5 ge ai ees savas oghet ig ace Beh S_ 9 P98 segs arate aes ies Neal ad ~ nt fs! oe eee ee well eh i er er ae aid ms ot) ~ ee ee eRe Yas Yate fa Tata had ae ella ._s ¥ al taek a ha A Se AA te nee 4 2.33.8 bb ee eb eb ee }_a_o._6. 5 _o_b_b_ See oe at hh te BE at ee tl ee Pe ae ee ee bb eee ee a od te te AP tee ae nt te a St ee oo we ee el tt ede had eal 2. ) AD A Rut ghee! yee pe ee £ VS (<7-s a el et at tae erst Fh ec de ee Pe t_6s 98 . Fae ob 6 ite a PaaS J aaeS, PF PP PD r Pe BP ae Fo PD 2A VEAP 59 eee nae seieiea tee. ST eerie Heater eree THE JOURNAL OF EXPERIMENTAL ZOOLOGY HDi D By WiuiiaM Ef). Caste Jacques Lors Harvard Univei “7 The Rockefeller Institute Epwin G. CoNKLIN Epmunp B. WILSON Princeton University Columbia University Cuarues B. DAVENPORT Tuomas H. Morgan Carnegie Institution Columbia University HERBERT S. JENNINGS GEORGE H. PARKER Johns Hopkins University Harvard University Frank R. LILLIE RAYMOND PEARL University of Chivago Johns Hopkins University and Ross G. HaArrRIson, Yale University Managing Editor | VOLUME 32 JANUARY—APRIL, 1921 | | THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA. ; i a \ . a < j } N if - « | A, ay \ 5 ae Aa inhvaSe } {, , _ ‘? ier ‘, , eg | < Aa j WY A ys cn % mice ; é f Sat PA ae rb df ee CONTENTS No. 1. JANUARY Ross G. Harrison. On relations of symmetry in transplanted limbs. One huncredvand: thintvestxaneuUees cys... a ie sete atlas ake es eens 1 JosePpH Hatut Bopine. Factors influencing the water content and the rate Of metabolism oficertain orthoptera. Six figures .,........0742.-.54.0. 137 CHARLES W. Merz anp Jost F. Nonrprz. Spermatogenesis in the fly, Asilus sericeus Say. Two plates (twenty-two figures) ..................0005. 165 No. 2. FEBRUARY Haroup H. Proucu. Further studies on the effect of temperature on cross- INCOVeLwMbhree-fromesre waa os ates eerie: Seas ee eee 187 W. E. BurGcr anp E. L. Burak. An explanation for the variations in the intensity of oxidation in the life-cycle. One figure.................... 203 Henry LAvRENS AND S. R. DetwiterR. Studies on the retina. The struc- ture of the retina of Alligator mississippiensis and its photochemical changes) “lhirteen timbres ere, 5s, cs. myers aise sod oer ae nee en eae 207 Wivsur WILuis SwincLe. The germ cells of anurans. I. The male sexual cycle of Rana catesbeiana larvae. Two text figures and fifteen plates (one -hundredvand thighy-one figures): 02.2.0). «5.8 de nosedevec dees nwes 235 J. A. DETLEFSEN AND E. Roperts. Studies on crossing over. I. The effect of selection on crossover values. Two text figures.................... 309 No. 3... APRIL RutxH B. Howiann. Experiments on the effect of removal of the pronephros of Amblystoma punctatum. Mwenty-three figures)... .. o26. 2202 4.. (000 Wm. A. Kepner AnD W. Cart WuttLock. Food reactions of Ameba proteus. Si plates. (twenty-one memes ie jee sce ccstencs koe ene sue sen cee cae 397 ALFRED O. Gross. The feeding habits and chemical sense of Nereis virens USP R NE SCs 22 « 2c eS et REE ok RE, cai Ne ee ame a 427 Lesure B. Arny anp W. J. Crozrer. On the natural history of Onchidium. 443 iil (kee | 15 ee i e fee He ee Pile 7 4 a4 - ; x ve als? . r, be Bee a ‘i. es | hee ge pow alg ‘9 awe : “a - i ti = s < js ya _ aE, % y Ki as j : / =i oe TA = is 1 wy) Pra se tr) Aleit’. Ris mays rhe ia i i ao ’ a oY ? ‘ 2 \ ie b Aba Gel, tae Se phe Bye ‘ ; ear mi i ; Aitia® \ +0 ay & HSS f ‘ i 7) ’ - 2 ‘ve , 5 - / : = Pee “st } + Wy x 7 ‘ a ‘" f 4 aha , 4 pe 3 sia" 3. 2 j \ ie { io 7 ie Ie £ ’ > ‘ $e é yoy ea: ~ aes : / j - het tat jy pps, iA one }- " yey oat | A LISP Sal 4 Z ! | ne ‘ , oe rac’ | es aha . } : : , ries Shain ee : : o- id) = ihe? < ey, iY? Sb sd pid te 8 \ 2 j 4 ; ee jhe ro j ix nny brah , ' ir “ : Pr ive j a ’ ; Ny : =v Jy F j PAln = ‘ of me i } + a8 : : | } ry ia é id * h sPeet my " : " Pray ‘ P * re 4 ’ ‘i or ‘ ol Milse - = 4 q ‘4 as r ih vai 3 4 ye } ? = ; * 1 - i a = i *, ' rs & JANUARY, 1921 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, NO. 1 rr oe? Geel, = iy = i > oi =a = Resumen por el autor, Ross G. Harrison, Yale University. Sohre las relaciones de simetria en los miembros transplantados. El autor ha llevado a cabo los siguientes experimentos con los esbozos de los miembros anteriores de Amblystoma: Trans- plante a la superficie lateral del cuerpo de otro embri6n; trans- plante al sitio normal después de extirpar el esbozo del miembro que le ocupaba; superposicién de un esbozo sobre otro después de separar su ectodermo; y transplante de medio esbozo en el sitio en que existia otro medio esbozo, extirpado previamente. Los injertos fueron obtenidos en el mismo lado del cuerpo o- en el lado opuesto e implantados con el eje dorso-ventral situado normalmente o invertido. Los dos primeros grupos de experi- mentos indican que cuando el eje dorso-ventral del miembro no esta invertido persiste la simetria prospectiva originaria, y que cuando se invierte, la simetria se invierte también (simetria enantiomorfica). La asimetria esta determinada: 1) Por la polarizacion del eje antero-posterior del esbozo y 2) Por la orientacién del esbozo respecto a la polarizacién dorso-ventral del ambiente orgdinico. Dos combinaciones (armé6nicas) pro- ducen miembros con la asimetria correspondiente al lado en que se colocaron; otras dos (desarménicas) producen miembros con asimetria opuesta. Se encuentran con frecuencia reduplicaciones el miembro primario sigue en este caso las reglas mencionadas, mientras que los miembros secundarios simulan sus imagenes producidas por un espejo, y a veces duplicados a su vez. Existe conformidad con las reglas de Bateson, las cuales sin embargo pueden enunciarse mas simplemente para incluir los supernumer- arios sencillos y dobles. En los experimentos con mitades de esbozos superpuestas, salvo ciertas excepciones, las combinaciones armonicas producen miembros sencillos y las desarménicas reduplicaciones de acuerdo con las reglas. El mesodermo del esbozo del miembro es un “‘sistema arm6nico equipotencial”’ y excepto para la determinacién de ciertas relaciones axiales, es autodiferenciable. Su forma, incluso sus relaciones simétricas, debe estar representada en su estructura intima. Translation by José F, Nonidez Cornell Medical College, New York AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, NOVEMBER 1 ON RELATIONS OF SYMMETRY IN TRANSPLANTED LIMBS ROSS G. HARRISON Osborn Zoological Laboratory, Yale University ONE HUNDRED AND THIRTY-SIX FIGURES CONTENTS OIG HOM... ::: < Peer sera eriee oOo ce ec R ie Seite ince fe 5 See oantaa tat Method svanduserminglocyeri ate Sie tyne ome rare oe ie eee ees eee ee General features of the development of the transplanted buds.............. XPS GIN ental ets <<, ., AeMPIeNreey wer Ane Oy see err ee cads a Slat a tall hiro ae A. Limb buds implanted in abnormal location—heterotopic trans- PLTGAtIONS eee ne eas ecco Pen een cle as be crea eae Ot PB OO 6. B. Limb th 8. 9. 10. 11. 12. . Homopleural transplantations, normal or dorsodorsal orien- FCO 1 RR ce, IL Te Ss ee is ep toe Reng LN eh 2 . Heteropleural transplantations, dorsodorsal orientation........ . Heteropleural transplantations, dorsoventral orientation........ . The shoulder-girdle in heterotopic transplantations............. Summary of the results of heterotopic transplantations......... buds implanted in natural location—orthotopic transplantation. Homopleural transplantations, dorsodorsal orientation.......... Homopleural transplantations, dorsoventral orientation......... Heteropleural transplantations, dorsodorsal orientation......... Heteropleural transplantations, dorsoventral orientation........ The shoulder-girdle in orthotopic transplantations.............. Summary of the results of orthotopic transplantations.......... C=cuperposed: limb. bildsuwmewer tte. 02 cares cc akin ods atl bioua. nemotes os 13. 14. 15. 16. 17. Homopleural transplantations, dorsodorsal orientation........... Homopleural transplantations, dorsoventral orientation......... Heteropleural transplantations, dorsodorsal orientation......... Heteropleural transplantations, dorsoventral orientation........ Discussion of experiments with superposed limb buds........... Dwiransplantation of halfipmusererne sso. she ce ate Seek sone ee ek 18. 19. 20. 21. 99 1 11 14 14 Ale 1g) 21 25 2d 29 29 30 31 43 55 57 2 ROSS G. HARRISON Generaldiscussions ic cckac:sescmie ce oek lo Shea RIO oles 5) oe ee 83 E. The rules of symmetry.............. He oA PE OF Seo ese 83 F. The mode of representation of symmetric relations in the limb TUGTNENG fo 28h. ee oo eco nd Fo a ee 85 G. Reduplication and the problem of polarity and heteromorphosis. 92 H. Form regulation and function in transplanted limbs..,.......... 102 Generalesummary. co cae soe ee ee en in eo 109 Iseirob references fe .2 2s oS Fe 4 ee occ es 0 ee er 115 Appendix—Histories of selected individual cases..............-.2...++00.00. 119 INTRODUCTION The circumstance that originally suggested the present study was the apparent difference in the results obtained by Streeter (07) and by Spemann (710) in their respective experiments with the amphibian ear vesicle. According to the original account of Streeter, the otocyst, when taken out of an embryo just after closure and replaced after having been rotated 180° on any of its axes, develops in normal posture, though a right vesicle placed on the left side remains true to its side of origin. According to Spemann, the inverted vesicle develops in inverted position, the rudiments of the constituent parts being localized, at the time of operation. Although subsequent work by Streeter (14) seems to have shown that the normal development of the inverted vesicles, found in his cases, was due to their regaining normal posture by rotation as a whole, the original divergence of results nevertheless had raised theoretical questions of great interest, which Spemann has ably discussed. The main question was whether we might have in the otic vesicle an ‘harmonic equipo- tential system’ with its future asymmetry in some way stamped upon its intimate structure. Though Spemann’s analysis an- swers the question in the negative, as far as the closed ear vesicle is concerned, it is nevertheless important to determine how far, if at all, systems of this kind are present in the embryo, for their study would throw light upon the question of the mode of rep- resentation of adult form characters in the germ, giving evidence from a new quarter with regard to the great problems of devel- opment which have usually been approached by way of experi- ments upon the unsegmented egg and the early stages of cleav- age. The method of embryonic transplantation obviously SYMMETRY IN TRANSPLANTED LIMBS 3 affords a means of studying this question in any organ or part that in the adult lacks a plane of symmetry. It was with this purpose in view that the present experiments with the limbs of Amblystoma were begun. Limb buds were implanted in both normal and abnormal location, oriented in various ways with respect to the main axes of the embryo-host, and the form and posture of the resulting limbs were studied. It became evident, after the first experiments were made, that the rudiment of the fore limb behaved differently from the audi- tory vesicle, no matter whether Streeter’s original interpretation or Spemann’s was accepted as correct. While it was found that a certain tendency did exist for inverted limb buds to rotate back to normal posture during development, this was not the usual result, nor did the rotation take place in the sense meant by Streeter in his later publication (14). Furthermore, many irregularities of development were produced by the operations, due largely to the power of the limb rudiment to duplicate itself by budding. On the other hand, it often occurred that buds transplanted from one side of the body to the other developed in harmony with their new surroundings, a right limb bud, for ex- ample, placed on the left side, giving rise to a normal left limb. The earlier experiments which were made in 1911 and 1912 led to no satisfactory general conclusion, so that publication of the work was deferred pending further investigation. Subsequently numerous additional experiments were made, in which more ef- fective precautions against regeneration of the limbs from the host were taken.2 The situation began to clear when in some of the cases in which the asymmetry of the limb was reversed by 1 Cf. Barfurth (94), who showed that supernumerary limbs could be produced in amphibians by regeneration after irregular amputation; Tornier (’05), who obtained multiple appendages by cutting into the limb bud of tadpoles; Braus (04, ’05, and ’09) and Harrison (’07), who found that transplanted limb buds frequently give rise to double limbs. 2 The first experiments were reported to the National Academy of Sciences in November, 1912, at the New Haven meeting. Later reports were made before the American Association of Anatomists in December, 1915 (’16), and before the American Society of Zoologists in the following year (’17 a). The main results have been stated somewhat more fully in the Proceedings of the National Acad- emy (715 and 717 b). 4 ROSS G. HARRISON transplantation it was observed that the reversal came about by a process of reduplication or twinning. By following closely the history of individual cases, it became evident that the double formation was not infrequently obscured by the preponderance of the reduplicating limb bud over the original, so that the former - grew into a member of opposite asymmetry, while the original bud was reduced to a mere spur or nodule, which might readily _ be overlooked. The tendency to produce duplicities thus proved to be even greater than the actual number of fully developed cases indicated. In other cases the reversal appeared to be more direct; at least, a limb of the side of origin often failed to appear as such on the surface, though slight irregularities in the early stages of development, coupled with an appreciable delay in the process, showed that some internal readjustment of the grafted tissue was taking place. It. seemed that the functional activity of the limb, when trans- planted to its normal environment, might accentuate the appar- ently anomalous results just described. In order to eliminate this factor, a series of experiments in which the limb bud was grafted on some other part of the body was undertaken. Here the proper nervous connections did not become established, func- tional activity was usually lacking or was at best but slightly developed, and the undisturbed effect, upon development, of the relative orientation of the tissues of graft and host could be ob- served. The latter experiments led to the formulation of the three following simple rules,? which hold for implantations either in normal or in abnormal location: Rule 1. A bud that is not inverted (dorsodorsal) gives rise to a limb of the side of origin of the bud, whether implanted on the same or on the opposite side of the body. Rule 2. An inverted bud (dorsoventral) gives rise to a limb of reversed asymmetry, whether implanted on the same or on the opposite side of the body. Rule 3. When double limbs arise, the original one (the one first to begin its development) has its asymmetry fixed in accord- 3 These rules were phrased somewhat differently in two preliminary communi- cations (717 a and 717 b). SYMMETRY IN TRANSPLANTED LIMBS o ance with rule 1 or 2, while the other is the mirror image of the first. Experiments previously reported‘ have shown that the limb bud is an ‘harmonic equipotential system,’> and additional experi- ments with inverted buds (p. 87) and with half buds (p. 83) confirm this result. We must assume, then, that the potencies of the cells of the limb bud to form the fore limb are in the last instance represented in their intimate structure and not merely in their arrangement. ‘The above rules show, however, that not all essential features are stamped upon the constituent elements of the rudiment at the time of transplantation. For example, the difference between the right bud and the left is not an abso- lute one, since a right limb bud upside down behaves like a left one right side up and vice versa. From this the conclusion has been drawn that the elements making up the limb bud are dif- ferentiated in an anteroposterior direction, 1.e., along the antero- posterior axis, but are not yet differentiated, at least not irre- versibly, along the dorsoventral axis at the period of development at which the transplantations are made. In this one respect the differentiation of the limb is dependent upon its orientation with reference to the dorsoventral axis of the embryo; otherwise as regards its specific form, the limb bud constitutes a self-differenti- ating system. These questions will be considered more fully in the concluding section (p. 85). : METHODS AND TERMINOLOGY All experiments were made upon embryos of Amblystoma punctatum in stages that have been previously defined.® In performing the operations the embryo which is to receive the implanted limb bud is first made ready. If the bud is to be placed in normal location, the wound is prepared as in the extir- pation experiments referred to above. A circular incision, hay- 4 Harrison, 718. > “Jedes kann Jedes und alles Einzelne steht in Harmonie zu einander.’’ (Driesch, ’02, p. 229.) See also: 799, p. 72; ’05, p. 679, and ’08 b, p. 120. 6 Harrison, 715 and ’18. 