NY ‘JJes}T Syuos -aid pooy 0} Aptunjsoddo usyM ‘A]Ae[NSo1 J9yyVAI suvodde qnq ‘owT} 94} jo javd odie] B IOF paTBa0U0d aI] 0} SuIVES q] ‘“sw0zj0q Ysnot uo uOWUIOD puB AIezITOS SI YSy sty, "[[NJ “wee OT 7B uUayey eo1yy, “Aunfur Aq peyetnuys usyM ‘1aApIs oind ysowye 04 uin} Avy ‘spuvq ojed smoys soumtjowuos fafqvesueyo ‘poy ‘][DJ 910M “Ue OT 3B UDye} vUG “ABP Aq ueppry ATesoyo Sel] ‘[vUINyoOU {pet YAIM poysem pure podiyg ‘Aep Aq spvey ynoqe Surpooyos 04 waary ssoeds usdo jo 1ayuenbeay ew A[peploep st ynq ‘setpoq ueyuUNs ynoqe usye} puB udes oq AB “STOoyos ul 10 A[suls ‘Avp Aq Sjoor JOAO UOUTUIOD A190 A SuUVNaY IT IT N (aodnoas pouuy-Mo][aA 10 Yysyyooyy) =esous I |-ueA voredorayo AP (todnois O1LOUL snjeydourdy (toddeus peA9-sse[y) snjejuenso G SnyyUBoBIIg (ysy Jorumbg) 9B5N}10} Z Sn} u900[0FT (ysy -JvOS MOT[AZ) SNolUl}IBul F snoued ([1B4-MOT]2 A) snansAryo pey) TIVNVO AUVINAWNITV 40 NOILIGNOO panuyuoj—e ATAVL ‘Wd ¢ NaMVL IVLOL Aqdugy | s[[nw7 TIVNVO AUVINAWNITV dO NOILIGNOO 1g sninAoQ WV ¢ Na@MVL sSalogds TIvLoL 563: THE COLORATION OF REEF FISHES ‘gorjlquenb [[eus ATOATYVIVdUIOD UI UBAS ‘POO} 9[GeYTJUEpT SUTUTV}UOD SUBOUT AT[BAL [[NJ W109} VY} 9[G"} SITY} UL pasn SV ¢ *pooy JayjO pey ouON “SMOTUUTUT pouTeyu0d ATT ‘UR QO'G 3B [OOYOS oUO WO USYe} o10M SuOUTTOOdsS Ayuomy, ‘Suoy Aep [[w@ 9aAoy ‘spooyos ur uouluI0D AOA "SYOBYS [V100 9Y} JO 19}[9YS oY} WOIJ JOO} MOF B UBY} BIOUT S90d WOpes {AEp Aq BATJOV AIO A *‘S19BUVI-JoII [VUINIP 918 BAOGE SoTDedS OM} BY} PUB STYT, “2A0qB asvo 94} UI SB ‘asnjoI AUIT[S ‘pesTUBSIOSIP JO [[NJ 9UT}SOqUT jo javd 19MO] OY, ‘Sseduods OM} puv oeVS][v Jo sotoeds vaIy} Suipnpour ‘pooy uajea AlYysely YJIM pes103 “ule C0'G 38 Udy} LIYJOUY ‘IUTJSOJUT JAMO] UT UOT}ISOdWIODVep JO 9dR4s peUBAPB UT POO} JO JUNOUIG dFIeT pBYy “Ue QO'G UIZB} 9UO ‘aBaTB o[qVYytyUop! ATIpwoL JO [[NJ “UU-e QO'OT puev 0¢'6 7B Udy} YORE 9UGQ “UOT}BUTUTeXE OIdODSOIOTU OU —ovs[8 YIM poly [om ApArey “Uw OQ'G 4B U9xe} GUO f UOUTUIOD ‘S¥SN4IOT, 42 sjurod pa}ejost Maj B YNOGe Avp Aq [oOoOYS ‘uOWIUIOD AOA JON ! 4 re (19uun yy) Jaqns xuBIRD snainsAayo I uopoyyedso.o1py SN4RIJI00 i! uopoyeeyy (ysg Jesue yoRtq) snj}eno1e Ssny}UBIBUIOg (suey on[q) Jake) pee cone) I SUNS TL (qnq9) XII} 8908 © snsoydayy THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 3 564 W..H. LONGLEY included which are insignificant in themselves, but gain interest through their relation to others Despite its brevity a number of trustworthy inferences may be drawn from the table. The five Haemulidae, which group includes Anisotremus, are noctyrnal, and given to schooling about coral heads or among gorgonians during the day. Brachygenys chrysargyreus belongs to the same family, schools regularly with its relatives at some of their rendezvous, and seatters with them at night throughout the open spaces. It may be taken after dark with a gill net as it swims along shore, where it never appears in daylight. It belongs to the bionomic group of which the grunts are notable members. The snappers (Neomaenis spp.) studied select food which differs markedly from that of grunts, but secure it at the same time. The stomachs of 87.1 per cent of the 208 specimens examined were full in the morning or empty late in the after- noon; yet it is clearly an understatement to say that that pro- portion of their feeding is done at night, for identifiable food was found in 85.7 per cent of those taken in the morning and in only 8.7 per cent of those captured twelve hours later. This is equiv- alent to saying that, if equal numbers were examined in the two cases, 90.8 per cent of the full specimens would occur among the former. Even this higher percentage fails to express the truth fully, for both the amount of food and the number of organisms con- tained in the fishes’ stomachs are greater in the morning than in the evening. The possibility that some whose stomachs con- tain food in the late afternoon have eaten nothing since morning must also be taken into consideration, but at present too little is known of the rate of digestion in the snappers to permit one to speak with assurance upon this point. The only observations which bear upon the matter are these, that in the three hours between 5.00 and 8.00 a.m. the proportion of empty stomachs in the total catch increased from 14.3 per cent to approximately 32.2 per cent although in the interest of accuracy a slight cor- rection is necessary, since the three species do not bear exactly the same ratio to one another in the two cases. THE COLORATION OF REEF FISHES 565 The feeding time of the yellow goatfish (Upeneus martinicus) seems to coincide with that of the grunts and snappers, though during the day individuals are sometimes seen stirring the sand with their barbels, as though searching for food. Holocentrus and Priacanthus may also be included in this group with slight probability of error. The number of these red fishes examined is small, but the results are consistent and a great mass of col- lateral evidence supports this interpretation. What fishes feed by day is not made clear by the tabulated record; but this may be supplemented by observations which permit one to state with some confidence that most Labridae, Scaridae and Chaetodontidae, to mention no others, are diurnal. From daylight to dark the activity of Iridio and Thalassoma is scarcely interrupted. Not one broken sea urchin is exposed upon the reefs, that they do not gather about and feed upon. The same is true of Lachnolaimus, except in so far as its com- parative rarity limits its appearance. Scarus may be seen floating listlessly near the bottom at daybreak, and combining forces with Sparisoma a dozen common species grub industri- ously upon it all day long. At dusk the activity of all the Scarids changes; all disappear, except as an occasional one in its nocturnal color-phase lies half-revealed beneath the imperfect shelter of coral, gorgonian or projecting stone. Darkness also modifies the behavior of the Labrids which, perhaps favored by their smaller size, disappear even more completely. Species of Thalassoma, Iridio and Xyricthys, three labrid genera, when confined in tanks, if the bottom be covered with loose sand to a sufficient depth, burrow into it and remain in hiding until the dawn. To burrow or partially cover themselves with loose material is not uncommon procedure among fishes. Gnathypops auri- frons and maxillosa use their jaws for digging and their mouths for carrying away excavated material from narrow pits they make and occupy. Halieuticthys aculeatus, a batfish dredged in 45 fathoms, backs into the sand and uses its pediculate pec- toral fins to throw an incomplete but almost impenetrable screen over its flattened body from its postero-lateral margins. Syno- 566 W. H. LONGLEY dus foetens lies flat, and may sink with one convulsive wriggle till only the dorsal surface of its head—or nothing—is visible beyond the depression beneath which it is buried. Flounders too, when resting, commonly bed themselves and diminish their visibility. In these various species each reaction, though their result be one, is performed in distinctive fashion. The Labrids commonly use the pectorals alone in swimming, but when they are about to bury themselves, all their trunk and tail muscles are called into play, and in an instant they