6 ROSS G. HARRISON ing the diameter of three and a half somites (ca. 0.9 mm.) and ventral to the third, fourth, fifth, and half of the sixth myotome, is made, and the dise of tissue, including both mesoderm and ectoderm, is lifted, after which the remaining mesoderm cells are carefully cleaned off. ‘This may all be done without injury to the pronephros, which lies immediately dorsal to the limb rudi- ment, though if this organ is injured or even extirpated, there is no noticeable effect on the subsequent development of the limb. The embryo thus prepared is held in readiness for the grafting, being secured in position by pieces of silver wire or glass rod bent into proper shape. The limb bud which is to be transplanted is removed from another embryo, as described above, care being taken in lifting it from its bed to take all of the mesoderm pos- sible. It is then transferred on the point of the scissors to the first embryo and fitted into the wound. The orientation of the bud is important and may be earried out as desired by noting the position of pigment markings in the ectoderm of the graft. After it has been properly placed, it is held in position for an hour or more by a single piece of glass rod, bent into such shape that it straddles the embryo, exerting a light pressure upon the grafted tissue. The healing of the wound takes place readily, though frequently a small area of underlying yolk may be left exposed on the border of the wound. ‘This usually heals in a day or two and does not seem to influence the result of the experi- ment, unless the yolk begins to disintegrate, in which case death of the embryo usually follows. At the time when the first experiments were made, the condi- tions necessary to prevent regeneration had not been determined, so that in a number of cases the extirpated area was too small or the wound bed was insufficiently cleaned of scattered meso- derm cells for the result to be conclusive. These experiments have not been included in the tabulations, but will be referred to separately in so far as theyare of special interest. In all of the later experiments the size and character of the wound in the host was such as to preclude regeneration of the limb from that source; the resulting limbs must, therefore, have arisen from the engrafted tissue. Even in the cases where the wound was SYMMETRY IN TRANSPLANTED LIMBS 7. not especially cleaned, there is no evidence, aside from certain exceptional cases, that the tissue of the wound bed displaces the transplanted bud, though the possibility of its participation in the make-up of the limb cannot be excluded, and it probably actually does take place to some extent. In transplanting the limb bud to a location other than the normal, the recipient embryo is first prepared as in the other experiments. A wound of proper size is made, usually in the flank just below the ventral border of the myotomes, and the bud grafted in the same manner as described above. In doing this it is well to avoid injury to the pronephric duct (p. 15). Three different factors regarding the placement of the limbs have been considered in these experiments, viz.: 1) location of the graft in the embryo; 2) the side of the body on which it is placed (whether the same from which it was taken or the oppo- site); and 3) orientation with respect to the cardinal points of the embryo. The experiments thus fall into eight categories, as follows (fig. 1): A. Limb buds placed in abnormal location—heterotopic transplantation. 1. On the same side of body as origin—homopleural (hom.). a. Dorsal border of limb bud dorsal with respect to embryo— dorsodorsal (dd.). b. Dorsal border of limb bud ventral with respect to embryo— dorsoventral (dv.). 2. On side of body opposite to origin—heteropleural (het.). a. Dorsal border of limb bud dorsal with respect to embryo— dorsodorsal (dd.), in which case the anteroposterior axis isreversed. b. Dorsal border of limb bud ventral with respect to embryo dorsoventral (dv.), in which case the anterior and posterior points of the graft correspond, respectively, to those of the embryo. B. Limb bud placed in natural location—orthotopic trans- plantation. The several categories under this head as under A. According to the rules stated on page 4, two of the combina- tions (homopleural dorsodorsal and heteropeural dorsoventral) yield limbs which are of the same side of the body as that on 8 ROSS G. HARRISON which they are placed (fig. 2), for the non-inverted limb bud from the same side (hom.dd.) does not have its prospective asym- metry changed while the inverted limb bud from the opposite side (het.dv.) does. The limbs which develop in these combina- D | | P RA). Vv \ ORTHOTOPIC D D D D D v D Vv PP RAJA P(A R pia PIA L PJA PIP L AJA Vv D Vv Dy Vv v Vv Vv HOM. HOM. HET. HET. D-D o-Vv D-D ov Fig. 1 Diagram showing the eight different operations. The outline of an Amblystoma embryo in the operating stage is shown above. The circles within it represent the limb bud, in the normal (orthotopic) and the abnormal (hetero- topic) location. The four circles below represent the four different ways in which limb buds may be oriented with reference to the cardinal points of the embryo; the letters (D, dorsal; V, ventral; A, anterior, and P, posterior) within the circles designate the original cardinal points of the transplanted limb, those outside the corresponding points of the embryo. The operations are represented to be on the right side. R, right limb bud; ZL, left limb bud; hom., homopleural; het., heteropleural. tions thus fit in with their surroundings; they have therefore been ealled harmonic. The other two combinations (homopleural dorsoventral and heteropleural dorsodorsal) give rise to limbs of the side opposite to that of their seat of implantation, for the inverted bud from the same side (hom.dv.) has its asymmetry reversed, while the non-inverted bud from the opposite side SYMMETRY IN TRANSPLANTED LIMBS 9 (het.dd.) remains as it was. The limbs which develop here are not primarily in harmony with their surroundings, so that these combinations have been termed disharmonic. “AIA--- MIRROR PLANE IT HOM. o-V / | | | 3 / \ ; . ' ' ' ; , / i i MIRROR PLARE II __ MIRROR PLARE I Fig. 2. Diagram showing the results of the four operations, heterotopic or orthotopic, represented as on the right side of the embryo. The circles indicate the transplanted limb buds, the letters having the same significance as in figure 1. Thus the two upper figures in the diagram represent homopleural, and the two lower ones heteropleural transplantations. The two on the left show the trans- planted bud in upright (dorsodorsal) orientation while the two on the left are inverted(dorsoventral). The limbs which develop are shown in profile, the ulnar border being uppermost (dorsal) in all which actually develop. A heavy outline indicates the primary member, a light outline the reduplicating one. It is to be noted, however, that the latter develop in by no means all cases, while the former may be resorbed in the heteropleural dorsoventral combination, leaving only the reduplicating member present. The broken outlines show the posture that the limb would have assumed,had it developed as a self-differentiating mem- ber totally independent of the influence of its surroundings. The transplanted limb bud is a flattened dise of tissue, and it is theoretically possible to make eight further combinations by placing the medial surface of the graft corresponding to the lateral surface of the embryo. This is impracticable, however, because 10 ROSS G. HARRISON the mesoderm would thus be brought to the surface and the ecto- derm buried beneath it. The same effect might be obtained, however, by transplanting the mesoderm alone. While the dif- ficulties in this procedure are great, they have now been in a large measure overcome. The positive experiments are too few in number to warrant any very definite statement, but they do indi- cate that it is immaterial which surface of the mesodermal dise faces laterally. A much greater variety of experiments could be had by experi- menting with positions intermediate between the upright and inverted positions, i.e., with limb buds turned, say, 90° instead of 180°. Such experiments may yield very interesting results, but as yet there has not been sufficient time to carry them out, nor has the effect of implanting the limb exactly in the midline been studied.’ The experiments with superposed buds were made in the same way as the above, except that the mesoderm of the host was not excised. In the case of half buds, more combinations are pos- sible, as described in the section dealing with this group. Both here and in the superposition experiments all possible positions with regard to the placement of the graft within the limitations stated above were experimented with. Relations of harmony and disharmony proved to be the same here as in the case of whole buds. The total number of cases of which records have been kept is 462. The analysis is based, however, upon the 271 individuals which yielded positive results. The identity of the individual cases has been maintained by rearing each in a container by itself and keeping a separate history of each. These histories consist in notes and in sketches made from time to time directly from the living specimens, mostly with the aid of the camera lucida. In dealing with so large a mass of material it has of course been necessary to select typical cases for presentation, and in order not to interrupt the continuity of the general account, the indi- vidual histories, as far as given, have been gathered together in an appendix. The main body of the paper has been divided in SYMMETRY IN TRANSPLANTED LIMBS ila accordance with the outline presented above. The larger groups of experiments have been considered apart from each other, and each subgroup is treated in a special section. The peculiar fea- tures of each of the larger groups have been considered at the beginning, and the results of the experiments summarized sep- arately at the end of each main section. The more general questions are treated in the final chapter. It has been thought best to provide numerous illustrations in order to avoid lengthy descriptions. Since it was not possible to keep a complete pictorial history of each case, those were se- lected for drawing that promised typical or otherwise interesting results. Unfortunately, however, it was not always possible to predict what the outcome of an experiment would be, so that some important cases were not drawn in early stages, while others of less interest were.’ GENERAL FEATURES OF THE DEVELOPMENT OF THE TRANSPLANTED BUDS The development of the transplanted limb buds must now be considered in comparison with normal development. When the normal limb bud appears it is a round prominence just below the pronephros. It soon becomes more sharply marked off from the background and begins to ‘point’ dorsoposteriorly.’ The radial border of the fore arm and hand is at first ventrolateral, then ven- tral, and the first digits to arise are the first and second. The third and fourth digits appear later on the dorsal border of the hand, so that there is never any difficulty in distinguishing the ulnar from the radial border unless the third and fourth digits are entirely suppressed. The palmar surface of the hand faces at first ventromedially and later medially. The transplanted limbs, both heterotopic and orthotopic, give evidence of their orientation early in development, inasmuch 7 Almost all of the preliminary sketches and many of the finished drawings were made by Miss Lisbeth Krause. The former, which were pencil sketches, had to be redrawn for reproduction. For this part of the work and also for a number of the original drawings I am indebted to Mr. A. Hemberger and Mr. H. D. Rhynedance. 8 Harrison, 718, p. 419. 12 ROSS G. HARRISON as their direction of ‘pointing’ is determined principally by the bud itself. In two of the combinations (homopleural dorsoven- tral and heteropleural dorsodorsal), they point anteriorly or dorsoanteriorly; in the other two (homopleural dorsodorsal and heteropleural dorsoventral) posteriorly or dorsoposteriorly like the normal. The subsequent development in the latter case is normal, but in the former there is a tendency for the limb to stick out more sharply to the side or to rotate more or less from the position in which it would be found were the position determined entirely by the orientation of the bud itself. Nevertheless, the palm tends to face ventromedially, or else the limb is so rotated that it faces more ventrally or anteriorly. In order to determine whether the limb is right or left, it is necessary to be able to dis- tinguish between the palm and the back of the hand, which is not always so simple as it might seem. It can usually be done, however, by noting the digits, which are frequently slightly flexed. When there is uncertainty, it 1s necessary to resort to sections, in which case there is no difficulty in distinguishing be- tween the two faces, because of the much greater thickness of the soft parts on the flexor surface of the skeleton. The duplicities that arise are of all grades and kinds, and occur in very different proportions in the several experiments. Some- times they make their appearance very early, sometimes late in development. In the orthotopic grafts reduplication is far more common when the developing limb and the substratum are of opposite sides. In such cases the doubling member nearly always appears as a bud posterior to the main limb, growing there into a limb of proper asymmetry. The extent of reduplication may include the whole limb from the shoulder down, or only certain of the digits. The duplicate limb is as if it were mirrored from the original in a plane which is perpendicular to the plane of the proximodistal axes of the two limbs® and which cuts the axes of the two limbs at their junction, at an angle which varies from almost 0° to 90°. In the former case the two members are almost parallel, in the latter they diverge in the opposite direction at almost 180°, the mirror plane bisecting the angle between them 9 Bateson, Materials for the Study of Variation, p. 479. x SYMMETRY IN TRANSPLANTED LIMBS . 13 (fig. 3). In the present paper the relation of the mirror plane to the long axis of the limb has not been taken into account for purposes of description, the relation only to the dorsopalmar and the radioulnar axes being stated; i.e., the degree of divergence of the two members is not taken into account. Thus, when the mirror plane is parallel to the radioulnar axis, the limb is said to A.DU eet) PR MP, (R) Fig. 3 Diagram showing mode of reduplication. PR, primary limb; P.DU, posterior reduplicating member; A.DU, anterior reduplicating member; WP;(R), primary (radial) mirror plane; 1/P2,(U), secondary (ulnar) mirror plane; / to 4, first to fourth digits, respectively. S, location of section shown in figure 4B. Dotted lines show the outlines of limbs as they would have been had there been no coalescence. be mirrored in a palmar or a dorsal plane, according as the palms or the backs of the hand face one another; when the plane is parallel to the dorsopalmar axis, the mirroring is in a radial or an ulnar plane, according as the radial or ulnar borders of the limb face one another (fig. 4, 4). Intermediate planes are de- scribed as radiodorsal, ulnopalmar, etc. (fig. 4, B). No attempt has been made for the present to measure accurately the angles of mirroring. It has been found, in agreement with Bateson, that fee. . ROSS G. HARRISON when there is a double reduplication, then the two mirror planes intersect at the bifurcation in a line perpendicular to the proximo- distal axes; 1.e., so that with reference to the radioulnar and dorso- palmar axes the planes of reflection face one another (fig. 4). Considerable deviation from this rule has, however, been noted in certain cases, and the amphibians do not seem to follow it with the same regularity as the arthropods, according to Bateson.!° MP2 (vu) PAL PAL MP1 (R) PAL Fig. 4 Diagram of reduplication, sectional view. In A the mirror planes are radial (MP) and ulnar (MP2), and a certain amount of coalescence between the primary and the anterior reduplicating members is shown, as in figure 3. In B the mirror planes are radiodorsal (MP,) and ulno pulnar (WP2). D, dorsal; PAL, palmar; R, radial; U, ulnar. EXPERIMENTAL A. Limb buds implanted in abnormal location—heterotopic transplantations In nearly all of the experiments in this group the limb bud was implanted on the flank of the embryo at the ventral border of the myotomes between the region of the fore and hind limbs. Ina few cases it was placed on the side of the head between the eye and the ear, but the grafts were absorbed in all of these except 10 Op, cit:, p. 552. SYMMETRY IN TRANSPLANTED LIMBS 15 one, which yielded an imperfect appendage. They need not be considered separately here, though a more extensive series of experiments of the latter type would probably yield different and more interesting results. The limb buds transplanted to the flank of the embryo are placed in an environment similar to that of the normal fore limb, as far as relations to the body wall and muscle plates are con- cerned, though they lack the specific blood supply and innerva- tion of the limb region. Consequently, a very high percentage TABLE 1 Heterotopic transplantations. Swmmary of experiments | ee pecans OPERATION = |= = ames sot | Oe MRS eeceent |e Waco hee le ext Homeddenes. scar 19 Pelee eS 6 30050) 4H 57e1 Gms Givasne.. 3:;- 0 eee 3l 12 0 00.0 11 91.7 1 8.3 EG Had Gar tess ac. oon eee 28 10 8 80.0 0 | 00.0 25 | 2020 IB IGE ihe otic RMSE Oc 60 16 C2) 286532). we \4ae8 8 | 50.0 HRotalis scabs as eee 138 45 |12 AS-i 189 |4020) |) 158 sa53 Average of percentages 32.3 33.9 33.9 1 Excluding all cases where death occurred prematurely or where the grafted limb was resorbed or remained rudimentary. Percentages in all tables have been calculated on the basis of positive experiments. 2 There is evidence that in this case thére was an error in the orientation of the bud and that it should therefore be classed in the group het. dd. of cases yielded only abortive limbs, and those that did develop rarely showed any functional activity. There is also greater difficulty in securing good healing of wounds in the intermediate region, so that a larger proportion of the cases died early. In many of these cases there is obviously some interference with the development of the pronephric duct, which becomes blocked. The secretion which accumulates causes the formation of a cyst of considerable size, which may interfere with the development of the limb bud. The results of the experiments are summarized in table 1. 11 Cf. Detwiler, 719 and ’20. YW fff f / / t G ify iff Li Uf ify MeO Lf Gi Wh ff veel if (MI AF YS = VILE JU fii PUAN”: Mi /[\\\\\WNS ( \ S N eee Se Pf Figs.5 to8 Heterotopic transplantation of fore limb; right limb to right side (hom.dd.). TR, transplanted limb. X 10. Fig. 5 Exp. Tr. E. 148, eight daysafter operation. Fig. 6 Same, twenty days after operation. Fig. 7 Same, twenty-eight days after operation, drawn from preserved specimen. Fig.8 Experiment Tr. E. 154, drawn from preserved specimen, killed twenty- two days after operation. ; 16 SYMMETRY IN TRANSPLANTED LIMBS 17 1. Homopleural transplantations, normal or dorsodorsal orien- tation. Nineteen cases were operated upon in this way (table 1). In all of the cases where observations are recorded (thirteen in number), the limbs, in the course of their development, gave evi- dence of their original orientation, in that they pointed posteri- orly or dorsoposteriorly when they first began to grow out (fig. 5). In the three cases that gave rise to single limbs they contin- Fig. 9 Heterotopic transplantation (hom.dd.), showing twin limbs from one implanted bud; PR, primary member; DU, reduplicating member. Exp. Tr. E. 182. X 10. ued their growth in this direction, developing almost exactly like the normal (figs. 6, 7, and 8). Likewise in the four cases that gave rise to double appendages, the transplanted buds first began to grow in a dorsoposterior direction, and only later did the re- duplicating buds appear on the anterior border of the original limb. The original bud developed in each case into a limb of the same side, and the reduplicating buds became limbs of oppo- site asymmetry (fig. 9). Histories of typical cases are given in the appendix (p. 119). 18 ROSS G. HARRISON (iy My } i S SSX Vou >) SS gi i W) ee, z, Hi We 7 nn an willl Co ae Ni yy SoS SSO AY SSS SSS [a ES SYMMETRY IN TRANSPLANTED LIMBS 19 2. Homopleural transplantations, inverted or dorsoventral orienta- tion. Thirty-one experiments of this kind were made, with re- sults as shown in table 1. Of the twelve cases yielding positive results, one” gave rise to a pair of limbs and the others to single limbs in which the asymmetry was reversed; i.e., the right limb bud when placed upside down on the right side of the body gave rise directly to a left limb. Even in the case which showed redu- plication the primary limb of the pair became reversed. In all of the cases where limbs resulted, the initial direction of point- ing was anterior or dorsoanterior (figs. 10 and 11); 1.e., nearly the opposite of normal. In four other cases this was also true. In only four cases is the direction of pointing recorded as poster- ior, and from these nothing definite was developed. All limbs which developed continued their growth in the same general direction, sometimes being directed more dorsally and sometimes more sharply anteriorly (figs. 12 to 17). They also showed the tendency to project more directly to the side than the normal limbs. The final posture assumed by these appendages varies considerably and does not seem to be dependent upon the degree of development attained by the appendage. Two cases, each having perfectly developed hands, exhibit the following extreme . conditions: One!’ is practically a perfect mirror image of the nor- mal right limb both as regards form and posture (fig. 18). The Figs. 10 to 17 Heterotopic transplantation of fore limb; right limb bud to right side inverted (hom.dv.), Exp. Tr. E. 219. N, normal limb, right side; 7'R, transplanted limb. X 10. Fig. 10 Dorsal view, five days after operation. Fig. 11 Lateral view, same age. Fig. 12 Dorsal view, eight days after operation. Fig. 18 Lateral view, same age. Fig. 14 Dorsal view, twelve days after operation. Fig. 15 Lateral view of limbs only, same age. Fig. 16 Dorsal view, sixteen days after operation. Fig. 17 Lateral view, same age. Fig. 18 Heterotopic transplantation; right limb to right side inverted (hom. dv.), Exp. Tr. E. 139; drawn from specimen preserved twenty-eight days after operation. X 10. 122 Tr. EH. 220. 13 Tr, BE. 139. 20 ROSS G. HARRISON upper arm runs dorso-anteriorly and laterally. The elbow bend is somewhat less than 90° and the fore arm and hand extend antero- ventrally and laterally. The extensor surface of the elbow-joint faces dorsally and slightly anteriorly and medially. The palm of the hand faces medially, anteriorly, and slightly ventrally. Fig. 19 Heterotopic transplantation (hom.dv.), Exp. Tr. E. 140; drawn from specimen "preserved twenty-eight days after operation. J'R, transplanted limb. x10] The other case has its upper arm transverse and horizontal, and its fore arm extends ventroposteriorly at an angle of less than 45° to the horizontal axis (fig. 19). The palm looks ventrally and anteriorly. In order to bring this limb into the position of the former, it would have to be rotated about the axis of the humerus 45° or more and then adducted dorsoanteriorly at the shoulder-joint through about 30°. The difference in position assumed by the limbs in the various cases is thus due to differ- 14 Tr. E. 140. SYMMETRY IN TRANSPLANTED LIMBS 21 ences in the amount of rotation, etc., undergone during the later stages of development. Histories of these cases are given in the appendix (p. 120). 3. Heteropleural transplantations, dorsodorsal orientation. Twenty-eight experiments in this class have been made (table 1). Five of these died prematurely, and in twelve the tissue was either resorbed or failed to develop beyond the nodule stage. In one case the bud developed into a stump about as long as the upper arm, but without digits. Two cases gave double limbs and eight developed into limbs which preserved their original prospective asymmetry. ‘Two other cases may belong in this cate- gory, one in which the original orientation of the bud is recorded as uncertain!® and another!’ in which it is recorded as dorsoven- tral probably by mistake. In the development of the limb buds in this group twenty-one, in addition to the two doubtful cases just mentioned, are recorded at first as pointing in an anterodorsal direction, thus preserving their original tendency in this respect. In the eight cases in which the pointing was slight and in the five in which no definite pointing was observed the limbs were abortive or resorbed. In the eight cases where single limbs of the side of origin de- veloped they retained their posture, developing as nearly exact mirror images of the normal fore limb of the side to which they were transplanted (figs. 20 to 23). The elbow-joint points dorso- anteriorly, though varying somewhat, and the palm of the hand faces ventrally, medially, and anteriorly (figs. 24 and 25). Indi- vidual cases show variations similar to those observed in the previous group. It is a striking fact that the general type of development is the same here in the heteropleural non-inverted buds as in the homopleural inverted bud, which shows that both the posture and the asymmetry of the limb depend upon some reaction between the bud and its new environment. (For case histories see p. 121.) The cases which showed reduplications, but two in number, differ considerably from one another. In the first!’ growth was slow and the resulting limb short with irregular reduplications str ee Ls. 26.7. ee Tes ees: 18 Tr. Ha 1L9. ROSS G. HARRISON N N \ Ss Shee Z\ 4: ti) ma 4 SYMMETRY IN TRANSPLANTED LIMBS 23 in the hand, so that right- or left-sidedness could not be deter- mined. In the other!® the limb developed promptly and formed a duplicate member (fig. 26), which was first seen at ten days and Fig. 26 Heterotopic transplantation (het.dd.), Exp. Tr. E. 127. Drawn from specimen preserved twenty days after operation. PR, primary; DU, redupli- cating member; / to 4, digits. Figs. 20 to 23 Heterotopic transplantation of fore limb; right limb bud to left side (het.dd.), Exp. Tr. E. 227. N, normal left limb; 7'R, transplanted limb. x 10. Figs. 20 and 21 Dorsal and lateral views, respectively, eight days after oper- ation. Fig. 22 Ventral view, thirteen days after operation. Fig. 23 Lateral view, seventeen days after operation. Figs. 24 and 25 Heterotopic transplantation (het.dd.), Exp. Tr. E. 107. Lat- eral and ventral views, respectively, of preserved specimen, twenty-six days after operation. X 10. edad Be ea) De Bf 24 ROSS G. HARRISON Wy HL | ’ 4 Uy WY UY YWYey/ Wp YU FE IDES Oe Lah SYMMETRY IN TRANSPLANTED LIMBS 25 which developed ultimately into a left limb, the mirror image of the primary member, having been reversed from the original prospective asymmetry of the transplanted bud. 4. Heteropleural transplantations, dorsoventral orientation. Sixty operations were done in this series. For some unknown reason a very large proportion (twenty cases) died prematurely and six- teen of the survivors yielded only abortive limb buds, leaving only twenty-four available for consideration. Eight of these are recorded as imperfect, six produced reduplications to some degree, and nine, single limbs of reversed asymmetry. Several of the latter were somewhat defective and others showed slight redu- plications. Several cases which are exceptional will be consid- ered below. In the cases where single limbs arose, development took place in a manner fundamentally like that of the limb buds normally oriented (hom. dd.). As the buds grew out, they began to point in a posterior direction, and so continuing, developed into limbs in normal posture (fig. 27). There was, however, less regularity than in the homopleural dorsodorsal group. ‘The direction of pointing was not always dorsoposterior, as in the normal limb, but was in many cases inclined more ventrally. There are rec- ords of pointing in all of the positive experiments and in many of the negative. In only three cases is the direction recorded Fig. 27 Heterotopic transplantation of fore limb; right limb bud to left side inverted (het.dv.), Exp. Tr. E. 193. Preserved specimen killed twenty-four days after operation. 10. Figs. 28 to 32 Heterotopic transplantation of fore limb; right limb bud to left side inverted (het.dv.), Exp. Tr. E. 217. N, normal left limb; TR, trans- planted limb; PR, primary member; DU, reduplicating member; / to 4, numbers of digits. Fig. 28 Dorsal view, five days after operation. Fig. 29 Lateral view, five days after operation. Fig. 30 Dorsal view, fifteen days after operation. Fig. 31 Lateral view of limbs, fifteen days after operation. Fig. 32. Limb showing beginning of reduplicating digits (DU) on ventro- anterior border (from a free-hand sketch nineteen days after operation). Figs. 33 and 34 Heterotopic transplantation (hef.dv.); right limb bud to left side. Exp. Tr. E. 163. Anomalous result. Primary member (PR) defective; reduplicating member (DU) reversed. Lateral and ventral aspects, respectively, drawn from specimen preserved thirty-nine days after operation. X 10. 26 ROSS G. HARRISON as dorsoanterior; one of these died early and the other two gave rise to imperfect limbs with indeterminate asymmetry.?° The individual cases in which limbs of opposite asymmetry developed were rather more irregular than in the preceding groups, though the best cases gave perfect reversed appendages. . In addition to the ones included in the tabulation, there is one other case that probably belongs in this category. It is one in which the orientation of the bud at the time of transplantation is recorded as uncertain.2! The limb that developed is a perfect one of reversed asymmetry in nearly the same posture as the nor- mal limb of the side to which it was transplanted. It showed an unusual amount of motility. In one case, included in the records of this group,” the transplanted bud developed into a normal limb of the side from which it was taken. It is believed, however, that a mistake was made in recording the operation in this case, and that probably in reality the orientation of the limb was not inverted. The direction of pointing, as observed on the third and fifth days after the operation when the limb bud is recorded as pointing anteriorly, is evidence, though not absolutely conclu- sive, that an error has been made. If this interpretation is cor- rect, the case would not be exceptional, but would accord with the eight cases described in the previous section. In the eight cases in which reduplications occurrred, the early stages of development were like the normal (figs. 28 and 29), the reduplicating buds not being noted until at least twelve days after the operation. Three individuals showed distinctly that the primary limb was of reversed asymmetry. In one case it was so imperfect that it could not be determined to which side it belonged, but the reduplicating limb was sufficiently devel- oped to show that it was of the same side as the bud was origin- ally, indicating that the original member was in all probability reversed. Another case? gave a limb with nearly symmetrical reduplication in the hand without anything to indicate which member was primary (figs. 31 and 32). Two long radial digits are present in the middle and two short ulnar digits on each side. Still another case gave a very peculiar result. The primary 20 Tr, EH. 108 and 203. 21Tr, EK. 109. 22. Beles 23°, Boe 24Tr, E. 163. SYMMETRY IN TRANSPLANTED LIMBS 27 limb bud developed into a long almost filiform structure, with out digits, that grew posteriorly on the ventral side of the body not far from the midline. Twenty days after the operation a sec- ond bud was noticed dorsal to the original, and this developed into a somewhat peculiarly placed limb. The upper arm runs transversely and the palm of the hand faces dorsomedially (figs. 33 and 34). This limb is clearly a left; 1.e., its original prospective asymmetry has been reversed. It therefore constitutes an ex- ception to the rules, not only because of the position of the hand, but also because of its particular asymmetry ; for the original (filiform) member should have been reversed (a left), and the second one reversed back again to the original asymmetry. How- ever, the fact that the latter developed at such a considerable dis- tance from the original member, might be regarded as indicating that it was beyond its sphere of influence, perhaps having been split apart from it at an early stage, and that it remained there- fore as of the same side. Several cases of regeneration after extirpation of half buds and of transplantation of half buds gave analogous results (fig. 132). 5. The shoulder-girdle in heterotopic transplantations. The limb-girdle in the hetrotopic transplantations is developed in more or less reduced condition, as was first shown by Braus ('09) in the anurans. Detwiler (718) has studied this question in Amblystoma, and has found that the degree of development of the girdle is dependent upon the size of the graft and the region from which it is taken, the scapula and suprascapula being local- ized in the tissue dorsal to the normal limb bud and the coracoid in that ventral to it.22 The form of the reduced girdle derived 25 Cf. Harrison, 718, p. 441 (Exp. Rem. E. 17 and H. R. E. 10), and page 135 of the present paper (Exp. H. R. E. 20). 26 It is a curious fact that in the embryo the limb-girdle has undoubtedly the character of a mosaic, without totipotence of its parts, while in the adult Triton, according to Tornier (’06), Fritsch (711), and Kurz (712), a small portion of the shoulder-girdle can regenerate the whole, including the fore limb. Ac- cording to the two last-named investigators, even if the whole girdle is removed, it will be regenerated together with the free appendage. Kurz has found that this holds for both shoulder and pelvic girdles but that removal of the sacral portion of the vertebral column prevents regeneration. In the anurans, accord- ing to Braus (’06), there is considerable variation in the regenerative powers of the limbs in early stages. 28 ROSS G. HARRISON from the usual round dise (limb bud) is roughly triangular, as shown in the figure of Detwiler’s model (his figure 28), with a ventral process projecting anteriorly, to be identified as a rudi- mentary coracoid, and a dorsal process, which includes the rudi- ment of the scapula. In the normally oriented grafts (homo- pleural dorsodorsal) these processes point anteriorly, with a single process projecting posteriorly slightly behind the glenoid cavity. This shows clearly in two cases.27 The question now arises whether the girdle follows the rules governing the asym- metry of the free limbs. The results, in the main indicate that such is the case, though the girdle developed is often so small and rudimentary, that it is not possible to determine to which ' side it belongs. In the inverted homopleural grafts, which give rise to reversed limbs, the girdle also seems to be reversed. ‘This is true in four cases out of the five examined in serial sections.?8 Among the heteropleural dorsodorsal transplantations, five cases have been examined in sections. In two of them?’ with well-developed glenoid cavity, the girdle cartilage is mostly ven- tral and posterior to the joint. This probably represents a cora- coid with asymmetry corresponding to that of the free limb. One case,*° with the cartilage projecting both anteriorly and pos- teriorly from the cavity, gives no evidence as to the side to which it belongs. One is too rudimentary,*' and one seems to have had its asymmetry reversed,” though the limb is not reversed. In the two dorsoventral heteropleural transplants which have been studied in sections, the side to which the girdle belongs cannot be determined. Other cases from among the earlier experiments, where in most instances the size of the transplanted bud was small, are inconclusive. On the whole, the cases where the asym- metry can be determined with any degree of certainty seem to follow the rules. Only a single case thus far examined is clearly exceptional. 27 Tr. EK. 148 and 154. $0'Tr Hs i20: 28 Tr, EK. 185, 186, 189, and 140. 3 Tr. Hele 29 Tr, HE. 124 and 169. 22D. HalOie SYMMETRY IN TRANSPLANTED LIMBS 29 6. Summary of the results of heterotopic transplantations. A survey of all the experiments in this group brings out the following facts: Implanted in dorsodorsal orientation, a limb bud gives rise to an appendage of its original prospective asymmetry, whether placed on the same or opposite side of the body. Such appen- dages have a normal posture when placed on the same side of the body from which they were taken, but when placed on the opposite side they mirror approximately the limb of that side, though they often become rotated to quite different postures. Implanted in inverted (dorsoventral) position, a limb bud gives rise to an appendage of reversed asymmetry whether placed on the same or opposite side of the body. When placed on the same side, such appendages mirror the normal limb of that side, but when grafted on the opposite side, they assume a posture approxi- mately identical with that of the limb of that side. Limbs implanted in any of the four positions here studied may produce reduplications. As far as it has been possible to deter- mine, the primary limb of the pair is then of the same side as a single limb would be according to the foregoing rules. The redup- licating limb has been found to be, with a single exception, the mirror image of the first. Limbs that are grafted in abnormal location have at best very incomplete function and are often apparently entirely immobile. They usually do not become so large as those that are implanted in normal location, and they show defects and evidences of atro- phy much more frequently. B. Limb buds implanted in natural location—orthotopic transplantation In these experiments the limb bud of the host was first removed and then either put back in place, or else a bud from another embryo was grafted into the wound. In all of the earlier cases the wound bed was not cleaned after removal of the bud, so that some cells from the host were left to mingle with the tissues of the transplanted limb rudiment. The later experiments, with 30 ROSS G. HARRISON but few exceptions, were carried out under precautions necessary and sufficient to preclude contamination of this kind: the extir- pated area was three and a half somites in diameter, and the bed of the wound was carefully scraped after removal of the bud.