plunge head foremost out of sight. That the behavior of three genera should be consistent each with the other, and yet be without observed parallel beyond the family, is significant. The ex- pedition with which the reaction is carried out, its regular re- currence at nightfall, the ease with which it may be evoked at any time of day by darkening the tank, and a tendency to perform it at a stated time, even in the absence of essential conditions, are also significant, for all alike emphasize its habitual character. Unfortunately it has not been noted in nature, but as that might occur only through some lucky accident, there can be little doubt that it is a normal response of the animals, and explains, at least in part their nightly disappearance. The Chaetodons, Angel fish and Tangs are diurnal. The most interesting record concerning them in the table is that regarding Pomacanthus arcuatus. This shows that in the very long in- testine of some fishes that ingest great quantities of food during the day much may remain many hours later. What may fix their feeding time, is not the presence or absence of material in the alimentary tract, but its apparent freshness. The grouped observations upon these fishes are consistent with the idea that they feed by day. They fit in well with what has been noted of their diurnal activities, and the fact that at night they are not on the open reef, but are more or less definitely sheltered among the heads. It does not appear that the activity of all fishes varies so dis- tinctly at different times. The yellow-tail and red grouper seem to feed when food is available, and to be governed by no other condition. From superficial agreement in habit with Epine- THE COLORATION OF REEF FISHES 567 phelus morio the same may be inferred for E. striatus and Mycteroperca venenosa. The chub (Kyphosus sectatrix) ap- parently schools by day in definite places, and one should expect to find it full in the morning and fasting in the late afternoon. At the moment, however, the records are too few to serve as a basis for sound judgement in the matter, and are not supple- mented by decisive observations. The nocturnal fishes fall into various subclasses according to the stations which they frequent by day. Some are rarely seen. Others congregate about the coral stacks, where all day long they lie idly at some little distance above the bottom; or swim leisurely through the openings between the heads, but in no sense seclude themselves in their recesses. Others, again, con- fine themselves less definitely to the region of the stacks, but, nevertheless, make such places distinct foci of their activity. Those whose concealment is so complete that under natural conditions one would scarcely suspect their presence, and assur- edly never appreciate their abundance, are the five red fishes. There are others that are rarely seen in the open. These are the green moray (Lycodontis funebris) and the ribbon-fish (Eques acuminatus). Of the former only one, and of the latter three have been encountered in the daytime; but these may not be signi- ficant observations, for not many of the morays have been taken at any time, and the other has also been obtained but rarely. Six were secured once when blasting at dusk, and one under similar conditions during the day. As collateral evidence tend- ing to justify the exclusion of the two species from the bionomic group indicated, it may be stated, that the spotted moray (L. moringa) is not uncommonly seen with its head protruding from holes in the coral, and that the widow-fish (EK. pulcher), though rarely met, seems also to have no aversion to appearing in the open when the light is strong. The second subclass of the nocturnal fishes includes the grunts and their allies, Anisotremus and Brachygenys, the two snappers (N. analis and apodus) and the yellow goatfish (U. martinicus). The gray snapper (N. griseus) represents the third subclass, and the chub may eventually find its place in this group. In 568 W. H. LONGLEY passing, it is worthy of note that there is a marked difference in the ratio of full to fasting specimens in N. apodus and N. griseus taken at 5.00 am. Empty stomachs are 3.6 per cent of the total examined in the former, while the proportion rises to 16.7 per cent in the latter. This may indicate that in nocturnal fishes irregularity in feeding is correlated with failure to conform strictly to the custom of schooling in closely restricted areas by day. There are also obvious lines of cleavage among the diurnal fishes. Some are surface feeders from whose environment the bottom colors are so remote as to be practically non-existent. Others, such as Microspathodon chrysurus and Eupomacentrus fuscus, haunt the coral stacks, swim freely in and out of their interstices, but seldom venture more than a-yard or two away. Finally, there are the wandering Labrids, Searids, etc., which differ from one another both in the horizontal and vertical limits of their range. Knowledge of these distinctions must precede appreciation of the correlation of color with habit which prevails among reef fishes. Two lists are displayed in table 3. The first includes species which may be observed moving freely in the mixed environment of the open reef. The second comprises others whose diurnal activity seems definitely centered in the coral stacks. Within the limit of present experience each is complete, and is ap- proximately correct, though later modification may appear desirable. Hence it is instructive to analyze both with reference to the distribution of sandy gray among their members, since the two environments differ widely in the proportion of this color they contain. The three blue-gray species differ sufficiently from the others to justify their exclusion from consideration in the present instance; yet to have passed them over without comment might have led to misunderstanding. Taking the remaining records as they are, we may say, then, that the color of three of 21 species in series II repeats that of sandy bottom. But in series I, which is only 50 per cent longer, there are almost five times as many which show evident adaptive color changes under the same con- ditions, or have permanent gray markings. 