*8 The results were somewhat different (proportionately) in the . TABLE 2 Orthotopic transplantations. Summary of experiments NUMBER OF SINGLE LIMBS SINGLE LIMBS EXPERIMENTS | NOT REVERSED REVERSED HADDALUELEIKELAE 2 OPERATION ————— Total Posi- | Num-| Per Num- Per Num- | Per ‘ tive ber cent ber | cent ber cent A. Wound bed cleaned and wound ' not less than 34 somites Moni. Sar pees ache 9 9 9 {100.0 C | 00.0 0 | 00.0 ROUT. iy; he satus, oe eco. 2 61 38 102) 26.3 iL 2:6:\| 2A geese et. da made ot eas 49 31 ] 3.2 52 16.7 | 25. sOme Het veer Ss or caiaoe vie, ase 26 16 O- | 00.0 | La ar9s88 1 6.3 gi Notre) Re 1 AS Sie Bee ora 145 94 20° | 21.3 fe 21. 022.3 | 5a) eae Average of percentages. 31.6 28.8 39.6 B. Wound bed not cleaned EL On Gi eee tad. ce 0 0 0 0 0 Hiomiidy2 2a ks.) tee B71 DH 194 | 95.0] Oo | 00.0 1 5.0 Fleteadale ci cee... eet eae 13 2) |) 154s osha 23a 8 | 61.5 Hetenas = eee. ee 21 15 O || OO#0) seri753.3 7 Aga Faye AL Se ea 75 48+}. 2t | 43.8)" das “\c29-9Nl 6 Saaee ‘ Including three cases in which the primary bud righted itself by rotation and the duplicate is disharmonic. * Limbs which became normal by rotation, including one case (I. E. 101) of hyperdactyly. ’ Normal by resorption of original member of pair. 4 One case included in which the posture of the limb was abnormal. two classes of experiments and have been summarized separately in table 2 (A and B). The differences will be taken up in connec- tion with the consideration of each of the subgroups. ?. Homopleural transplantations, dorsodorsal orientation. This is in reality merely a control experiment and is a test of the effect 38 Harrison, 715 and ’18, p. 422. 4 SYMMETRY IN TRANSPLANTED LIMBS 31 of the operation as such on the development of the limb. A fore limb bud is carefully excised and either replaced in the same wound or else engrafted in normal position in another embryo from which the limb bud had been previously removed. Only nine individuals were operated upon, in all of which the wounds were carefully cleaned. Normal limbs developed in all cases, though they were slightly retarded in the earlier stages of development in comparison with the unoperated limb of the _ opposite side. In six of the cases the pronephros was removed and in the other three it was left in. No difference was noted between the two sets. It may be safely concluded that the effect of the operation itself upon normal development is prac- tically negligible. 8. Homopleural transplantations, dorsoventral orientation. In some of the cases of this series, as in the last, the limb bud was simply lifted and replaced after rotation through 180°. In the others the wound bed was first prepared in one embryo and the bud taken from another. The latter method is preferable and it was employed in all the later experiments. The total number of experiments is one hundred and four, of which sixty-one were with cleaned wounds of proper size. The latter will be considered first, since the conditions of experimenta- tion are more definitely known and there can be no doubt that the limbs were derived exclusively from the transplanted tissue. Leaving out of consideration the twenty-three cases which died prematurely or gave rise merely to abortive or rudimentary limbs, there are thirty-eight cases which yielded positive results, as recorded in table 2A, The single limbs are in the minority and are of two kinds, reversed and non-reversed. The most re- markable case** (history on p. 124), which really gives the clue to the interpretation of the experiments of this group, is the one in which a limb of reversed asymmetry developed, a right limb on the left side, perfectly normal in form, function, and posture, as far as the last is possible on the wrong side of the body (figs. 39 to 41). The shoulder-girdle of this limb is also reversed and i) IB De THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, No. 1 By ROSS G. HARRISON 8 eis Hip) ( ae Yi YY) 35 a e ——- o di Yi i lt Ah Wes ; i 1} ily hey yy y\ 4 iw ye SYMMETRY IN TRANSPLANTED LIMBS 33 is quite separate from the rudimentary girdle developed from the tissues of the host (p. 59). The ten cases in which normal non-reversed limbs developed are clearly contrary to the rule (p. 4). The records of these cases show that the end result is reached by a process of rotation at the shoulder-joint during development (figs. 56 to 58). They will be considered below (p. 40). The reduplicated limbs, of which there were twenty-seven, fall, like the single, into two groups. In the first the original bud developed into a limb of reversed asymmetry, while in the second it is not reversed. Observations upon the earlier stages of the operated limbs show that in those cases which give reduplications, as well as in the case of the simple limb with reversed asymmetry (figs. 35, 42, 43, 49, 51, and 52), the original direction of pointing is either dorsal, anterior, or dorsoanterior, and more sharply lateral than normal. Likewise in the case of those that develop into single non-reversed limbs, the first pointing is more sharply lateral than normal, and also more dorsal, though only two are recorded as pointing slightly anteriorly from the dorsal direction. This shows that the original tendencies of growth, immanent in the bud at the time of transplantation, are by no means inactive when it is in its new position. One or the other of two conse- quences of this growth tendency now ensues, indicating a sort of antagonistic reaction between the organization of the transplanted rudiment and that of the surrounding parts. The limb either continues to grow in an anterior or anterodorsal direction, in Figs. 35 to 41 Orthotopie transplantation; left limb to left side inverted (hom.dv.), resulting in a normal right limb onthe operated side. Exp. I. E. 64. TR, transplanted limb. X 10. Fig. 35 Lateral view, five days after operation. Fig. 36 Ventral view, ten days after operation. Fig. 37 Dorsal view, sixteen days after operation; transplanted limb covered by gills. Fig. 38 Lateral view of limb, sixteen days after operation. Fig. 39. Dorsal view, twenty-three days after operation. Fig. 40 Ventral view, twenty-three days after operation. Fig. 41 Lateral view, twenty-three days after operation. 35 T. E. 49 and 94. 34 << SS WS SSSaxq_ ROSS G. HARRISON Figs. 42 to48 Orthotopic transplantation; left limb bud to left side inverted (hom.dv.), resulting in duplicate limbs. Exp. I. E.60. N, normal right limb bud; TR, transplanted bud; PR, primary member; DU, reduplicating member. X 10. Fig. Fig. Fig. Fig. Fig. Fig. Fig. 42 Dorsal view, five days after Uperation. Lateral view, five days after operation. Dorsal view, seven days after operation. Dorsal view, twelve days after operation. Lateral view, twelve days after operation. Lateral view of specimen preserved eighteen days after operation. Ventral view of limb, preserved specimen. SYMMETRY IN TRANSPLANTED LIMBS 35 which case its asymmetry is reversed (figs. 36 to 39), just as in the corresponding class of heterotopic transplantations, or it gradually rotates towards its normal position while retaining its original prospective asymmetry (figs. 56 to 59). In the former alternative duplicate limbs are nearly always formed. Only in the one case, referred to above, did a perfect single limb arise. In the other alternative single limbs usually arise, though some of the cases of reduplication certainly belong to this group. In the duplicities belonging to the first group the original limb bud continues to grow in an anterior direction and ultimately becomes a reversed limb. After a time a reduplicating bud appears on the posterior border of the original bud (fig. 44) and in the clearer cases grows into a homopleural limb in approxi- mately normal posture (figs. 45 to 48). The original bud becomes a reversed limb which, together with the reduplicating member, may form an almost symmetrical complex. Twenty-four of the thirty-one** cases of reduplicated limbs are probably of this type. Fifteen are certainly so,*7 and in three others*® that are very similar all that is lacking to place them unequivocally in this group is a definite observation as to which bud was the primary one; six more cases*? may also be inter- preted in the same manner, though they are not sufficiently clear to insure that this is the only possible interpretation. The degree of reduplication varies here, as in the other groups of experiments, from the condition where almost the whole arm is involved to that in which the hand is only partly double. In three cases*® the anterior bud was much reduced (p. 49), the posterior bud becoming a somewhat irregular homopleural limb. In eleven cases there is only one reduplicating appendage, which is always posterior to the primary (figs. 43 to 48), while in the remaining twelve" there are evidences of further doubling, usu- 36 Four cases are considered here which are not included in the tabulation on account of the fact that the wound was only 3 somites in diameter (I. E. 39, 41, 44 and 45). 37 J. EK. 48, 60, 62, 63, 66, 72, 74, 75, 81, 85, 87, 89, 91, 92, and 96. 387. BK. 44, 45, and 52. 39 T. EK. 39, 68, 70, 93, 100, 102. 40 T, EB. 92, 93, 100. 417, KE. 39, 45, 62, 63, 66, 72, 75, 81, 85, 87, 91, and 93. 36 ROSS G. HARRISON MULL pp fp RYY é WWM UY LY) YS Ug: ' 7 ayy NS X Ss SSX i} i SYMMETRY IN TRANSPLANTED LIMBS 37 ally on the anterior side of the original (figs. 49 to 55 and 61). When the latter condition arises and the anterior reduplicating member is sufficiently developed, it is seen that it, too, is mirrored from the original member and is homopleural (fig. 55). In one case there are three complete hands, one of which has two of its digits doubled.” The plane from which the posterior redupli- eating member is mirrored in the final form of the limb varies from radial to dorsal (figs. 3 and 4) and is usually intermediate between these two extremes (p. 13). Nineteen cases follow this rule, three are indeterminate and there is only one positive exceptional case, in which the mirror plane is ulnodorsal.**? When there is also an anterior reduplicating member, it is generally mirrored from a plane 180° around the limb axis from the first; i.e., ulnar, ulnopalmar, or palmar. The reduplications belonging to the second group are more restricted and less certain of diagnosis. The limb bud retains its original prospective asymmetry, reaching an approximately nor- mal position by rotation, and reduplication is much less extensive, involving in most cases the digits only (fig. 62). Three cases“ almost certainly belong to this group, and there may be two others.” | Of the two remaining cases of reduplication, one died too young; in the second** the supernumerary limb was of the same side as the primary and was quite distinct from it. This is a very unusual condition, but the transplanted bud in this case was Figs. 49 to 55 Orthotopic transplantation; left limb bud to left side inverted (hom.dv.), resulting in limb with two reduplicating members. Exp. I. E. 63. N, normal right limb bud; TR, transplanted left bud; PR, primary limb; A.DU, anterior, and P. DU, posterior reduplicating members. X 10. Fig. 49 Dorsal view, four days after operation. Fig. 50 Lateral view, four days after operation. Fig. 51 Dorsal view, seven days after operation. Fig. 52 Lateral view, seven days after operation. Fig. 53 Dorsal view, ten days after operation. Fig. 54 Lateral view, ten days after operation. Fig. 55 Dorsal view of specimen preserved seventeen days after operation. Gills (BR) removed to show limb. 1 to 3, numbers of digits. 27.E.87. “1. E.72. “1. EB. 86,88,90. “I.E. 41,59. “I.E. 38. 38 ROSS G. HARRISON Z YH, Wij y WV \ aah \\ i) LEY DEEL IVI 7/., \\ SALSA ES = SEL 7 Why \ a) ‘ : YD) NN NAN Z| ) SYMMETRY IN TRANSPLANTED LIMBS 39 larger than usual (four somites in diameter), and it is possible that the reduplicating bud, growing from near its anterior border, was uninfluenced by the primary limb and hence was not mirrored. There remain for consideration those cases in which a single non-reversed limb developed. As in the other cases, the limb bud in these showed at first the consequences of abnormal orien- tation. When first observed it pointed more sharply laterally and more dorsally (less posteriorly) than normal. Two even pointed dorsally and slightly anteriorly. In the course of devel- opment the limb gradually changed its posture and ultimately came to a perfectly normal posture by a process of rotation at the shoulder-joint (figs. 56 to 59). Ten such cases were obtained,*7 though in several of them?’ it is possible that reversal may have been brought about by early reduplication and suppression of the original bud, as deseribed in the next section (p. 49). In one of these*? a supernumerary radial digit was present, but this is to be regarded as a case of hyperdactyly rather than one of mirrored reduplication. In one case*® the limb which originally developed showed irregularities in the digits. The arm was then ampu- tated above the elbow, and the appendage which regenerated was in every respect normal. ‘This case is of considerable interest in showing that the abnormal condition which produces reduplica- tion is not necessarily stamped upon the whole structure, but may be due to some local mechanical disturbance. In reviewing this group of experiments, it is clear that the first two results, 1.e., single reversed limbs and most of the reduplica- tions, come under the same scheme. ‘There is a primary reversal of asymmetry, without reduplication in the first case and accom- Figs. 56 to 59 Orthotopic transplantation; left limb bud inverted (hom.dv.). Limb reaches normal posture by rotation. Exp. I. E. 49. N, normal (right) limb; 7, transplanted (left) limb. Fig. 56 Eleven days after operation. Fig. 57 Twenty-one days after operation. Fig. 58 Thirty-eight days after operation. Fig. 59 Preserved specimen, killed at thirty-nine days. 47 J. E. 49, 55, 69, 71, 73, 77, 84, 94, 99, and 101. gig Gul Be bs 48 For instance, I. E. 77. SOOT, Swill: 40 ROSS G. HARRISON panied by reduplication in the second. In the latter the second- ary bud, being the mirror image of the other, is again reversed back to the original prospective asymmetry of the transplanted bud. This then occupies an approximately normal position and may function to a considerable extent as a normal limb, though impeded by the connection with its mate. These two results are. directly comparable to those of the heterotopic transplantations of the corresponding class (fig. 2). The third result, in which normal homopleural limbs develop, reaching their normal position gradually by rotation, is funda- mentally different. Here no reversal occurs; the limb bud begins its development as a self-differentiating system, but later, under the stress of the changed relation to its environment, it comes again into normal posture. What determines whether the limb bud shall reverse its asym- metry or rotate back to its normal posture? The earlier experi- ments of this series afforded no satisfactory answer to this ques- tion. It was certainly not due to the size of the wound, the mode of preparation of the wound, the presence or absence of the pronephros, or the age of the embryo. What seemed most likely was that there were minor accidental differences in the amount of rotation to which the limb bud was subjected at the time of operation. It was conceivable, for instance, that if the dise were rotated anteriorly around the dorsal semicircumference of the wound a little less than 180° (fig. 60), the reversing effect of its organic environment might be lessened and the rotation back to normal position facilitated; in this case a normal non- reversed limb would result. If, on the other hand, the grafted bud were rotated 180° or shghtly more, the reversing effect might be at a maximum and rotation most impeded, in which case a heteropleural limb or twin limbs would arise. Experiments made in the spring of 1917 had for their main purpose the testing of this hypothesis. Operations were done in pairs; in one case the limb disc was rotated about the dorsal circumference in a posteroanterior direction slightly more than 180° and in the other slightly less, extremes being probably not more than 190° and 170°, respectively (see histories on pp. 125-6). SYMMETRY IN TRANSPLANTED LIMBS 41 The results are not altogether conclusive, though they point to the correctness of the hypothesis. Twenty-three operations were done, of which seventeen yielded positive results. Ten cases harmonize with the hypothesis; four are doubtful, and three are contrary to it (figs. 61 and 62). When the difficulty of ex- actly estimating the degree of rotation is considered, many ap- parent exceptions must be expected, and a far greater number of experiments would be necessary to eliminate statistically the effect of this uncertainty. As a matter of fact, the records of the cases classed as surely exceptional give evidence that the amount D A 180° °° P Vv Fig. 60 Diagram showing difference in amount of rotation in two sets of experiments. The circle represents the left limb bud and the arrow the direction of rotation. A,D,P,V, the direction of the cardinal points of the embryonic body, anterior, dorsal, posterior, and ventral, respectively. of rotation at the time of operation was probably not correctly estimated, for the first direction of pointing (p. 11) in all of these cases is not according to expectation. The thirty-seven cases in which the wound bed was not entirely cleaned of mesoderm may now be considered. These show a marked contrast to those with cleaned wounds, inasmuch as there are very few reduplications and a very large proportion of normal non-reversed limbs. Thus out of twenty cases which yielded positive results eighteen or 90 per cent are normal, as compared with 23.8 per cent (ten cases) in the clean-wound class. Only one case (5 per cent) had a reduplicated limb, as compared with thirty-one duplicities (73.8 per cent) in the clean-wound class. ROSS G. HARRISON N : Ey =) )\ ea Gg Lt —— WWE: LEE Y, Sree Ss = oe Br Lish—_> = Pent te SSS ! == — = eee ZB \\ Ls es Ml) pp Ut |p py phils ps) tft PUL! 244 try fy — eee Yi WEE ; RES Ssascwe SSsSSS>—] = — Sas —S== —————— — aD {! as = : mn ae Reeeee aw SRS WARS WH MAY UNO : = SBE ee Fig. 61 Orthotopic transplantation; left limb bud to left side (hom.dv.), rotated slightly more than 180° from its normal position, 7.e. P to A’ in fig. 60. Exp. I. E. 85. Primary member (PR) is a right; posterior reduplicating mem- ber (P.DU) is a nearly normal left; anterior reduplicating member (A.DU) partly coalesced with primary. ; Fig. 62 Orthotopic transplantation; left limb bud to left side (hom.dv.), rotated slightly less than 180° (i.e. P to A in fig. 60). Exp. I. E. 86. The trans- planted limb is primarily a left, having reached its normal posture by rotation; second (2’) and third (3’) digits, reduplicated. X 10. SYMMETRY IN TRANSPLANTED LIMBS 43 Since these differences can scarcely be accounted for on the ground of different degree of rotation of the limb buds at operation (p. 40), it would seem that the few mesoderm cells remaining in the wound bed must have exerted some influence upon the develop- ing limb. This does not mean that the limbs which do develop in such cases arise solely by a process of regeneration from the host. In fact, the rate of development, which is only slightly retarded below the normal, precludes such an interpretation. What probably does take place is an intermingling of cells from the host and the graft, with the result that the former, acting in the same sense as the environment with which they are in har- monic relation, counteract the tendency of the inverted elements to reverse their asymmetry. This was, however not shown to the same degree in the corresponding experiments with super- posed limbs (p. 65). 9. Heteropleural transplantations, dorsodorsal orientation. For- ty-nine cases were operated upon in this way and thirty-one lived long enough to yield definite results (table 2). By far the largest number of these (twenty-five) developed reduplications of one kind or other. Five cases gave rise to limbs with reversed asymmetry, 1.e., to limbs which developed to fit their new sur- roundings, though one of these was considerably underdeveloped. One yielded a somewhat imperfect non-reversed limb and four were rudimentary. These results seem altogether divergent from the corresponding heterotopic transplantations. An examina- tion of them shows, however, that fundamentally they accord with the latter, complete agreement being modified, by a second factor, which may suppress the original bud in favor of the reduplicating member. The normal environment of the trans- planted bud and the concomitant normal functioning seem to facilitate this transformation. Moreover, there is no hard and fast line between the different results just enumerated, and the individual cases may be taken as forming a series, beginning with the single non-reversed and ending with the single reversed limb. The reduplications are intermediate. They will be considered in this order. 44 ROSS G. HARRISON == SSS SS SSS SSS 65 66 A : | =<, Sa SSS S \ “ LLL = Wy = . - LE SS i= Ny Zc coe ——— =e: \ »\ = == ; SS = Sot = WY = ~ SS een a Zz — 10. Fig. 78 Dorsal view, six days after operation. Reduplicating bud already more massive, though less prominent, than the primary. Fig. 79 Lateral view, six days after operation. Fig. 80 Dorsal view, eight days after operation. Fig. 81 Ventral view, fifteen days after operation. Fig. 81A_ Lateral view of limb. Fig. 81B_ Dorsal view of same. Fig. 82. Dorsal view, thirty-three days after operation. Fig. 82A Ventral view of transplanted limb. Fig. 82B Ventral view of normal right limb. 51 52 ROSS G. HARRISON My ify y a. iN Wi, ie A . H il Gees / Nee’ S Z ee ! SS. tay ,, es) d =e FZ Y Se, aos } = A \— ay} Sn oe ee tw fs yi \\' ' f Figs. 83t085 Exp. R. E. 77. ing member (DU) which has become a normal left limb. X 10. Orthotopic transplantation; right limb bud to left side (het.dd.). Primary member (PR) reduced to a small spur on the reduplicat- Fig. 83 Dorsal view, eight days after operation. planted bud already visible. X 10. Fig. 84 Dorsal view, eleven days after operation.., than primary. Fig. 85 Ventral view, twenty-six days after operation. Figs. 86 to 89 Oriiotopic transplantation; right limb bud to left side (het.dd.). Reduplication of trans- Reduplicating bud larger Exp. R. E. 69. Primary member (PR) reduced to a nodule on the reduplicating one (DU). X 10. Fig. 86 Dorsal view, nine days after operation. Fig. 87 Dorsal view, twelve days after operation. Fig. 88 Dorsal view, sixteen days after operation. Fig. 89 Ventral view, twenty-six days after operation. 53 SYMMETRY IN TRANSPLANTED LIMBS 54 ROSS G. HARRISON teen in number, in thirteen of which limbs developed. The distribution of these in the various groups does not show any significant differences from the cases with cleaned wounds. Eight (61.5 per cent) gave reduplications and three (23.1 per cent) Figs. 9) to 92. Orthotopic transplantation; right limb to left side (het.dd.). Exp. R. E.95. Primary bud (PR) entirely obliterated; the reduplicating member (DU) a normal left limb. N, normal right limb. X 10. Fig. 90 Dorsal view, ten days after operation; the primary bud (PR) shows as a slight nodule. Fig. 91. Dorsal view, thirteen days after operation. Fig. 92 Dorsal view, thirty-three days after operation. Owing to weakness of wrist and hand extensors, the larva has difficulty in bringing its hand to nor- mal posture. SYMMETRY IN TRANSPLANTED LIMBS 55 developed into reversed limbs. In at least two of the individuals reversal was brought about by reduplication. The third is un- certain. Of the two cases (15.4 per cent) recorded as develop- ing without reversal, only one is clear. The other died at fifteen days and was lost, so that the notes made from the living speci- men could not be verified. 10. Heteropleural transplantations, dorsoventral orientation. Twenty-six experiments were made in this group. In five out of the twenty-three individuals that lived the transplanted tissue was resorbed, and in two others the resulting appendage was im- perfect or rudimentary, so that sixteen positive cases are available. Single limbs with reversed asymmetry developed in fifteen, and only one gave rise to a duplicate structure (table 2). This group of cases shows that from the first the transplanted limb buds behave differently from those implanted in dorsodorsal orientation. When they begin to become prominent, they point dorsoposteriorly in most cases, though sometimes more sharply dorsally and frequently more laterally than the normal bud (fig. 93). As the bud grows, it thus occupies a nearly normal posi- tion, though it may continue for some time to project more sharply to the side or more dorsally than the normal limb (figs. 94 and 96). When the third and fourth digits develop, they are, however, not formed on the ventral border of the appendage, as they would be if the original asymmetry were preserved, but they come in on the dorsal border, just as in the normal hand of the side to which they were transplanted (figs. 2, 95, 98, 99, and 102). The palm of the hand, as in the normal individual, faces the body of the larva. Besides the one case in which reduplication actu- ally occurred, there were three others in which slight indications of doubling appeared, only to disappear later, the more ventrally lying bud soon being resorbed. Histories of typical cases are given on page 128. | In all of these cases there was some retardation of development, and in some’ it was very marked. A somewhat greater amount of tissue is lost by disintegration when the limb is placed dorso- ventrally than when placed otherwise, since the bud does not BSW Geka gO. 56 ROSS G. HARRISON fit into the wound so exactly. Besides this there seems to be a time factor involved in the reversal, which would indicate that the dorsoventral axis of the limb elements is slightly differenti- ated, though not irreversibly so. <= Figs. 93 to 95 Orthotopic transplantation; right limb to left side (het.dv.). Exp. R. E. 80. Transplanted limb (7'R) becomes a normal left. N, normal right limb; 7 to 4, numbers of digits. Fig. 93 Dorsal view, six days after operation. Transplanted bud much smaller than normal. Fig. 94 Dorsal view, fourteen days after operation. Fig. 95 Ventral view, forty-two days after operation. — SYMMETRY IN TRANSPLANTED LIMBS ol The sole ease in which a double appendage resulted®® is inter- esting, inasmuch as it shows that the primary bud grows into a reversed limb, while the reduplicating bud has the original asym- metry (figs. 103 to 105). This is the opposite of the result ob- tained when the bud is implanted in dorsodorsal orientation. (History on p. 129.) In the experiments with wounds not cleaned the proportion of reduplications is considerably larger—seven out of fifteen, or 46.7 per cent, as against one case in sixteen in the clean-wound group. There were eight cases (53.3 per cent) in which normal limbs with reversal of asymmetry developed, as against fifteen (94 per cent) in the case of the clean-wound experiments. 11. The shoulder-girdle in orthotopic transplantations. ‘The above account has dealt only with the external features a the limb. The shoulder-girdle is likewise of interest. As the heterotopic transplantations show, a small portion of the girdle surrounding the glenoid cavity always develops in connec- tion with the grafted limb. After extirpation of the limb bud, however, the outlying regions of the girdle, including the supra- scapula and portions of the procoracoid and coracoid, develop from cells that are left in the host.°° It was to have been expected, therefore, that relations of harmony or disharmony would mani- fest themselves in the shoulder-girdle in orthotopic grafts. Study of serial sections of some of the cases shows that this is usually the case. Twelve individuals, belonging to three different groups have been examined in this way. The three harmonic grafts (het.dv) all show girdles that are normal, except that they are somewhat underdeveloped. ‘There has obviously been a union of host and graft tissues to form a normal whole, in spite of the fact that the transplanted bud was from the opposite side of the body. The nine disharmonie grafts all show some form of irregularity, and in nearly all cases there is some sort of double girdle with reversal of the part that is derived from the graft. The condi- tion of the girdle is complicated by the reduplication of the free 59 R. E. 98. 60 Cf. Detwiler, 718, p. 503, and Harrison, ’18, p. 429. 58 Wi # ii } fe pl 100 ae SSS SERS —— ROSS G. HARRISON lr “ 102°. 5 Z LY if 4 SYMMETRY IN TRANSPLANTED LIMBS 59 limb which takes place in most cases (table 2). It is more readily understood in the two cases in which a single limb of opposite asymmetry is present. In the first of these," in which a limb bud from the same side of the body was implanted in inverted position (p. 32, figs. 39 to 41), there are two entirely distinct shoulder-girdles. The anterior one has no connection with the other and is undoubtedly derived from the host, having the characteristics of girdles which develop after extirpation of the limb bud. The scapula and su- prascapula are already joined in cartilaginous union with the procoracoid, but the coracoid is connected with the latter by ligament only. ‘The girdle belonging to the transplanted limb is mainly posterior to the other, though there is some overlapping. It is large to have developed from a transplanted bud, but it has the characteristics of such. There is a distinct procoracoid process as well as a large coracoid, both of which project pos- teriorly from the glenoid cavity. This girdle is clearly reversed, as is the transplanted limb which is connected with it. The other single disharmonic limb is the one developed from a bud taken from the opposite side of the body.” The limb itself is atrophic (fig. 64). The girdle is double, but the ventral parts of the two members are fused. The suprascapula, which is single and belongs to the host, is not connected with the rest. The Figs. 96 to 99 Orthotopic transplantation; right limb to left side (het.dv.). Exp. R. E. 107. Fig. 96 Dorsal view, eight days after operation. Transplanted bud (TR) smaller than normal (V) and more pointed. Fig. 97 Dorsal view, thirteen days after operation. Fig. 98 Lateral view, thirteen days after operation. Fig. 99 Dorsal view, nineteen days after operation. ° Fig. 99A Lateral view of transplanted limb. / to 3, numbers of digits. Figs. 100 to 102 Orthotopic transplantation; right limb bud to left side (het.dv.). Exp. R. E.116. Transplanted limb (7R) becomes anormal left. X 10. Fig. 100 Dorsal view, seven days after operation. Transplanted bud only slightly smaller than normal. Fig. 101. Dorsal view, thirteen days after operation. Fig. 102 Lateral view, same age. 317. E. 64. oR Sie 60 ROSS G. HARRISON SS EEE / GU Yf Yi if / I, Figs. 103 to 105 Orthotopic transplantation; right limb bud to left side (het.dv.). Exp. R. E. 98. Reduplication, the primary (PR) member being a left (reversed). DU, reduplicating member. X 10. Fig. 103 Dorsal view, nine days after operation. Fig. 104 Lateral view, same age. Fig. 105 Dorsal view, preserved specimen, age fifteen days. SYMMETRY IN TRANSPLANTED LIMBS 61 ventral part, which forms the glenoid cavity, is in fore and aft symmetry, with a coracoid and procoracoid process pointing in each direction. ‘The posterior half of this cartilage has almost certainly developed in connection with the grafted limb and is reversed, while the anterior half is derived from the host. In the disharmonic cases which have reduplicated limbs, the shoulder-girdles are on the whole less regular, owing to the com- plex articulations of the double appendages. One of them” _(hom.dv) is, however, similar to the one first described in having two entirely separate girdles, one derived from the host and one from the graft. The suprascapula, procoracoid, and coracoid of the former are separate chondrifications, situated directly opposite the corresponding parts of the normal limb. The girdle of the transplanted limb has a broad flat glenoid cavity for articulation with the massive humerus. There is a large coracoid running ventrally from the joint, though without any very well- marked procoracoid. This girdle is placed some distance pos- terior to that of the host. Another of these cases (hom.dv) is more like the second case described above, inasmuch as the dorsal element (suprascapula) is separate, while the two coracoids (from host and graft, respectively) are fused. The procoracoid of the host is a separate cartilage in this case. ‘Two other cases” are of the same general type with fused coracoids, though they are rather too young to show all characteristics. Again, two others** have two scapulae with coracoids fused. There is only one case*’ that shows in sections practically no sign of doubling of the girdle, though even in this the coracoid region is thicker than normal and the glenoid cavity is large in correspondence with the more massive humerus. To sum up: The shoulder-girdle in orthotopically grafted limbs is derived in part from the host and in part from the transplanted tissue. The former portion retains its normal asym- & 7, E. 81. 647. E.. 93. 8 T. EK. 68 (hom. dv.) and R. E. 129 (het dd.). 66 R. E. 77 (het. dv.) and R. E. 96 (het. dv.). 877. EK. 60 (hom. dv.). 62 ROSS G. HARRISON metry, while the latter behaves in accordance with the rules governing the asymmetry of transplanted limbs. In the dis- harmonic combinations the portions of the girdles derived, re- spectively, from the two sources may fuse together or may remain entirely separate. In the harmonic combinations they unite to form a single normal girdle. 12. Summary of the results of orthotopic transplantations. The orthotopic transplantations develop according to the same rules as the heterotopic. In the homopleural dorsodorsal and the heteropleural dorsoventral groups rules 1 and 2 (p. 4) are very closely followed. In the former the limb buds, being right side up, retain their normal asymmetry; and in the latter, being upside down, they reverse it. In both groups this results in limbs which correspond to the side on which they are implanted (harmonic combinations). In the other two groups he primary single limbs which develop do not correspond to the organic environment, since the homopleural graft, when placed upside down, becomes re- versed, and the heteropleural graft right side up retains its origi- nal prospective asymmetry. In these combinations, which have been called disharmonic, single limbs are, however, the exception. It is here that rule 3 comes into play. Reduplica- tions occurred in 71.1 per cent of the cases in the homopleural dor- soventral group and in 80.6 in the heteropleural dorsodorsal. The former includes only one case of single limb reversed. In this class are also five cases of reversed single limbs, which are fundamentally the same as reduplications, the original limb having been suppressed or resorbed. The disharmonic relation thus augments immensely the tendency to reduplicate. In the case of the heterotopic grafts, on the contrary, the greater pro- portion of reduplications occurs in the harmonic combinations. This curious fact will be discussed below (p. 107). The ten cases of non-reversed single limbs which resulted from homopleural inverted buds are, as already pointed out, exceptional in that the limb regained its normal posture gradually during development by rotation at the base. SYMMETRY IN TRANSPLANTED LIMBS 63 C. Superposed limb buds In the preceding study of transplanted limbs certain experi- ments were described, which showed that the mesoderm from two limb buds, when fused together; would develop into a single normal limb. At first larger than normal, the size of such a limb is soon regulated. In the former communication only those experiments were considered in which the orientation of the superposed bud was normal (hom.dd). The effect of the orienta- tion of the graft will now be taken up. TABLE 3 Superposed limbs. Summary of results NORMAL SINGLE | WITH REDUCED | REDUPLICATED REDUPLICATION NUMBER OF NORMAL EXPERIMENTS SINGLE LIMBS OPERATION Posi- | Num- P Num- Pe Num- P Total ave ee seat ber cent ; bee Baw lorena ame ios. aks sae 5 5 5 100 0 00 0 00 Ta liniie Cohan eae ne S a 1 20 0 00 4 80 LEIS (Ol oe ee em ts 6 5 0) CO 1 20 4 80 JBI. (0 N7ty Sane eee 9 5 5 160 0 00 0 00 ioelicr ees... ceil Be Oe fel 55 | 5 8") "40 The experiments are summarized in table 3. There were twenty-five operations, of which twenty are available for the analysis. Two of the combinations, the ones which the ordi- nary transplantations have shown to be harmonic (homopleural dorsodorsal and heteropleural dorsoventral) yielded only normal appendages (ten cases). The two disharmonic’ combinations (homopleural dorsoventral and heteropleural dorsodorsal) yielded reduplications in nine cases out of ten. One case, in which one member of the duplicate limb was reduced to a spur, is included among the reduplications. aah,” 13. Homopleural transplantations, dorsodorsal orientation. In this group development went forward with a minimum of dis- turbance. The only abnormal feature to note is the large size of the double bud in certain individuals. In several of the cases THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, No. 1 64 ROSS G. HARRISON 20, Dp) Ew : AL, < Lo 109 1098 \ | SYMMETRY IN TRANSPLANTED LIMBS 65 the difference from normal size persisted for twelve or more days, ®8 gradually diminishing during that period (fig. 106). In others the difference was less marked, though in all some difference in favor of the limb on the operated side was noted. 