569 THE COLORATION OF REEF FISHES sninqtos _snqeliys uO[NWIB asvyd z0joo Avis oAtydepy snjeydoutdy totum d ayedto uoTnuleeyyT | -TjuBv pynoys suo sv ueazjo se avodde jou seop 4ynq aseyd 10joo Avis aarydepe elied ‘poAvidsip Ajoyersdordde st O11oUL uv ‘aAotoq JT ‘sey yorym “ysy AvIS IO AYBS V uo[Nuev_T | YOryM osvyd 1009 ABIS B SBP] snjeydoutdy UINULOJSO1IBUL SN4BIIys UO[NULOR FT uopoyavyy UIN}VIUT[OABT sn}B][900 UO[NWIv FT UMOIG YIM popurq !ABrH wopoyovyy snosnj snqeaystdeo snajusovmodny uopoyavy yD sdouv900 SnulyBoVl[y Avis ysmniq AjesieT Joqnid xuvieyg snoidsiesA1y9 pues oI M UBITO sXuaskyovig | uo posed pooy 0} sured possorddns MOoT[oA [JIM WOU epneol{[as ; oe -wedg ‘suoiser yeyidt1000 : pue yejuo1y woody ATIOT SNOTUISATA -193sod puv A][vijueA SUT SUOIJIJOIB snuoijostuy | -pvy yseM MOT[OA YITM ‘ABD SnUIe[BO MOT[OA oq Avut soovdsisyur 4Yyst] jo party} soddy ST[I}VXBS STIVITIO ‘youyq 10 UMOIG YIvp YIM popueq {Avis on[_ jnpyopnqy sky Qoljesuy Syavuroy soroadg SyIVUrldy setoedg Il Ssarwas I Saluas Se puqoy pajspijuos fo saysyf faas fo sdnowb om) ur fivsb fipuvs fo uoyngr.ysiqg , € HIAV.L LONGLEY W. H. 570 SOTIOS OM} OY} JO SIOQUIOUT OFVAIAT ay} UdeMjJoq SjJoedset 9UIOS UT 9} ¥vIPeUTIE}UT Ayjeor st soroeds siyy, ‘osvyd Avis oatydepy ‘suy Sq uodn Mo]JoA Jasu0IjS YIM pusB ‘spuBq IeyIep qNoyyIM IO YQIM ‘moT[ed YSstuMOIg ATWOUTUIOD SI 4[ ‘“wojjoq Avis IVAO YuoUTYsN{pBe 97}¥ -11doiddv uv Sutyeur jo ojqudvo st soroods sty, Snosiis STUSBUIOD NT snpode STUIBULOIN SsT[vue STUBBUIOON, snansAayo uopoyyedsoro1yy BSULIOUL STJUOPODATT oBvsnj104 snijus00[0F] I9JIOOTS Sn} Uad0[0 FT STUOISUODSE snai}U900[0FT asvyd 10[00 Avid aATJdeps uv BABY Swadsaaniy pus wpsiovbpjiqgo pwosiwdy (seroeds aay) euostiedg (soroeds 9A) SNIBIG ‘ds snivosopnesg snjenoie snyyUuBoBUlOg ‘uy [BpNBd 19A0D 0} ATIO -ajsod Sutpuvdxe pueq snansAryo [eroyey MOT[PA fABIs ong sninAdQ BSOUITIOA OLlOUW “WT SB OUIeG Bo1ddoO.1aqyO ATW SNUIIXBUL osvyd 10j09 Avis oAT}depy SNUITB[OUYIVT snjeIpel Oyen) | u10940q evuuldljnovur Avis IAAO IO[OO UT SUa}YSTT OIpUy osvyd 10joo Avis aATJdepy | sN}ezIIAIG OPT SYIVULOY Il Saluas saroadg syreuley setvedg panuyuogo—e WIAVL I saluas 571 THE COLORATION OF REEF FISHES SNOTUT} ABUL snoued snyequento snyyUuBoRlid I0]oo yey} Aq peyeuUrIUIOp u10}30q I9AO pue ‘aseyd ABS SI UT ydaox9 paAdesqo usveq Jou sey douUBpUNe 9AT}BIeduI0D aqyidsap ynq ‘asueyo r0[09 jo 1aMmod paylvyy “apis oy} uo sjods yiep oe1yy {Avr eseyd qoJoo Atis sAydupy 010UL “FT JO YVY} SoTquioselr asey “w0jJ0q ApuBS 1dA0 uMOYS asvyd 10[00 AvAS B SLT S[BI10} Bul SulA[Jopun oq} Jo apvys oyy 0} AT}OUTYSIP AIBA SaATaS -w9y} Ysn{pB sazis [][v 97e4S siy} up ‘usezyed papurq 8 oUINSSB (SUO] JOO OAT IO INOJ) JSasIB] oY} UaAD ‘UI04 -jOq «vou S$}Se1 puv syuUIS WI uay AA “SjoAey,T aaddn 10 a} BIPSULIO}UL 7B Waes AT[BNS sn}epnoeuL snoued ¢) (“BUIISSIpT} TU pu® snptytu * 7) “anl ‘snyetose jiq BUIOSS¥BY TL, snjedoy BINA TL yaks) hun ccey9) SsIyqnoy, BpnoBl1eq suoeidydg 572 W. H. LONGLEY It may be urged that the fact that these lists are incomplete, or may reflect some unconscious prejudice of their compiler, detracts from their significance. But, whatever may be the case regarding the second suggestion, the first isinvalid. Farther observation will merely emphasize the disproportionate occur- rence of gray in the two series. Though this is not the dominant color among the heads and close about them, it occurs in both places, as well as upon the open reef. Hence perfect contrast between the two groups may scarcely be anticipated. There- fore, no conclusion seems more legitimate than that we have in the distribution of gray markings and color phases an additional instance of correlation of color with habit among fishes. Brown is so common in the environment of the species of both series that it is scarcely worth while attempting to secure definite suggestion regarding its meaning by reexamining them. Other lists might be compiled that would yield results comparable with those above, but their presentation seems unnecessary. Wholly aside from the frequent appearance of brown upon the fishes and in their environment, it is interesting to note that many species show a brown phase inits presence. ‘To limit one’s choice of examples arbitrarily, the following may be cited from the forms mentioned in table 3: E. morio and striatus, I. bivitta- tus and maculipinna, L. maximus, M. venenosa, Sparisoma abild- gaardi and flavescens, 8. barracuda, T. hepatus, N. griseus, and N. apodus. But, if brown may replace gray as an essential self-color, as it does in L. maximus and N. griseus when they change their environment, no functional conspicuousness may - be ascribed to either, under the conditions in which it appears. Nor may this be imputed more rationally to any quality of contrast inherent in a combination of the two colors, such as appears in E. striatus (fig. 2); for the relative intensity of the two elements in the fish’s pattern may vary continuously, as the proportion of light to dark in its environment changes with its change of position. Color photographs alone can convey the impression an observer receives in the field. Although those of the common sort seem to show, that the phases at the ends of the graded series are obliterative, since THE COLORATION OF REEF FISHES 573 it appears that either would be more conspicuous than the other, if displayed in its stead. But the extreme phases of coloration are not essentially different from the intermediate ones, and may be separated in time by no more than five seconds, and, in space, by as many feet. Is the dark fish under the head less immune from attack than when it is a-yard from shelter, that it should assume a more conspicuous pattern as it moves from beneath it? Or is this contrastive design in brown and gray a warning combination, whose serviceableness dimin- ishes as the fish moves still farther into the open? It seems impossible to answer such questions in the affirmative; yet, if all are aspects of obliterative coloration, no combination of brown and gray that may appear upon the reef fishes in an unchangeable pattern may be assigned to another category, un- til it is proven that its possessor’s normal range lies beyond the bounds within which the changeable forms show comparable phases. Yellow is not of common occurrence in large patches upon the reefs, so it is virtually impossible to make extended observations upon its effect in evoking yellow phases, which, by the way, are not frequently encountered among the fishes, in spite of the fact that yellow marks or washes appear in abundance. The red parrot fish (8. abildgaardi), with its notable power of color change and capacity of adjustment to light and dark back- grounds, in its darkest phase is distinctly washed with yellow over the whole top of its head. The immature blue-head (T. bifasciatus) has a gray phase with no yellow, assumed over sand, and a yellow phase which it resumes when it returns to more variegated surroundings. There are also indications that the porgy (Calamus arctifrons) dons and doffs its yellow with reference to the character of the underlying bottom. Thus yel- low may occur in combination with brown and gray, colors to which no conspicuousness attaches in themselves. In some species it is suppressed, when its bearer passes into an environ- ment to which the color is foreign, and reappears only upon its departure from that place Hence in its bionomic relations it seems to differ in no wise from the colors already considered. 