14. Homopleural transplantations, dorsoventral orientation. Four out of the five cases in this group gave rise to reduplications in the grafted limb. The reduplications vary. One®? is a typical case of radial mir- roring of the lower part of the forearm and hand (fig. 107). Another” is similar, except that the anterior member is itself reduplicated, the hand being a nearly symmetrical complex with four digits. These two individuals are in every respect like those cases of reduplication resulting from simple inverted limb buds, in which the primary member is reversed and is accom- panied by a non-reversed twin which takes up the normal posi- tion. In two other cases” the reduplication is less and is of a character not necessarily attributable to disharmonic combina- tion, though there is nothing to indicate that it is not due to such a cause. Principally the digits are involved (fig. 108). In the remaining case” a normal limb developed. This one and possibly also the two foregoing are analogous to those cases of simple transplantation in which the inverted limb bud develops into a normal limb without reversal by means of rotation (p. 40). Fig. 106 Superposed limb bud; right limb bud to right side, normal position (hom.dd.). Exp. 8. E. 3. Normal limb (7R) on operated side. N, normal left limb. Preserved specimen, ventral view, eighteen days after operation. X 10. Fig. 107 Superposed limb bud; right limb to right side inverted (hom.dv.). Exp. 8. E. 18. Reduplicated appendage (TR) on operated side. Preserved specimen, ventral view, eighteen days after operation. X 10. Fig. 108 Superposed limb bud; right limb bud to right side inverted (hom.dv.) Exp. 8S. E. 9. Operated limb (TR) normal except for reduplication of second digit. X 10. Fig. 109 Superposed limb bud; right limb bud to left side (het.dd.). Exp. S. E.6. Reduplication with heteropleural member reduced to spur (S). X 10. Fig. 109A Outline of limb bud from above. Five days after operation (free- hand sketch). Fig. 109B Same, eleven days after (free-hand sketch). cS Ht. 3. BoD. 1, 18: WSs Bie2. 1§. E, 9 and 14, 725. E. 10. 66 ROSS G. HARRISON Sita es UCT M0, ions a SYMMETRY IN TRANSPLANTED LIMBS 67 15. Heteropleural transplantations, dorsodorsal orientation. In this group the results of five experiments all fall clearly within the same category. A normal appendage in approximately normal position developed, and this had a reduplicating member attached to its radial border. The differences between the cases consist in the extent of reduplication and the degree of development attained by the disharmonic (anterior) member. The most extreme cases are one” in which the heteropleural member is reduced to a spur (fig. 109), and one” in which there is almost complete doub- ling from just below the shoulder down, the heteropleural member being, however, atrophic (figs. 110 to 113). Two of the three remaining cases” are very similar to one another, the anterior member with two digits arising from near the elbow. In the third individual the hand is reduplicated’® externally, and the whole arm is somewhat shorter and thicker, indicating some degree of internal reduplication. 16. Heteropleural transplantations, dorsoventral orientation. The five cases in this class all gave normal limbs (figs. 117 and 118). Three of them showed slight indication of doubling (figs. 114 and 115) in the early stages of development (four or five days after operation), but the more ventral-lying prominence had dis- appeared at the time of the next observation in each individual Figs. 110 to 113 Superposed limb bud; left to right side (het.dd.). Exp. S. E.12. Reduplication. WN, normal left limb; 7R, operated limb; HET, hetero- pleural member; HOM, homopleural member. Fig. 110 Dorsal view, five days after operation. Fig. 111 Lateral view, same age. Fig. 112 Ventral view, ten days after operation. Fig. 118 Ventral view, seventeen days after operation. Figs. 114 to 119 Superposed limb bud; left to right side (het.dv.). Exp. S. E. 11. Operated limb (7'R) of large size; N, normal unoperated limb. X 10. Fig. 114 Dorsal view, five days after operation. Fig. 115 Lateral view, same age. A distinct nodule or bud (DU) on ventral border of limb was soon afterward resorbed. Fig. 116 Ventral view, ten days after operation. Fig. 117 Lateral view, same age. Fig. 118 Dorsal view, seventeen days after operation. Fig. 119 Lateral view of limb, same age. eS, Hs 6. Soi. Ae 7% S. E. 8 and 21. #8, E. 15. 68 ROSS G. HARRISON / (figs. 116 and 117). A sixth case’? is inconclusive owing to gen- eral weakness of embryo, but it is not inconsistent with the results in the other cases. A typical history is given in the appen- dix, (pz 31). Though more than the normal amount of material is present in the composite limb bud, in two of the cases of this group the developing limb is recorded at first as slightly smaller than the normal. In one case no difference in size was noted, while in two the limb on the operated side is noted as larger. In the other harmonic combination (homopleural dorsodorsal) all cases showed the limb on the operated side to be somewhat larger in size. 17. Discussion of experiments with superposed limb buds. The principal differences between these experiments and those of simple transplantation are that in the former the tissue available for the formation of the lmb is approximately double in amount, and there is a mixture of tissues having two different or- ientations except in the one group, homopleural dorsodorsal. In the harmonic combinations the amount of tissue is so regulated that after a time size-differences disappear. The amount of tis- sue is, moreover, never quite double that of the normal limb be- cause of the material lost by the operation and of the general retardation of growth due to the same cause. In the case of the heteropleural dorsoventral grafts, which are classed as har- monic, some readjustment must be necessary, as shown by the amount of retardation. In all of the disharmonic combinations there is a mixture of tis- sues differently oriented and with different prospective meaning as regards the particular asymmetry of the future limb. The twin limbs that arise are, therefore, not necessarily due to redu- plication by budding, as they must be in the simple transplanta- tions, but probably in part at least to the circumstance that one of the pair develops out of the original limb bud, while the other is from the transplanted tissue. LES 135m bop SYMMETRY IN TRANSPLANTED LIMBS 69 D. Transplantation of half buds Partly as a further test of the question of equipotentiality and partly to study more thoroughly the effect of harmonic and dis- harmonic combinations, a series of experiments with half limb buds was instituted. Instead of removing the whole circular disc comprising the limb rudiment, a semicircular piece was cut out, the wound bed carefully cleaned, and the removed portion replaced by a piece of similar size and shape from another limb bud. Considering only vertical and horizontal halves and re- placing vertical only with vertical and horizontal only with hori- zontal, there are sixteen different experiments possible, which have been numbered in the diagram (fig. 120) from 1 to 16. There are five different pairs of attributes, which appear as alternatives in the operations. Thus the transplanted half bud is either—1) homopleural (hom.) or heteropleural (het.); 2) up- right (dd.) or inverted (dv.) ; 3) homogeneous (homogen.) or hetero- geneous (heterogen.); 4) vertical (vert.) or horizontal (horiz.); 5) anterior (ant.), dorsal (dors.) or posterior (post.), ventral (vent.). This aggregation would consist of 2° or thirty-two classes, were it possible to combine the attributes of operation independently without restriction, as would be the case were the pieces rectangu- lar. Since, however, they are semicircular they fit in only half the cases, and the total is therefore reduced to sixteen. All of the possible experiments have been performed. If both halves of the dise are considered movable, further pos- sibilities open up. There would then be thirty-two different com- binations, which, however, in eight cases would be practically identical with the experiments where the whole disc is transplanted. None of these experiments have been performed, since the tech- nical difficulties would be at least doubled, and, as far as the study of either of the questions at issue is concerned, they would offer no advantage over those in which only one half of the bud is transplanted. Again, were the limb disc homogeneous, either or both halves could be turned inside out and then one hundred and twenty-eight different combinations would be possible. These are precluded, as in the case of the whole discs, by the 70 ROSS G. HARRISON impossibility of grafting successfully pieces with the mesoderm turned toward the outside and by the difficulty of handling pieces of mesoderm free from ectoderm without disturbing their arrangement. Perhaps we may consider ourselves fortunate in being subject to such restrictions. HOM.DV 4 HET.DD (A LIRYA g 9 HET.DV Fig. 120 Diagram showing the sixteen possible combinations (1 to 16) obtain- able by transplanting half limb buds. The shaded area signifies the stationary half, the clear area the transplanted half. R, right; L, left; D, Dorsal; V, ven- tral; A, anterior; P, posterior. The operations are represented as on the right side of the embryo. Returning to the experiments actually carried out (fig. 120), we find that four of them consist merely in replacing the excised piece with another of exactly the same kind in normal orientation. These serve, therefore, as controls for testing the effect of the operation as such on the further course of development. It is also seen that half of the combinations are harmonic and half dis- harmonic (p. 8). Half are of course homogeneous or com- SYMMETRY IN TRANSPLANTED LIMBS 71 posed of two similar halves, while the other half are heterogene- ous. Of the former, six belong to the disharmonic group and only two to the harmonic, while of the latter the reverse is the case, a circumstance that affects the proportionate results of the experiments. The effect of removal of the various halves of the limb rudiment has already been described (Harrison, 718). As shown by such experiments, any half of the limb bud can give rise to a whole limb, though quantitatively the material is eecentrically distrib- uted, there being more limb-forming tissue in the dorsal and anterior halves than in the ventral and posterior halves, respec- tively. Accordingly, four of the homogeneous combinations would have somewhat less than the normal amount of tissue, while four would have a little more. In the later experiments an attempt was made to compensate for this by not cutting the area exactly in half. Owing to the large number of combinations in the experiments, it has not been possible to perform a sufficient number of each, for accurate statistical treatment. The number is sufficient, how- ever, to compare the more comprehensive groups; for instance the homogeneous with the heterogeneous and the harmonic with the disharmonic. Seventy-nine operations were done, sixty-eight healing success- fully. Badly defective limbs developed in but four cases, so that sixty-four remain for the purpose of the analysis. These experi- ments are summarized in table 4. From the results of transplanted whole limbs we should expect the following to take place; the harmonic combinations should give rise to simple normal limbs, the disharmonic to reduplica- tions. The homogeneity or heterogeneity of the combination should not be expected to make any difference in view of the other tests of the equipotentiality of the system. These expectations were in the main realized, probably in fifty-five out of the sixty- four cases (85.9 per cent) (table 7). There are, however, sources of confusion, which in certain cases make several interpretations possible, and which for this and other reasons must not be over- looked. For example, it is known from experiments with whole 12, ROSS G. HARRISON limb buds, that a normal limb may arise from a disharmonic com- bination by the suppression of the original bud or by its reduction to a mere excrescence on the reduplicating member, which latter may develop into a normal limb. Eight of the ten normal cases which would otherwise appear anomalous may certainly be thus explained, and possibly the remaining two. It has been found TABLE 4 Transplantation of half limb buds. Summary of results of actual experiments OPERATION RESULTING LIMB = | Designa- | Nor- 2 Side of : Direction tion of = mal | Re- N ee oe vane Composition ee (eae No byre dupli- pee Dead aus half tion 1 | hom. | dd | heterogen. | vertical | ant. 2 0 0 0 1 2 | hom. } dd | heterogen. | vertical | post. 2 0 0 @ 1 3 | hom. | dd | heterogen. | horiz. dors. | 2 0 0 0 0 4 | hom. | dd | heterogen. | horiz. vent. | 2 0 0 0 0 5 | hom. | dv. | homogen.. | vertical | ant. 0 2 2 0 1 6 | hom. | dv | homogen. | vertical | post. | 0 0 oe n0 2 7 | hom. | dv | homogen. horiz. dors. 0 2 2 0 0 8 | hom. | dv | homogen. horiz. vent. | 0 0 4 1 0 9 | het. dd | homogen. vertical | ant. 2 2 0 3 1 10 | het. dd | homogen. vertical | post. | 0 1 SEO 0 11 | het. dd | heterogen. | horiz. dors. 0 0 5} 0 1 12 | het. dd | heterogen. | horiz. vent. | 0 0 4 0 1 13 | het. dy | heterogen. | vertical | ant. 4 0 1 0 1 14 | het. dv | heterogen. | vertical | post. 4 0 0) 0 2 15 | het. dv | homogen. }_ horiz. dors. 5 0 2 0 0 16 | het. dv | homogen. | horiz. vent. | 6 0 0 0 1 Total number of cases, 79; positive cases, 64... .| 29 7 i 28 atl 1 Includes one case of anomalous reduplication. 2 Includes two cases of anomalous reduplication. also that almost any transplantation or even simple defect ex- periment may sometimes bring about reduplication. The three anomalous reduplications, being slight, are probably of this class. A further source of error might arise from the circumstance, that either the grafted or the stationary half may in certain cases be solely responsible for the limb that develops; for it is known, on the one hand, that any graft may be resorbed and, on the other, SYMMETRY IN TRANSPLANTED LIMBS 73 that when half the disc is excised, complete suppression of development may sometimes result, probably through accidental injury to the remaining part. The result of the former contin- gency would be confusing, owing to the development of a normal limb in place of a reduplication. . In connection with these questions it must also be borne in mind that the cases of union of two disharmonic halves differ, with respect to the disharmony of the combination, from those in which the limb-bud has been transplanted as a whole. In the former only one half of the rudiment is involved, the other being in all cases harmonic with the surrounding tissues. We are, therefore, dealing with a rudiment that is disharmonic in itself, while in the case of the whole limb the transplanted bud is harmonic in itself, though disharmonic with respect to the organ- ismasawhole. This might possibly give rise to some differences in the results in the two classes of experiments. In view of these considerations, we should not expect the trans- plantation of half buds to give such clear-cut results as the experiments with whole ones. On the other hand, it must not be overlooked that the sources of confusion above enumerated, while accounting for nearly all of the anomalies, also render less cogent the cases which conform to the rules. Nevertheless, after taking all circumstances into consideration, it can scarcely be doubted, that the experiments with half discs do afford a valuable confirmation of the results obtained from the other experiments. 18. Homopleural transplantations, dorsodorsal orientation. The eight cases of homopleural grafts in upright orientation (hom.dd), two involving each half of the bud, all resulted in normal limbs, as was to have been expected, for this operation is nothing more than replacing an excised portion with one exactly similar. Only slight retardation of development is recorded in some of the cases. 19. Homopleural transplantations, dorsoventral orientation. The nineteen experiments with homopleural grafts in inverted posi- tion (hom.dv) resulted, in accordance with expectation, in a large number (fifteen) of duplicities’® (figs. 121 and 122). The 78H. BE. 29 and 31. 74 ROSS G. HARRISON remaining four cases were normal. In the early stages, however, all of the latter gave evidence of reduplication (fig. 123). The limb bud, when it first appeared, showed two distinct nodules or prominences, one of which developed into a normal limb. In Figs. 121 and 122* Transplantation of half limb bud (comb. 6, fig. 120); pos- terior right to anterior right (hom.dv.). Exp. H. E. 31. Partial reduplication of hand, mirror plane being radiodorsal. Arm and medial hand homopleural. 1 to 4, numbers of digits of main hand; 2’, 3’, digits of reduplicating member. Fig. 121 Outline of normal (N) and operated (7R) buds from above, nine days after operation (free-hand sketch). Fig. 122. Ventral view, preserved specimen twenty-one days old. X 10. Figs. 123 and 124 Transplantation of half limb bud (comb. 7, fig. 120); dorsal right to ventral right (hom.dv.). Exp. H. E.2. Operated limb slightly thicker, reduplicating member reduced to a nodule (S). Fig. 123 Outline of operated limb, dorsal view eight days after operation (free-hand sketch). Fig. 124 Ventral view preserved specimen, twenty days old. X 10. SYMMETRY IN TRANSPLANTED LIMBS 75 three cases the other prominence persisted also, at the elbow in one?’ in the form of a spur (fig. 124), and as a nodule at the shoulder in two others.8° In the remaining case*! all external traces of reduplication disappeared. On the other hand, two of the cases of reduplication are of an anomalous nature and cannot be regarded as conforming to the rule. Both of these were experiments in which the anterior half of the limb bud was replaced by a posterior half. We should expect in such a case to find posteriorly a homopleural member developed out of the stationary portion of the bud, while anteri- orly there should be a reversed limb which might itself be redu- plicated. . The opposite is, however, true. In both cases the an- terior member is not reversed. The posterior member is reversed (a left) in one case® (fig. 126) and in the other* it is itself double, the anterior portion being reversed and the posterior homopleural (fies i25)., ; The operated limb in all of these cases was composed of two homogeneous halves. Histories of typical cases are given in the appendix (p. 132). 