574 Ween LONGLEN The study of blue has yielded results that are unsurpassed in suggestiveness, but it is much more difficult in some respects than other equally important undertakings, and has been neg- lected in proportion. This color seems to occur in nature in prominent markings, or as a self-color, upon no fishes which lie close upon the bottom, although Mast’s (14) flounders distinctly developed it under experimental conditions. Similarly, it is wanting upon those living just beneath the surface film, except as there are bluish reflections from the silvery lateral stripe of some of he Esocidae, for example. It appears in a great variety of tints and shades, ranging from the blue-gray of Abudefduf saxatilis and Ocyurus chrysurus through the pale blue of Gnathypops aurifrons and Searus caeruleus to the richer blue of Thalassoma bifasciatus. It even attains the blue-black sometimes worn by Teuthis caeruleus and constituting the permanent ground color of a large Pseudoscarus that seems undescribed by systematists. Kyphosus sectatrix, Caranx ruber and Elacatinus oceanops com- plete the list. It is noteworthy that a number of these fishes, and particularly the blue-gray types, have one habit in common that is as dis- tinctive as their color. Abudefduf is equally at home breaking the water’s surface above or near the coral stacks on quiet days, or deeper down among the heads themselves. Caranx and Ocyurus pursue schools of minnows and make the water fairly boil about them in calm weather. But the former may enter one’s traps upon the bottom on occasion, and the latter feeds commonly upon Crustacea that never leave it. Kyphosus, which is distinctly darker and less blue than the others, has the same vertical range, but is neither seen at the surface as fre- quently, nor appears to come at all commonly into as shallow water. At Tortugas full-grown specimens appear by day, typi- cally at mid-level or slightly lower, in water approximately 20 feet deep, over one small patch of broken bottom, or about a mass of sunken wreckage. It seems more than accident that four out of ten species grouped by color should react habitually in a manner uncommon among THE COLORATION OF REEF FISHES 575 fishes as a whole. There are 52 species, for example, listed in table 3. Of these eight are included in the group under con- sideration. Among the forty-four remaining there is only one whose vertical range is coextensive with those above. It is questionable, indeed, whether this much should be conceded, for the gray snapper has been observed only twice actually break- ing the surface as the others do. Each occasion was in the very early morning when the water was teeming with Annelids— the palolo worms—in their annual swarm. But even if the record be accepted without qualification, the proportion in the two cases stands as 40 to 2.27 per cent, or, in other words, the reaction in point occurs more than seventeen times as frequently among fishes of the blue series as it does among others selected at random. There are additional observations which tend to support the hypothesis that a correlation exists between the habit of swim- ming well above the bottom and the development of blue pigment. Gnathypops aurifrons (Jordan and Thompson, ’04), a species known from only one specimen, taken at the Tortugas, is not very common, but one sometimes chances upon a colony of these interesting creatures in water 8 to 10 feet deep, or even shallower. They seem to spend a large part of their day float- ing inactively well up over their burrows. When undisturbed, they maintain themselves at an angle of 70 to 80 degrees with the horizontal for indefinite periods. Upon being alarmed they retreat into their shelters tail foremost, and remain in these tiny vertical pits until the disturbance has subsided. Even if un- molested they seem to withdraw toward dusk, and probably remain under cover all night. Hence, in spite of their fossorial and tubicolous instincts, it would appear that they are pre- eminently fishes of the middle depths, living in moderately shallow, open water, above the bare bottom in which all but one of some dozens of burrows observed were located. Thalassoma bifasciatus and Iridio bivittatus occur so commonly together, that of them, if ever, one might say, ‘“‘Here are two animals living in the same environment, but differing in color and pattern, therefore, it is perfectly apparent, that if one type 576 W. H. LONGLEY of coloration is protective, the other is not.’’ Yet it appears that the ranges of the two species differ both in horizontal and vertical extent. ; The Iridio occurs on the turtle-grass flats, to whose color it is able to adjust itself, but the Thalassoma does not range over the larger areas of this character. Not one has been taken among scores of Iridioes caught with the seine on grassy bottoms, nor do they come about baits'set out in such places. In other respects the horizontal ranges of the two specise seem to coincide. It was a long time before difference in their vertical distribution was noticed which was sufficient to suggest an appeal to experi- ment, but when this was undertaken it was demonstrated with ease that the Thalassoma rises readily to higher levels than the Iridio. If food be placed on coral heads of different height, Thalassoma will arrive first at the higher stations and may be the only visitor, although Iridioes swim about at lower levels. Again, if in a tank filled with water to a depth of 15 inches a vertical partition a foot high be erected, and specimens of the two species be placed on one side, a Thalassoma will swim over the obstruction first, and may recross twenty times, before the first Iridio finds its way over. Each experiment, therefore, clearly confirms the suggestion that led to their performance. But though there is correlation between blue color and a fish’s habit of swimming at intermediate levels in water of moderate depth, its meaning may not be apparent, for Wallace (’77) writes, and the explanation is approved by Poulton (’90), that “look- ind down on the dark back of a fish it is almost invisible, while, to an enemy looking up from below, the light under surface would be equally invisible against the light of the clouds and sky.”’ However, at a depth of 15 to 20 feet in clearest water, under a tropical sky, one does not see the fishes above against a mere light or whitish background. The sky seems to have fallen, for one stands beneath a dome of blue, paling toward the zenith and deepening toward the horizon. Little is visible at 20 feet. The ‘atmosphere’ of the painter is thick; it is apparent almost at arm’s length, and softens the outlines of all but the nearest objects. THE COLORATION OF REEF FISHES VATA In this Lilliputian world one has a demonstration of the effect of color and pattern which supersedes all argument. Fishes come from nowhere and vanish into nothingness, not because they have gone beyond the range of vision, but have wheeled at right angles. The’ secret involved in their disappearance is largely this, that, as they turn, foreshortening reduces, even to zero, tell-tale lateral markings upon which the eye had been fixed. Blue-gray bodies, or blue-gray bands upon bodies of other colors, are utterly resolved into the blue-gray haze which surrounds them, and one is wholly unable to perceive the outlines of objects easily within visible distance, as the distinctness of certain markings testifies. Figure 8 illustrates the effect of differential visibility of two colors combined in simple fashion. It shows, in other words, the mode of operation of Thayer’s ‘ruptive pattern,’ when it appears upon a countershaded body. It is so efficient in the case of Abudefduf, that when the fish is above the level of the observer’s eye, its silhouette becomes indistinct, or is inter- rupted, at a distance of 4 or 5 yards, through the assimilation of some of its color elements with the watery background against which it appears. If it were wholly of the gray color, suitably countershaded, it would be almost invisible at that distance, except as its eye might reveal its position. Some simply colored species of the upper levels, when appropriately viewed, are, in- deed, the veriest ghosts of fishes. Tylosurus is a notable ex- ample, and Sphyraena, though a large fish, has defied the camera times without number, largely on account of its lack of contrast with its background. At this point it may appear to the reader, that the presence of the dark bars of Abudefduf, which prove so clearly that its partial invisibility is attained well within the limit of vision, is inconsistent with the argument that is being developed. For if natural selection guides the evolution of such characters, why, it may be asked, should the fish retain these marks, if its visi- bility might be farther diminished by their suppression. This fair question may be answered most readily by calling attention 578 W. H. LONGLEY to some differences in habit upon the part of the three species last mentioned. The coloration of Tylosurus raphidoma is simple and appar- ently unchangeable. Its surroundings are equally simple and almost as invariable, for it has not been observed at any time more than a few inches beneath the surface, where it may lie for considerable periods, scarcely covered and almost motion- less. Sphyraena has much in common with Tylosurus, but sometimes sinks to the bottom. As has been noted, under such conditions it changes in shade if necessary, and assimilates itself with the substratum. The vertical range of Abudefduf exceeds that of Sphyraena, and its relations with the bottom are more intimate, yet, in spite of the fact that its color is inconstant, definite adaptive color changes have not been observed in the species The fish bears instead a relatively stable color com- bination, which is compounded of characteristic tones from its environment. There is, therefore, no inconsistency in Abudefduf’s retention of its dark bands. Its coloration, like that of most other fishes as yet studied carefully in their relation to a complex environ- ment, is a patent compromise. Its range is extreme, and as a result the vicissitudes to which it is exposed are many. But the risks it runs are well distributed; for its pigmentation is such that it is at once both more and less distinctly visible than it should be, if it were all of any one of the several colors perfectly suited to a limited portion of its range. To conclude, there is undoubtedly correlation between at least the lighter blues and the probability of being seen from below against an aqueous background. Moreover, the interpretation which it seems necessary to place upon this fact is in no wise opposed by the appearance of others in combination with ‘water colors’ in one pattern. There is little novelty in the facts concerning the distribution of green among the fishes studied. They are added in the inter- est of completeness and are thoroughly consistent with what has gone before. This color appears upon surface-swimmers, or upon bottom fishes in correlation with the occurrence of green THE COLORATION OF REEF FISHES 579 in their environment. It dominates the coloration of such types as Atherina, Stolephorus, Tylosurus and Hemirhamphus, which typically occur at high levels. Among representatives of this group at Tortugas the simple green color is distinctly interrupted only by a silvery lateral stripe, which seems to occur nowhere except upon fishes of this habit, and suggests that pattern, no less than color, may not be wholly fortuitious. The correlation of green with its presence in the normal habitat of the animals displaying it is best shown by study of those seined upon reef flats covered by turtle grass. These are not of clear and uniform green color. The plant’s flat, narrow leaves may be too short, and the vertical branches of the rootstock from which they spring too far apart for the sand to be completely concealed. In each tuft the older, outer leaves, whose death and detachment approach, are brown. While they retain their connection, or lie scattered among the others, their contribution to the color complex is important. In this composite environment of which greenness is, never- theless, the distinctive quality, fishes have been taken as shown in the following table. The table includes twenty-four species. Eleven of these are wholly or largely of an unchangeable green color, or have defi- nite green phases, which are assumed in the midst of green sur- roundings. That this should be true of so large a proportion of the total catch among the grass, is perhaps more than might have been anticipated. It certainly demonstrates that green, like the other colors, is distributed according to system, for with the exception of one group there is probably no other in which it occurs with such frequency. Yet the statement above is mechanical and expresses the truth imperfectly; the facts cer- tainly merit more careful analysis. These twenty-four species are not members of one bionomic association. Some of them may be excluded from consideration after brief review of the evidence. Synodus and the flounder may both be seined on sandy bottoms adjacent to the grass flats. Both are accustomed to bury them- selves more or less completely when resting. Both aremarked THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 23, NO. 3 LONGLEY Wisw i. 580 soov[d 19440 UI UVES SOUITJIUIOS JIB OZIS OUTS oy} jo Suouoeds Sunox “A[UOUTUIOD IoYyeI Uy], “4[Npe sv oures ‘mor[eA puv AvaAB-on[g a1oyM ‘SodL1yS [VUIPN}ISUO] MOIIVU UT JOATIS puw MOT[OX u10}40q ApuBS JaAO UdYe} SoUIT} ‘SS¥Id oY} SUOW ddUBpUNB UI pouTes av Ssoseyd UMOIG ‘sosvyd Avid puw uMOIG ‘Ud0I5 UT suvedde fasuRYyo I0J0O Jo 1amod poyreyy -a8]a9 U90S 4O “ATUOWUIOD I9y}VI UaYyV I N I 1 WL -9UI0S 8ZIS OWS JO saTDeds auIRG pue usels yO PAOGB SB ISIMIOY}O /UddIS YYSTT a1 MOSTO ‘1OJOD asuByo 04 AFI[IGB’ Jo doUepTAS OU fuseI3 yIeq aL1IYMOS[A PAALsqo ‘s78Q ssvis oy} uodn ATUOWUIOD 9yIMb udyey 10 UVEeG “Ysy UseIS vB AT[BIYUOSS SSLIS 9]}1N} OY} SUOWB UIYe} SoZIS [TY “W10}}0q Jo $3.08 ‘sosvyd umoiq puv Avis ‘udeIs fasuByd 10[00 Jo tamod oB1eT petezyunodus you ynq ‘A[peywedear uayey, u90q JOU SFT I[@ JSow]e 19A0 ATOpIM sosuvy aLOYMOSTO S1N990 :ATJUENbeIZ UsyBy, ‘“Sodr43s [BUIPNyIsuo] AvAS 10 on] JUTeT YIM UMOIG Aq wou W0}}0q ApUBS UO OSTR UayV} 9ZIS oUIES oy} JO Sotoeds ouTEY ‘ponds Y4SU9] UT YOur ouo ynoge suouTIedS [[VUIS OMT, ‘UMOIG 10 AVIS IoyIVp YLM poyoop ABI OUIT} OUIBS OY} JV UdYe} ‘Sddo YIM o[vU puB o[vUey ‘suoUTOeds OMY, ‘“Yysyodid Avis puv uMOIg SUOTSBI0O JO IJoOqUINU B UO UdYB], ‘“Spuvq UMOIG YIM YSTMO]I A udyB} VU “UMOIG YAIBp AOA “SUOT YOUT 9UO YnoQe ‘T[RUIG UdYv} OMT, ‘“SpBoy [B109 JNOqGe puB Su10}}0q AYOOL UO UOMIUIOD ALIA “UOT}VAOTOD UT a[quIavA {KuUITq popuvq ABS puv UMOIG ‘[[BUIg SHUVN GAY ‘ant ‘sninsAiyo snandog ‘Ant ‘stuseuds STUBBUIOIN ‘Ant ‘snprdsiy snyjuBoRBUoyy “Ant ‘sTu1oolty sfrydoyoe'y ‘Ant ‘styepneorq sArydoyoe'y WYOSITY] OPTI] Bye TATC OMIT “Ant ‘TZaruny{d wopnuee yy ‘Ant “ds tapunoypy umJ1oA9 sAy{o1oIyAAIOD “Ant ‘SUOIJIJOIB SNUIETBD ‘ds viuy Ta100U STOvJOW Salogds soy sspib-3]}.1N}] UO pauras saysry b ATAVL = 581 THE COLORATION OF REEF FISHES sqyey ssvasd oy} uodn sv [jam sv ‘a1oys ‘UMOIG 10 ABIS JoyIep YIM poyreur ‘ARID sesvyd Avis pu’ udeis fuautoeds 9uG uMorq ‘usuTIeds auG Ivou w10}}0q Apues ‘aveTo IaAO ATUOWIUIOD poulog ‘m10}}0q ApUBS UO OS[R UDyBT, “UMOIG 10 Avis IOYIVp YIM poyrvur ‘ABI d19Y MOST WOUTUIOD {AVIS PUB UMOIG ‘YISUI| UI SOUT G*[-Y'[ Use} SudUTTDedS [RIBARG IIIYMOSTI UOYB} JOU {S}VY ssvis oY} UO UOTIUIOD “YASUI UI SaTOUT G°z YNOq*e 03 dn ‘usyvy SazIs IIV ‘pues Av13 UO 4SAI 0} SOUIOD IO ‘19AO SUTIMS YS oY U9YM ABIG SUIN} 9DBJANS [BSIOP !UddI4) a.1IY MOST poinoes jou {syvpy ssvas oy} uo A]Juonbasj udyBy, ‘A[SNO9UBIULISUI JSOUI[V OpBUI SI UddaIS 0} quousnipy ‘soseyd 10yjo pue Avis ‘M9013 YIM osuUvYD 1O[OD Jo 1aMod yvoId AIOA SOSsassog S}Vy 94} WO UOUUIOD ST YSy STYyy !Joor uodo 944 ‘soszyd Avis puv ude YJIM OsUBYD IOTOD Jo 19M0d JFIR] SBET a1OYMOSTS USYV] OUONY “SHd9 YJIM SoTVUI puB So[VUla] 9INYVUL popnpoul youVD ~“AviB wAOJTUN Jsouye urn} AvuUI puB ‘asuRYD IOT[OD Jo 1amo0d as1v] oAvY SusUITDZdS UMOAS-j[ey eUI0G “ABI ATUOUTUIOD Vd¥FJINS [ESIOP YJIM ‘UMOIG IO U90I4) d1IYMISTO pojou JON ‘949 oY} YSnosyy od114s UMOIG MOIIBU “UddL Ajuo usutoeds sug, ‘pat pue Avis ‘UMOIG YIIM poyIeUl ‘[]BUIg I9A0 WOWULOD ST SUIDSOABH “SG “ant ‘WINII9UTD BUIABISAK ‘ds sAyqousy ‘Ant ‘snyedoay styynoey, “ant ‘sue}a0j snpousg ‘Ant ‘epnovaiegq vuovisydg Ilejsuods soprorsydg xvysAu0|doy Burostiedg “Ant ({) Sudoseavy euostiedg AByovul BUIOYSOYdIg Isouof vuroysoydig ‘ds vuordi00g 582 W. H. LONGLEY with colors which are repeated upon other fishes and upon some Crustacea (Portunus ventralis and Gonodactylus oerstedii) which may be taken in the same sandy region. They do not belong to the grass-flat fauna strictly defined, but are apparently swept up and gathered in from minor barren patches, which may be wholly bare. With perfect fairness both may be dis- regarded in the present connection. Xystaema should be rejected with them. Its colors are much the same as theirs; young specimens are common near shore, and others that are full grown may be seen frequently idling over sandy bottom along the beach during the day. Actaeis is no more justly considered a member of the group we are attempting to define, for it seems to be much more abundant in other places. Of those remaining Sphyraena at least may be seen and has been taken as commonly elsewhere, in the same early stages of develop- ment, with less efficient implements. This revision, every step in which is justifiable, leaves, then, nineteen species, of which eleven (57.9 per cent) repeat, or are capable of repeating the green color characterizing the environment in which they were secured. There is another correction that may be applied. Fishes frequently encountered are, upon the average at least, more justly considered typical members of the local fauna than those that appear in the haul only rarely. Upon this basis, Amia, Scorpaena, Teuthis and Xyricthys may be, or must be denied consideration until more complete records of their distribution are available. It may be that one of these or even more should be retained, but the relative frequency with which green occurs upon the fishes of the grass flats may probably be measured more accurately if all four are dropped, than if all are included in the calculation. To prune the list no farther, although such action would not be wholly unreasonable, there remain fifteen species which may be regarded tentatively as characteristic of the locality in which they were taken. Ten (66.6 per cent) of these repeat the dis- tinctive color of their environment. Except among surface fishes green occurs in no such ratio, and if these be excluded in THE COLORATION OF REEF FISHES 583 addition to the present group, it is probably impossible to name as many green fishes as are mentioned here, though the entire fish fauna of the Tortugas be drawn upon. Since this includes more than two hundred species (Jordan and Thompson, ’04), the statement above is highly significant. There is no reason to believe that all typical fishes of the grass flats are green. However, one fair inference may be drawn from the facts presented, i.e., the more nearly a species is restricted to these places at a given stage of its development, the more probable it is that its pigments include that color. It is appar- ent, therefore, that there is intimate correlation between the colors and habits of tropical reef-fishes. It is scarcely necessary to repeat that this fact, which has been demonstrated in the individual cases of red, gray, the lighter blues and green, is of the utmost importance in its bearing upon the problematic significance of animal coloration. The working hypothesis enunciated in the introduction seems adequately sustained. Much more has been done, indeed, than test it by applying it to the reef fishes, but positive results in this phase of the investigation support the suggestion of the obliterative effect of color and pattern, which flows from the all but universal existence of countershading and the common occurrence of adaptive color changes within the group. DISCUSSION OF RESULTS Fishes which range by day amid the most varied surroundings display the greatest variety of colors. ‘They may also possess the best developed means of defence. In this event there would be secondary correlation of vivid color combinations with armament and ‘distastefulness.’ Whether this is so must be determined before appraising the fact above as a confirmation and extension of the partial truth, or a demonstration of error existing in the color hypotheses that postulate conspicuousness. In the diversity and richness of their coloration no large group of fishes surpasses the Labrids and Scarids. These are, there- fore, fit subjects for the present inquiry. If their distasteful- 584 W. H. LONGLEY ness is fictitious, this should appear from the analysis of the stomach contents of potential enemies. It was with this idea in mind that the feeding habits of snappers were examined more closely than those of other species, for none seemed more likely to exact its toll from unprotected forms. From the snappers’ stomachs several perfectly recognizable specimens of Iridio bivittatus were taken. The jaws and pharyn- geal teeth of fishes of this genus, and probably of this species, are found very commonly with vertebrae and other structures which are slowly digested. Four specimens of Scarus punctula- tus which were only slightly macerated were secured. What appeared to be remains of the same species was obtained in a number of cases. Jaws and pharyngeal teeth, differing sufficiently among themselves to suggest that others of the genus are freely eaten, are frequently recovered, and one Scarus croicensis was doubtfully identified. Sparisoma hoplomystax was represented by one specimen, and another in an advanced stage of de- composition was assigned to this species. If this determination is incorrect another must be added to the list of edible forms. One Sparisoma flavescens was also obtained. Five species of the Labrids and Searids in Jordan and Ever- mann’s Fishes of North and Middle America were described from types, or were known to the authors only from specimens found in the stomachs of snappers or groupers. When these are added to the five mentioned in the preceding paragraph it makes a total of more than 11 per cent of edible forms in the two families. Though many of the Scarids and some of the Labrids when full grown are too large to be preyed upon by the snappers the list presented is certainly capable of extension. It already includes some brilliantly colored types, hence it seems incredible that these creatures are protected by distastefulness advertised by warning coloration. It is also clear that they are not secure from attack at night, and doubtful whether they ever enjoy immunity unaccounted for by their speed, alertness and incon- spicuousness, for some at least wander far from the shelter of the heads, and do not instinctively fly to cover when alarmed. 