20. Heteropleural transplantations, dorsodorsal orientation. This combination, being disharmonic, yielded out of seventeen cases twelve duplicities (figs. 127 and 129) and three limbs that became normal by reduction of the reduplicating member (fig. 130). In two individuals* normal limbs resulted without exter- nal evidence of incipient doubling, and two of the reduplications, in one of which both members are of the same side in linear series, are of an anomalous nature. This makes four cases out of sev- enteen that do not follow the rule. Two of the combinations are heterogeneous; all of these conform to the rule except one of the anomalous reduplications. The other three non-conforming cases belong in the homogeneous class, and it is interesting that all of the normal cases in this group resulted from the combina- tion of two like halves. MAS (, 1, 2 a Jal 18, 16} 80 H. BK. 18 and 21. CM & [al ae su H. BH. 4. 84H. R. E. 43 and 44. 76 ROSS G. HARRISON Fig. 125 Transplantation of half limb bud (comb. 6, fig. 120); posterior half right to anterior right (hom.dv.). Exp. H. E. 5. Two limbs, the posterior of which has a double hand. Anterior member an almost normal right; of the two parts of the posterior member, the anterior one is a left hand and the posterior, aright. X 10. Fig. 126 Same operation (comb. 6, fig. 120) (hom.dv.). Exp. H. E.13. Ulnar reduplication! HOM, homopleural hand; / to 3, digits of same; HET, hetero- pleural hand; 1’ to 3’, digits of same; N, normal (unoperated) left limb xX 10. Fig. 127. Transplantation of half limb bud (comb. 10, fig. 120) ; posterior half right to anterior left (het.dd.). Exp. H.R. E.1. Double hand. HOM, homo- pleural hand with digits (7 to 4); HET, heteropleural hand with digits. (2’ to Be o< OL SYMMETRY IN TRANSPLANTED LIMBS Ut! The double limbs are of various degree and kind. The least - involved is one in which only the first digit is doubled. In this individual the ventral half of the limb was replaced by a ventral half of the opposite side, and in all probability very little limb material was actually transplanted, since, in the embryo from which the graft was taken, the operated limb developed almost asrapidly asthe normal. In five other cases* (fig. 127) the whole hand is involved, with indications that in three of these at least’’ the internal reduplication extends farther proximally. In two cases the fore arm and hand®s (figs. 128 and 129) are externally double, and in one,*? which was not fully developed when pre- served, doubling would probably have shown from near the shoulder down. In two of the individuals®? there are secondary reduplications. In the two anomalous cases"! (figs. 1381 and 132) there are two entirely separate limbs. Histories are given on page 134. Unfortunately, external observation does not always reveal the relations of each of the two halves of the bud to the developing members. 21. Heteropleural transplantations, dorsoventral orientation. Out of twenty-two successful experiments in this group nineteen resulted in the development of normal limbs (fig. 133), which is according to rule, and only three gave rise to reduplications (fig. 134). Two of the latter’? involved only the radial digits, in which paimar reduplication was present, the limbs being other- wise normal. In the remaining one a bifurcated appendage arose, but the dorsal branch remained merely as a spur attached to the main limb, which was normal though undersized and with slight syndactyly. In one case,*? which has been classed as normal, a filamentous appendage probably not a limb, developed a short distance ven- tral to the main limb, which was normal though slightly shorter. SARS Bi 30: 90H. R. EH, 21 and 47. 86 HW. R. E. 1, 15, 46, 47, and 48. 1H. R. E. 9 and 20. 87H. R. EB. 15, 46, and 47. 2A. 2. LOvand 1G: 88H. R. E. 5 and 21. iy Re Bez: aOR. B28 78 ROSS G. HARRISON This group of experiments is interesting because two of the combinations (ventral half in place of dorsal and dorsal in place of ventral) are homogeneous. Out of thirteen such cases, normal limbs developed in eleven. For histories of representative cases see appendix (p. 136). 13O Figs. 128 and 129 Transplantation of half limb bud (comb. 11, fig. 120) ; dorsal half right to dorsal left (het.dd.). Exp. H. R. E.5. HOM, homopleural member; HET, heteropleural member. Fig. 128 Outline of limb from above, ten days after operation (free-hand sketch). Fig. 129 Preserved specimen lateral view, nineteen days old. X 10. Fig. 130 Transplantation of half limb bud (comb. 9, fig. 120); anterior half left limb bud to posterior right (het.dd.). Exp. H. R. E.11. Normal limb with small spur (S). X 10. 22. Discussion of experiments with half buds. In order to es- tablish the conclusion stated in the introduction to this section, that it is the harmony or disharmony of the half-and-half combi- SYMMETRY IN TRANSPLANTED LIMBS 79 nation and not one of the particular qualities of the operation that determines whether normal or reduplicated limbs arise, it will be necessary to examine the numerical results of the experiments more carefully. If we take the actual figures of the experiments and examine the qualities in pairs, we find the actual number of each class and 132 Fig. 131 Transplantation of half limb bud (comb. 10, fig. 120); posterior half left side to anterior right (het.dd.). Exp. H. R. E.9. Anterior member a right (homopleural), the posterior one a left (heteropleural), but imperfect. Ventral view of specimen preserved forty days after operation. X 10. Fig. 131A Lateral view of limbs of same. Fig. 132 Transplantation of half limb bud (comb. 11, fig. 120); dorsal half left side to dorsal right (het.dd.). Exp. H. R. E. 20. Two right limbs, the ante- rior one imperfect. X 10. 80 ROSS G. HARRISON the proportion of normal results to be as given in table 5, column 6. ‘Normal by resorption,’ being fundamentally the same as reduplication, is classed as such. Fig. 133 Transplantations of half limb bud (comb. 16, fig. 120); ventral half left side to dorsal right (het.dv.). Exp. H. R. E. 36. Normal limb. Fig. 134 Transplantation of half limb bud (comb. 18, fig. 120); anterior half left side to anterior right (het.dv.). Exp. H. R. E. 10. N, normal left arm; TR, grafted arm with palmar reduplication of .two digits (DU). X 10. The one thing that stands out is the great difference between the results of the harmonic and those of the disharmonic combi- nations. In the case of none of the other attributes of opera- tion is there anything like the same difference between those of a pair, though in the case of homogeneity vs. heterogeneity the difference is considerable (37.1 vs. 64 per cent). SYMMETRY IN TRANSPLANTED LIMBS 81 However, the comparisons cannot be accurately made without the same number of experiments in each class, unless made by means of percentages. This is quite obvious, for instance, in the case of the homopleural transplantations with dorsodorsal orientation, which all result in normal limbs. The relatively TABLE 5 Transplantation of half buds. Comparison of pairs of qualities of operation ° NUMBER OF CASES ASAE | pall eee PAIRS OF QUALITIES COMPARED |Normal| = ee FOR INEQUAL- Norma! by nie Tctal ae SLE SE ea “tion | cated? “en omopleuralo. ... 2. cee ae 8 4 11 23 | 34.8 50.0 Heteropleurils.. 22. epeneee sear Al 3 13 37 | «56.8 50.2 Worsodorsale 542 ch. SAE el | nO 3 10 23 | 43.5 56.25 DonsoventGall.): 2)... ea ae eee 19 4 14 ay |) Ged! 43.9 Homogeneous... .))) 92). See eee 13 7 15 30) ode Heth Hetenozeneous). 4. . = = aocnete ae, 16 0 9 25 | 64.0 72.5 WETTIG Mle ae sicc-cns Mente nokta 14 5 8 2 ole9 53). 09 EMOUENZ ONG ete ce) kes sc Nore hele NLD 2 16 3a | 45.5 46.8 Anterior or dorsal half trans- JST STG ea? 15 6 11 382 | 45.45 50.2 Posterior or ventral half trans- | , plantede a eeaees |e hee 14 1 13 28 | 50.0 50.0 Harmonic (hom. dd and het. dv) | 27 0 3 30 | 90.0 93.9 Disharmonic (hom. dv 2nd het. | | | Casey ee As) eet el eee DA NTE. 21 30 (Bey 6.25 1 The four cases of anomalous reduplication have been omitted from this tabulation. small number of cases in this group affects the record for homo- pleural transplantations by reducing considerably the number of cases that would have developed normally, thereby giving undue weight to the larger number of dorsoventral cases which result in reduplications. Likewise the dorsodorsal vs. dorsoven- tral record is influenced by the relatively small number of homo- pleural cases. $2 ROSS G. HARRISON On this account the operations in each class have been reduced to a common basis. While the probable error of these figures is in most cases large, the comparisons resulting therefrom are no doubt much more reliable than those resulting from the figures of the actual experiments. They are given in the last column of table 5. In examining this table we find that there is little or no asso- ciation between the experimental results and the following quali- ties of operation: homopleural vs. heteropleural, dorsodorsal vs. dorsoventral, vertical vs. horizontal, anterior and dorsal vs. posterior and ventral, the deviation from total lack of associa- tion (50 per cent) being in the most extreme case but 6.1 per cent. When we examine the figures with reference to the pair, homo- geneity vs. heterogeneity, we find that there is a much wider dif- ference (27.7 per cent as compared with 72.5). This would have to be regarded as a significant difference but, as will be seen below it is only secondarily so. The marked association between the harmonic combinations and normal development (93.9 per cent) and the very small proportion of normal development (6.25 per cent) in the disharmonic group, show that it is largely this pair of attributes that determines whether development will be normal or not. This quality of harmony or disharmony, however, is not like the simple qualities of side of origin, orientation, or direc- tion of the incision, but is itself a combination of two of them. Those that are harmonic are the homopleural dorsodorsal and the heteropleural dorsoventral combinations, the other two being disharmonic, as in the experiments with whole limb buds. _ When we consider the homogeneous and heterogeneous com- binations, we find them unevenly distributed with respect to the harmonic and disharmonic. This is on account of the restric- tion of operation due to the semicircular shape of the trans- planted pieces, which makes half of the combinations impossible of execution. Were these all possible, there would be complete symmetry in the aggregation as a whole. In reality, it will be recalled, six of the homogeneous combinations are disharmonic, while only two are harmonic. On the hypothesis that it is the harmony of the combination that determines normal develop- SYMMETRY IN TRANSPLANTED LIMBS 83 ment, and with an equal number of experiments in all of the six- teen possible classes, the expectation would be that only 25 per cent of the homogeneous and 75 per cent of the heterogeneous would be normal. This corresponds closely to the figures 27.7 and 72.9, respectively, found in table 5. As regards the question of equipotentiality, the results of these experiments are equally striking. The two homogeneous com- binations which, according to expectation, should yield normal limbs did so. Thus two ventral halves yielded normal limbs in all six experiments, as did two dorsal halves in five out of seven. In three cases of disharmonic homogeneous combination normal limbs developed by resorption of the reduplicating bud; two of these were from two anterior halves and one from two posterior. ‘Two further cases of normal limbs from two anterior halves developed without external evidence of resorption. While the last five, if interpreted according to the rules, can only be accepted as evidence of equipotentiality in so far as they show that a whole limb can develop out of a single half bud, the others show that two half buds which are alike except that they are from opposite sides of the body may give rise, when harmonic, to a single normal limb. GENERAL DISCUSSION In this section the following questions will be considered: 1) the foundation of the rules of symmetry; 2) the mode of repre- sentation of symmetric relations in the limb rudiment; 3) the formation of reduplications; and 4) form regulation and function in transplanted limbs. E. The rules of symmetry. The validity of the rules of symmetry which have already been stated in the introduction (p. 4) will best be realized by considering the results of the several experiments in tabular form (table 6). Conformity is shown most strikingly in the heterotopic group, where there is only a single apparent excep- tion in forty-five cases; and this exception, as already pointed out, is probably due to an error in recording the operation. 84 ROSS G. HARRISON In the orthotopic group the lowest percentage of conformity (65.8) is found in the inverted homopleural buds (hom.dv), where the exceptions are due entirely to adjustment by rota- tion of the limb as a whole. In the superposed buds the sole exception is due probably to the same cause. The exceptional TABLE 6 Showing conformity to rules in the several experiments : OPERATION RESULTING LIMB Conforming * Side of | O,jen-| Harmonic to rules ISUTE GES Type of experiment origin of an or dis-_ ; Total graft harmonic Aun Be oo PeLeent Whole bud heterotopic..| hom. | dd | harm. 7 |100.0) 0 7 Whole bud heterotopic..| hom. | dv | disharm. | 12 |100.0} 0 12 Whole bud heterotopic. .| het. dd | disharm.| 10 |100.6) 0 10 Whole bud heterotopic..| het. dv | harm. 15. | 9358) <2.(?)) 6.3.02)) 36 Whole bud orthotopic...| hom. | dd | harm. 9 |100.0} 0 9 Whole bud orthotopic...| hom. | dv | disharm.| 25 | 65.8/13 [34.2 38 Whole bud orthotopie...| het. dd | disharm. | 31 /100.0} 0 31 Whole bud orthotopic...| het. dv | harm. 16 {100.0} 0 16 Superposed buds. ......| hom. | dd | harm. 5 |100.0) 0 5 Superposed buds.......| hom. | dv | disharm. 4 | 80.0) 1 {20.0 5 Superposed buds....... het dd | disharm. 5 |100.0| 0 ~5 Superposed buds....... het. dv | harm. 5 |1C0.0} 0 5 Half buds). a s.cccaes,. ....2-|, Hom dd | harm. 8 |100.0) 0 8 Halfbuds. 5.5. 544... +. >| som dv | disharm. | 15 {100.0} 0! 15 Hate buds. .o. eee. 0... | sete dd-|disharm. | 13 | 86.7] 2! |13.3 15 Halt buds teak. o2.: | hett dv | harm. 19 | 86.4; 3 /13.6 22 Wotala SRE eee eck COs oak accion 199 | 90.9/20 Ort 219 Average Ofspercentaress ali serie, . nents < o2ais 94.5 5.5 1 Four cases of anomalous reduplication have been omitted from this tabula- tion, inasmuch as they cannot be classified either as conforming or as exceptional. cases arising after transplantation of half buds have already been discussed, and, as pointed out above, there are in this group of experiments obvious disturbing factors which might readily account for the exceptions. Taking the experiments as a whole, 90.9 per cent conform and 9.1 per cent are exceptional, but if allowance is made for the difference in the number of experiments in each class, assuming that each is a fair sample of what would SYMMETRY IN TRANSPLANTED LIMBS 85 occur in a large number of cases, then but 5.5 per cent are exceptional. The behavior of transplanted limb buds in accordance with the above rules indicates that the posture and the asymmetry of the limb is determined neither by the limb itself nor by its surroundings exclusively, but by an interaction between the two. This is best described by the assumption, that in the stages exper- imented upon the anteroposterior axial differentiation is already determined within the limb bud, while the ventrodorsal axis (prob- ably radio-ulnar of the grown limb) is determined by its orien- tation with reference to the surrounding tissues of the host (fig. 135). In a given place a right limb bud upside down thus be- haves like a left limb bud right side up and vice versa (fig. 2). It is scarcely necessary to point out that this is not a gravity effect, for the embryo lies on its side during the period when the dorsoventral axis of the limb is determined, ‘upside down’ being used here merely with reference to the cardinal points of the embryo itself. What the nature of the influence exerted by the organic envi- ronment may be, has not been determined. Whether it acts upon the intimate structure of the limb bud or directly upon the differ- entiating systems contained therein, without affecting the inti- mate structure as a whole, cannot be answered from the present data (p. 101). The influence is not sharply localized, for it is the same both in the limb region itself and elsewhere along the flank of the embryo, so that it is probably an effect of the axial differentiation of the tissue elements themselves. It is possible that light may be thrown upon this question by transplanting the limb bud to the dorsal or to the ventral midline of the embryo. F, The mode of representation of symmetric relations in the limb rudiment The question whether the adult parts are localized in the germ, forming a mosaic, must be answered in the negative for the limb bud, as used in the experiments, i.e., if we consider as such a dise of tissue, three and a half somites in diameter, cen- tering ventral to the fourth myotome, and leave out of account the outlying regions from which certain portions of the shoulder- 86 | ROSS G. HARRISON girdle develop. This conclusion is based upon the following evidence derived from the experiments: 1) After extirpation of any half of the limb bud, a complete normal limb may develop from the remaining half; 2) fusion of two limb buds by super- position is followed, if the combination is harmonic, by the devel- opment of a single normal limb, which at first is usually larger than normal, but in which there is rapid regulation of size; 3) Vv HOM.DV HETDV Fig. 135 Diagram to show determination of asymmetry of limb. The circles represent the limb bud, the squares the surrounding part of the embryo. A,D, P,V, the cardinal points of the embryo—anterior, dorsal, posterior, and ventral, respectively. The heavy arrows represent the determining axes, i.e., the antero- posterior axis of the bud and the dorsoventral axis of the surrounding parts; UL, future ulnar border; D/, approximate direction of outgrowth. The smaller arrows show the other axes of bud and surroundings, respectively, which are not effective in determining the axes of the definitive limb. SYMMETRY IN TRANSPLANTED LIMBS 87 a normal limb usually develops out of two like halves, i.e., two dorsal, or two ventral halves, if properly oriented, when the opposite half is entirely missing; 4) after inversion of the limb bud the material that normally would have formed the radial half of the limb gives rise to the ulnar half and vice versa, so that prac- tically no part of the bud has the same fate that it would have had if it had been left in place; 5) the inoculation of mesoderm from the limb bud under the skin of the flank of another embryo may result in the formation of a normal limb, although the inoc- ulated tissue is badly disarranged by the operation. According to all tests that have been applied, the embryonic limb rudiment constitutes, therefore, an harmonic equipotential system, though, as a whole, it is self-differentiating except for the determination of its dorsoventral axis. The term ‘harmonic equipotential system’ is employed here, as defined by Driesch, in the sense that the potencies of all parts of the system are the same, the constitu- ent cells being totipotent.