585 THE COLORATION OF REEF FISHES ‘(6 ‘SY ‘fF 93R][q) o10Ys rvou u10}}0q ApuBs 19A0 sieddvus Avi3 ayy sev snonoidsuoour se A][nJ sures 41 404 ‘uMoys AjLB9]O o1v 9]Suv IOT1a4s0d 83178 4ods 94} puv uy [esaop oy} Jo aseq 94} 78 JOTOo YIwp jo aspnus sy} uoao 10; ‘ooJted St snoo} oy, ‘Sur}jos [eordéy v ul ysy Sty} SmoYs “¢ oIndy ‘TTT a4°eI1g 8 pavysoy poetolod AyaAt}00q401d oq Avur sotoads sty} yey uoTo0uUN00 ToY}OUB Ul soyvys prvysioy | peuTUIo}op ATIIOJOVISIVVS Uaeq Jou sey SUOT}TpUoD Jey Jepun ynq ‘afqevesuvyo st usoqqyed SJ] “saojoo cures oY} AT]eIyUeSse YIM ynq ‘poyxreu A]JUeAyIP St y[npe oy, UOT}B4SOD [B10 UT poseSua aU ‘syovuojs § 1eddeus woay UOYE) S[VNPIATpUr vely, “spvay oyy SUOUIE 4J9[D PROT B UT ATA [MJ Ul pejou usuttoeds ougy “Kup Aq uedo oy} ul uses useq jou svy jeA “uouIUI0D AIOA SI soroads STU, | yovyq Yoru104s Staddeus wos uoyey o10M suouloeds uMOIS-[[NJ voy, ‘SUOT}Ip ~W09 J9q}0 Japun ABM oUTeS ay} UT 9}RI0d0 AvUT Spueq Yyaep sz YF MOYS Soz¥]d quaT[ooxe 8 paeysioy “41 SUIPUNOLINS 1oyVM BY. UT 4SOT ST pUB SaAlOSsstIp IO[OD punoss Avid-an][q 8}r MOTAq WOly PoMOTA STYSY oY} Woy VY} ‘S908 IAAIOSqO ayy SB ‘Moys sydeasoqyoyg | | Se a = ee ee ee | SHUVNaY a a ee eee oe sauids joun pun josiop abot -yaed ut MOT[PA SUT ‘spueq yovyq opIm OM} “punois AaTaATIG sauids [pun pun Jossop abun) ‘40d PRIT puv spuvq youTq ‘oz AA adi1ys MoT[ax ‘AIOATIQ jods [epneo youtq ‘sodrays OM} YIM ‘MOTIOX Sjods youlq omy “oprwog sauids wojnosado ~ald ‘MOTTex yystaq Ayoryo $n}e]]900 wopoyaryy snjersideo uopojavyD soskio xuwie9 SNOTUIGITA Snue1jOSsIUy BpNBol][IS BIULy STIBIT[IO sug ‘ond yY311q Aportyo Apog sxyorposuy (ST[I}exus) snj}eUlsieUr popu Youyq puew MoT[a A jnpyepnqy NOIMLdIMOsad § duVHDIaUL Salogds soysy snonaidsuo), fo 181) paypjouun uy $ HIaVL LONGLEY Wire 586 snpode *NV jo Sunod oy} JOU SI opvuUr ST QoUdIOJOI YOTYM OF WIIOF oY} JVY} “19ABMOY ‘aINs JOU WR T ‘opeys ul sosuvyo aaAtdepe yourjstp Awa sjiqryxe soddvus Avis 4npv oy, 4ST] SIY} UT SoTDeds oY} Jo JSOUI UBY} SNONdIdsUOD sso] 41 potopts -u0d OYM ‘pavystoy Aq pojou AyyIGuiueA “stoddeus Aq uszvo ATuoww0s A19A sy ‘sosvyd uMOIq puB UdeIs ‘Avis OATYdEpB sUTT IO[OO ules ey} Yonut jo ovsye Suoure osvyd snovovarjo uv ul uses Ayyuenb -alj sour sy “Avis oped 04 youyq Woy sosuvA SoapqviavA AOA IOTOD eZIS WNIpsU jo xIS posi03stp uswumtoeds peddvas, ouo {s}unis jo sotveds 10y}0 pue siy} syRo stuqouny styUOpOOAT ‘avozo yoA You SI Ssutpunod -ins s}t Aq poaT[orjuod oie SaSUBYD IO[OD SYS oY} YoryM 0} Yuo, -xo OYJ, “UWOSIoyor “yy 7B BOUT BY} UI puw Yyoop sy} ynoq*e suoUT -roeds Aq podvydstp sv 1oyjyouy ‘sjoor oy} uodn osvyd ouo ueyy d1OUL SUIMOYS poAdosqo Useq JOU SBYy yNq ‘S9}zBqS (6§0,) PUPSUMOT, se ‘10JOO s}I oBduByo 03 APTIqe peytvu seyFY “ABp oy} Bulnp SUBIUOSI0S BSuouw pue spvoey ynoqe spfooyos fiepeesy [vUINJDON JU9}X9 JUIOS 0} IO[OD S}I Sutsuvyo jo siqedey peadesqo vary} 10 OM ATUO ‘sBSNzAOT, 4B WOUTUIOD JON JYSI] JO VANOS oY} OF UOT}LIOL JUBISUOD OU SUTeIUTLIY “SooBjans [Bt}UVA pUB [BSLOp JO epeYs oY} UI G hes ag ‘ca bent t dein " iit a te IY ' Ray Ph hae eas ce wh Se SUBJECT AND AUTHOR INDEX GENTS on the chromatophores of the brook trout Salvelinus fontinalis Miteh- ill. The action of various pharmaco- logical and other chemical................ Amblystoma larvae. Photomechanical changes in the retina of normal and trans- planted eyesiOfessnacns- sass oe: Amblystoma tigrinum larvae to light and darkness. The reactions of the melano- HOLES Ofer cea Prey voies:5 Sictecvers sree Animal coloration. Studies upon the bio- logical significance of Antiopa in intermittent light and in con- tinuous light of different illuminations, and its bearing on the ‘*‘continuous action theory” of orientation. The rate of loco- MOONMMOVANeESSaseec-)1e ale eels eee Applanatus Kennel. Aster: a reversible gelation PaWevouieh on: Microdissection studies. II. The cell Auditory sensory epithelium in the formation of the stapedial plate. Therdleofthe..... RISTLE inheritance in Drosophila. Selection Brook trout Salvelinus fontinalis Mitchill. The action of various pharmacological and other chemical agents on the chro- matophores of the Cae. The free-martin; a study of the action of sex hormones in the foetal life o Cell aster; a reversible gelation phenomenon. Microdissection studies. II. CHAMBERS, JR., Roperr. Microdissection studies. II. The cell aster; a reversible gelation phenomenon CHAPIN, CATHERINE L. «2 cielo rece Da on the eye of Prorhynchus ap- planatus Kennel. Effects of light and 147 147 453 147 507 225 519 603 Darkness. The reactions of the melanophores of Amblystoma tigrinum larvae to light EG | pope eee ET RAPE aan SORE ORG 195 Didinium nasutum with especial reference to their significance. Conjugation and en- CY StMENn GIN 907.0104 cas oh cere aera ieee 335 Douey, Jr., Wrii1AM L. The rate of loco- motion in Vanessa antiopa in intermittent light and in continuous light of different illuminations and its bearing on the “continuous action theory”’ of orientation. 507 Drosophila. II. Selection. Bristle inherit- ATICONITAS iris re tiots av erele eitue, roe oe erelemaarect 109 NCYSTMENT in Didinium nasutum with EK especial reference to their significance. Conjugationsandies. epee ieee 335 Environment on sex. Studies on sex in the hermaphrodite molluse Crepidula plana. De: Influence Obs asecctes cr ero tae he Sako e ras erate 225 Epithelium in the formation of the stapedial plate. The réle of the auditory sensory.. 85 Eyes of Amblysoma larvae. Photomechan- ical changes in the retina of normal and transplanted sare Oey aeRO COMETS sade ees 71 Eye of Prorhynchus applanatus Kennel. Effects of light and darkness on the...... 519 OETAL free-martins. A microscopic study of the reproductive system of.......... 53 Foetal life of cattle. The free-martin; a study of the action of sex hormones in the...... ay Free-martins. A microscopic study of the reproductive system of foetal............. 453 Free-martin, a a study of the action of sex hor- mones in the foetal life of cattle. The .. 371 Hrog, tadpoles: The photokinetiec reactions 0 ne eRe SOR OaGaO DEO cere eTA oo 00 cls ame 361 Function with alterations in pigmentation. Evidences associating pineal gland...... 207 ELATION phenomenon. Microdissection studies. II. The cell aster:areversible. 483 Gland function with alterations in pigmenta- tion. Evidences associating pineal....... 207 Gouup, Harugey N. Studies on sex in the hermaphrodite moilluse Crepidula plana. 1. History of the sexual cycle............ 1 Gouup, Haruey N. Studies on sex in the hermaphrodite molluse Crepidula pinDe: II. Influence of environment on sex.... 225 ANCE, Rosert T. Studies on a race of H paramoecium possessing extra contrac- tile vacuoles. I. An account of the morphology, physiology, genetics and eytology of this new race................-- 287 Hermaphrodite mollusc Crepidula plana. II. Influence of environment on sex. Studiesioniseximitheweres ce... seer cee 225 Hibernation. The spleen during............. 277 Hormones in the foetal life of cattle. The free-martin; a study of the action of sex... 371 NHERITANCE in Drosophila. II. Selec- LION. MO DISbLE se eile re cle yee letec isthe a ees 109 Intermittent light and in continuous light of different illuminations, and its bearing on the “continuous action theory” of orientation. Vanessa antiopa in The rate of locomotion in 604 Kee Effects of light and darkness on the eye of Prorhynchus applanatus.... 519 Kepner, Wn. A. and FosHerr, A. M. Effects of light and darkness on the eye of Pro- rhynchus applanatus Kennel............ 519 ARVAE to light and darkness. The reac- tions of the melanophores of Amblystoma TLOT UU 234.42 <2 ye oo es aioe arco imeoe eae tale 195 LAURENS, Henry. The reaction of the mel- anophores of Amblystoma tigrinum lar- vae to light and darkness...............-- 195 Laurens, HENRY AND WILLIAMS, J. W. Photomechanical changes in the retina of normal and transplanted eyes of Ambly- stoma larvae? : hee. ee ctlset cn iain oe nieinie 71 Light and in continuous light of different illuminations, and its bearing on the “continuous action theory”’ of orientation. The rate of locomotion in Vanessa antiopa IMEUNCEHIMGGbOM Ge cise = peickeste sterols cceis eae 507 Light and darkness on the eye of Prorhynchus applanatus Kennel. Effects of........... 519 Light and darkness. The reactions of the melanophores of Amblystoma tigrinum LTE VAGHE OMIM Oo cute set « ctteserai dys oleae a eitlensuacnteraye so 195 Light.’ Reactions of the whip-tail scorpion to 251 Liniin, Frank R. The free-martin; a study of the action of sex hormones in the foetal itis Gi. (C;Z1htel Fees GAAS cneich a eiciee aula os sie 371 Locomotion in Vanessa antiopa in intermittent light and in continuous light of different illuminations, and its bearing on the “continuous action theory’’of orientation. alterna tev Ob seoys spe. mesic crs taie’s,dyere exepeyey erate cuca 507 Lonatery, W. H. Studies upon the biological significance of animal coloration........ 533 Lowe, Joun N. The action of various phar- macological and other chemical agents on the chromatophores of the brook trout Salvelinus fontinalis Mitechill............ 147 ACDOWELL, Epwrn Carterton. Bristle inheritance in Drosophila. II. Selection 109 Mann, FranxK C. and Dries, Detta. The spleen during hibernation................ 277 Mast, S. O. Conjugation and encystment in Didinium nasutum with especial reference tortheimsigniheance : 5. cea- cs site dele see ee 335 McCorp, Carry’Pratr AND ALLEN, FLoyD P. Evidences associating pineal gland function with alterations in pigmenta- LOST en st sz cVipasieee aitapa bo. susseyare Sine Al iece vanes 207 Melanophores of Amblystoma tigrinum larvae to light and darkness. The reactions of EERO RE easiest ava a/ni co oy SPIE A «Sota Sa TNS 195 Microdissection studies II. The cell aster: a reversible gelation phenomenon........... 483 Microscopic study of the reproductive system of foetal free-martins. A................. 453 Molluse Crepidula plana. 1. History of the cycle. Studies on sex in the hermaphro- ATES Pelee ap cheteidah aoe: o cote 0.4/0 essen sioeTEME ela arerere)s 1 Molluse Crepidula plana. II. Influence of environment on sex. Studies on sex in the hermaphrodite... .0 ic. seem sccm 225 ASUTUM with especial reference to their significance. _ Conjugation and encyst- pealcyern Gow DIKoh tsk: 160 See eames cAlar Seeley Aare 335 ARAMOECIUM possessing extra contrac- tile vacuoles. I. An account of the morphology, physiology, geneties and cytology of this new race. Studies on a TARO Of ere rewire ome eye att tiscegert ie es oia nee 287 Patren, BrapLtEy M. Reactions of the whip- CALM BCOMPION tO Lighten ane. eo see eeeteee 251 Pharmacological and other chemical agents on the chromataphores of the brook trout Salvelinus fontinalis Mitchill. The ac- LION OL VATIONS) Nokisieeie ee niecn aicleeie ae Oe 147 INDEX Photokinetic reactions of frog tadpoles. The 361 Photomechanical changes in the retina of nor- mal and transplanted eyes of Ambly- stoma) lar vactae a hate nee 71 Pigmentation. Evidences associating pineal gland function with alterations in........ 207 Pineal gland function with alterations in pig- mentation. Evidences associating...... 207 Plana. I. History of the sexual cycle. Studies on sex in the hermaphrodite mollusei@repidulawai ee wosee eee 1 Plana. II. Influence of environment on sex. Studies on sex in the hermaphrodite . molluse'@repidulapecs-e +e nes sea 225 Prorhynchus applanatus Kennel. Effects of light and darkness on the eye of....... 519 ATE of locomotion in Vanessa antiopa in intermittent light and in continuous light of different illuminations, and as bearing on the‘‘continuous action theory” of orientationty elibetsat yn. «sok sulin 507 Reactions of frog tadpoles. The photokinetic.. 361 Reactions of the melanophores of Ambly- stoma tigrinum larvae to light and dark- ness Phe... epee aoe. ok ee 195 Reactions of the whip-tail scorpion to light... 251 REAGAN, FRANKLIN PEARCE. The role of the auditory sensory epithelium in the formation of the stapedial plate........... 85 Reproductive system of foetal free-martins. A microscopic study of the................ 453 Retina of normal and transplanted eyes of Amblystoma larvae. Photomechanical changes'in the:23...\)s see Se ee oa Reversible gelation phenomenon. Microdis- section studies II. Thecell aster: a....... 483 ALVELINUS fontinalis Mitchill. The action of various pharmacological and other chemical agents on the chromato- phores of the brook trout................. 147 Scorpion tolight. Reactions ofthe whip-tail.. 251 Selection. Bristle inheritance in Drosophila. Sensory epithelium in the formation of the stapedial plate. The réle of theauditory.. 85 Sex in the hermaphrodite molluse Crepidula plana. I. History of the sexual cycle. Studies On 52... Aa epee iene ate 1 Sex in the hermaphrodite molluse Crepidula plana. II. Influence of environment on sex. “Studies /oni.).< aise ese sieve ules 225 Sex hormones in the foetal life of cattle. The free-martin; a study of the action of...... 371 Spleen during hibernation The............. 277 Stapedial plate. The réle of the auditory sensory epithelium in the formation of the. 85 ADPOLES. The photokinetie reactions OF Prog eee eee ahs ee ee ne 361 Tigrinum larvae to light and darkness. The reactions of the melanophores of Am- Dlystomiaiseeeree wire sca nchaelt ane aerate 195 Trout Salvelinus fontinalis Mitchill. The action of various pharmacological and other chemical agents on the chromato- phoresiofsine brook»: ...¢)/... asses 147 ACUOLES. I. An account of the mor- phology, physiology, genetics and eytol- ogy of this new race. Studies on arace of paramoecium possessi ng extra contractile. 287 Vanessa antiopa in intermittent light and in continuous light of different illuminations and its bearing on the ‘‘continuous action theory’”’ of orientation. The rate of locomiothomaAm <0 .60 cisco ineieeieeem teeta 507.2% +3 HIP-TAIL scorpion to light. Reactions OFHO TEs Js cbasdecietactee Oh elsares Se . 251 a evn | HY bY fr M nt Si vik a Nh 127 AL, Fee te ; rat SS Aer See {x} of ote tt ra . ‘* > aa ~ BS See 5 eee RYH axey ens s og te aio oe taletaigtere’s ~ pre Saye Fy Sah