*%* Its use does not imply that the writer attaches to the existence of such systems the same signifi- cance as Driesch, who considers them as constituting a proof of the ‘autonomy of life.’ Even without this, however, and even though the actual system may not reach the abstract perfection demanded by its definition, 1t remains as a useful conception in experimental morphogenesis. The existence of the equipotential system necessitates, in fact, the assumption of some sort of mo- lecular hypothesis for the representation of adult form in the germ, and herein lies its importance in connection with the pres- ent study.” In particular, we must look to the constitution of % The concept ‘harmonic equipotential system’ is defined by Driesch (’05, p. 679) as follows: ‘‘Bekanntlich nenne ich harmonisch-iquipotentielle Systeme solehe Formganze, bei denen eine Differenzierungs- oder Wachstumsgesamt- leistung in ihren Einzelheiten jeweils einzelnen Elementen des Ausgangsganzen zufaillt, derart, dass jedes Einzelne dieses Ganzen jedes Einzelne jener Leistung vermag, alles Einzelne aber derart in Harmonie steht, dass die Leistung selbst ein Ganzes ist.’? The bearing on the question of vitalism is discussed in various papers, especially: ’99, p. 99; ’01, p. 170; ’08 b, p. 138. % Child has expressed skepticism as to the very existence of equipotential systems; for instance: ‘‘I think we may say that there is at present no valid evidence for the belief that any living system which is undergoing regulation or development in nature is at any given time an equipotential system”’ (’11, p. 306). Cf. also Child, ’08. 88 ROSS G. HARRISON the elementary units of the limb bud, rather than to their ar- rangement, for the representation of those relations of symmetry that the experiments here described have revealed.** Inother words, it is the intimate protoplasmic structure that underlies symmetry. In an equipotential system without axial differentiation, it is most natural to assume that the elements themselves are iso- tropic.°7 Axial differentiation would then result from the grad- ual modification of these units by reaction with other elements of the system or through external influences. These differentia- tions with reference to directions in space may be referred arbi- trarily to three axes crossing one another at right angles. They are geometrically of four grades, according to the number of axes along which polarization has taken place. Taking the models used in stereochemistry to show the spatial relations of the atom groups in certain carbon compounds, we may represent the above four conditions of the elements of the organism or system by four figures (fig. 136) in which the groups that determine the axial relations are situated at the four angles of a tetrahedron. At the center of each tetrahedron we might by analogy assume a carbon atom linked to the four groups oecu- pying the angles of the figure, though this is not necessary for the present purpose. By hypothesis the groups at the angles are supposed to be at first all alike (fig. 136, 1). If one of them should be changed by some reaction, the structure of the molecule would become polarized (fig. 136, 2), and if all the molecules should assume approximately the same orientation, the system which they constitute would show a similar polarity. If two of 98 The question whether relations of symmetry of the organism are to be based upon symmetrical relations of the intimate protoplasmic structure is answered in the affirmative by Driesch (’08 a, p. 144): ‘‘Wir miissen also alle Symmetrie und auch alle Wirkungen, die von fusseren #aktoren ausgehen und sich auf Symmetrie beziehen, auf priformierte, gerichtete Elemente des ‘Protoplasmas’ beziehen und kénnen in jenen Wirkungen nur richtende und umordnende Geschehnisse sehen. 97 To avoid misunderstanding, it should be stated that when we speak of equi- potentiality and isotropy, we do not lose sight of the fact that the system in its entirety is heterogeneous. SYMMETRY IN TRANSPLANTED LIMBS 89 the groups become differently modified, then the structure be- comes bilaterally symmetrical (fig. 186, 3). And, finally, if three become modified, so that all four are different, then the arrange- ment becomes asymmetrical (fig. 136, 4 and 5) as in the ease of optically active substances with an asymmetric carbon atom. In the last phase there are two kindsof individuals, which are exactly alike in every respect, except that they are the mirror images of RIGHT Fig. 136 Diagram to show hypothetical progressive differentiation of the structural units. 1) condition of isotropy; 2) polarization with reference to one axis; 3) bilateral symmetry (two axes differentiated); 4 and 5) condition of com- plete asymmetry (three axes differentiated) giving right and left enantiomorphs. one another—in other words, rights and lefts. This is expressed in aggregate form in the right- and left-handed crystals corre- sponding, respectively, to the dextro- and laevo-rotatory forms of otherwise identical substances. The experiments with limbs show that the bud at the time of transplantation is in either the second or the third phase, probably the former. There must be a differentiation along the antero- posterior axis, because if this is reversed the limb shows it by growing in a direction nearly opposite the normal. The medio- 90 ROSS G. HARRISON lateral axis is probably not differentiated, though in the absence of sufficient experiments in reversal of this axis, it is better to make no definite assumption regarding the intimate structure in relation to it. The dorsoventral axis is at most but slightly differentiated, and if it is at all, then the differentiation is revers- ible.°8 As already pointed out (p. 55), there is some ground for the latter assumption, for it has been observed that after transplantations in the heteropleural dorsoventral position (the harmonic combination in which the dorsoventral but not the anteroposterior axis is reversed) the adjustment of the tissues of the limb bud is apparently not immediate, but involves a time factor, probably not entirely accounted for by the effect of the operation as such. Whatever the character of this dorsoventral differentiation may be, itisnevertheless very slight in comparison with the anteroposterior differentiation, which has become irre- versible by the time the stage in question is reached. If we could experiment over a wide enough range of stages, it should be possible to determine the time limits of the above phases of axial differentiation of the limb rudiment. At present, however, there are no data bearing upon the question, for in the earliest stages in which the transplantation of limbs has been carried out (embryo with wide open medullary folds), as shown by Detwiler (18), the limb bud follows in its development the same rules as here formulated. Lest the foregoing scheme seem too formal, it may be pointed out that the model has been chosen to explain solely the relatively simple characters of polarity and symmetry. Upon this as a basis, further experimentation may yield facts from which the mode of representation of more specific form features may be determined. There is nothing in such a scheme inconsistent with the fact that the cell itself is not a homogeneous system, for the model is supposed to represent only that constituent of the system which determines the adult character in question. °8 This is perhaps odd in view of the facts brought out by Przibram (’10b), showing that dorsoventral differentiation is very marked in the animal organiza- tion, more so, for instance, than the anteroposterior differentiation. SYMMETRY IN TRANSPLANTED LIMBS 91 The point which it is desired to emphasize is that in an organic ‘equipotential system’ there must be some intimate structural basis for adult characters in the units that make up the embryonic rudiment.*? It cannot be in the arrangement of these units; for in that case marked disturbances of development would be pro- duced by such operations as removing half of the rudiment, fusing two buds together, combining two like halves, inverting the dorsoventral axis, or inoculating masses of mesoderm cells from the limb rudiment under the skin of the flank; and yet normal development may follow any of these procedures. These experiments yield, of course, no information concerning the localization in the cell of the representatives of the adult form characters in question. The system here dealt with is a pluri- cellular one, but it is interesting to find that in the most thorough and careful studies of polarity and symmetry in the egg, the basis of these properties is found to be in the cytoplasm of the egg cell. Lillie (06, ’09) shows that the polarity of the Chaetopterus egg must be located in the ground-substance, because any amount of shifting of visible granulations in the egg, such as yolk, oil droplets, pigment, etc., has no effect on the polarity of the result- ing embryo. With this conclusion the work of Morgan and his collaborators (08 b, ’09, 710) on the centrifuged eggs of various animals, more particularly Arbacia and Cumingia, is in substan- tial accord. Conklin (716, ’17), in his study of Crepidula, con- cludes that it is the spongioplasmic framework of the egg-cell that determines its polarity, though he does not consider how this quality is determined in relation to the intimate structure of the spongioplasm. To the extent that Conklin places the seat of polarity in the more viscid rather than in the more fluid constit- uent of the cytoplasm, he takes issue with Lillie, but in the main, there is agreement between these two investigators. Lillie, how- ever, goes a step further when he says (’09, p. 77): ‘‘The existence of polarity and bilaterality in an optically homogeneous medium, and the persistence of both as to orientation under experimental conditions that seriously modify the quantitative relations of the oriented medium in different regions (as, for instance, when 93'Cf. Drieseh, 1. ¢c. Q? ROSS G. HARRISON the yolk granules are packed closely into the small cell of the two-celled stage of Chaetopterus) seem to me to argue for a molecular basis of the fundamental principle of vital organi- zation.”’ Morgan, likewise, takes this view when he says (’09, p. 114), “These considerations incline us to the view that there exist in the molecular constitution of the egg the potential factors of symmetry.”’ The scheme outlined above is in harmony with this concept. On the other hand, Child (18, ’15), as also Della Valle (713) rejects all such hypotheses, basing the phenomena of axial differ- entiation upon the occurrence of gradients, which, according to Child, are primarily of a functional (metabolic) nature. It seems to the present writer that such gradients may well be an expres- sion of the polarity rather than its cause. G. Reduplication and the problem of polarity and heteromorphosis The reduplications which have been observed in the various experiments have already been described sufficiently for the pres- ent discussion (pp. 35, 45, 65, 73). The salient facts are: 1) that the duplicate is the mirror image of the original limb; 2) that more than one secondary member may arise by budding from the same primary bud, in which case both of the former stand in some relation of symmetry to the original; 3) that the secondary appendages themselves may be doubled, forming a more or less symmetrical pair. There are a few exceptional cases, where two members of the same side stand in linear series, but probably these have arisen only where the two rudiments are far enough apart not to influence one another.!°° 100 Several cases are to be considered here. One (H. R. E. 10-) is a case in which the anterior half of the limb bud was removed. Two limbs developed, one clearly from the remaining posterior half and the other probably from the anterior border of the wound (cf. Harrison ’18, p. 441). The operation was done on the left side and both limbs were lefts, the posterior one being somewhat defective. In another case, which had an early history similar to the above, the posterior member was very defective and it was impossible to determine whether it was aright oraleft. A third case (H. R. E. 20) is the one figured on page 79. This is not a case of regeneration, but one in which the anterior member probably developed from the grafted half, while the posterior member may have developed from the stationary ventral half. Both are attached to the same shoulder-girdle, but there are two separate glenoid cavities. SYMMETRY IN TRANSPLANTED LIMBS 93 Bateson, in his ‘‘ Materials for the Study of Variation” (’94), has given an exhaustive review of the literature relating to super- numerary parts, in which the limbs are fully considered. In this treatise he has made a masterly analysis of the available material, particularly with reference to the appendages of arthropods. The phase of the problem which is especially relevant to the pres- ent discussion is that concerning what Bateson calls minor sym- metries, in which the supernumeraries are in some way symmetri- cal with respect to themselves or to the normal appendages with which they are associated. The other class of supernumeraries, in which two identical appendages stand in simple succession to one another are, according to Bateson, practically unknown, and even those that have been described are considered by him to be of somewhat doubtful nature, though many cases of simple hyperdactyly would seem to belong in this category. The symmetrical extra appendages fall into two groups: 1) those in which there is a pair of extra members symmetrical with themselves, arising from the normal appendage with which one of the supernumeraries appears to have a definite relation of symmetry, and, 2) those in which the single supernumerary is symmetrical with respect to the normal. The former condition Bateson considers to be the more usual, and, in fact, he accepts the existence of the latter with a certain skepticism which seems unnecessary.'" It is true that many cases that apparently fall within the latter group may upon closer examination be found to belong in the former, but the converse isalso true, as will be shown below (p. 97). Bateson has devoted special attention to the first group, and, on the basis of about one hundred and twenty cases in insects and a considerable number in the Crustacea, he has formulated the following rules,! showing the relation of the supernumerary appendages to each other and to the original member: I. The long axes of the normal appendage and of the two extra appendages are in one plane: of the two extra appendages one is there- fore nearer to the axis of the normal appendage and the other is remoter from it. 101 Op, cit. p. 5389 and 553. Consider, however, in this connection, the clear case described by Bender (’06). 102 Op. cit. p. 479. 94 ROSS G. HARRISON II. The nearer of the two extra appendages is in structure and posi- tion formed as the image of the normal appendage in a plane mirror placed between the normal appendage and the nearer one, at right angles to the plane of the three axes; and the remoter appendage is the image of the nearer in a plane mirror similarly placed between the two extra appendages. Transverse sections of the three appendages taken at homologous points are thus images of each other in parallel mirrors. In the vertebrates Bateson marshals a large amount of mate- rial, of which about fifty cases are in amphibians.’ At the time Bateson’s book was written, however, little or nothing was known regarding the origin of supernumera y appendages in either the arthropods or the vertebrates. Since then a large amount of experimental evidence has accumulated to show that they may be formed by superregeneration, especially by regeneration from complex or irregular wound surfaces.!°% The evidence all cor- roborates Bateson’s main gene. alization regarding the relation of symmetry of supernumerary limbs, and there are practically no exceptions.!” The importance of double supernumeraries (Bruchdreifachbil- dung, la doppia rigenerazione inversa, see p. 95) is emphasized in the papers by Emmel (’07) and Della Valle (’13), and this con- ception is given prominence in Przibram’s more general discus- sion of the question (’09, p. 234). 103 See Bateson (pp. 554-5) for a discussion of the older literature. 104 In the amphibians the investigations of Barfurth (’94), Giard (’95), Tornier (97, ’00, ’05, 706), Lissitzky (’10), Fritsch (11), Kurz (’12), Della Valle (’13), and others have added much to our knowledge of the subject. In the crustaceans Przibram (’02), Reed (’04), Zeleny (’05), and Emmel (’07) have reported experi- ments which, though not so numerous, are none the less important. The more recent literature is fully discussed in many of these papers, especially in those of Lissitzky, Fritsch, and Della Valle, to which the reader is referred for details. 10 A remarkable exceptional case has recently been described by Dawson (20) in a lobster, in which there is an extra pair of chelipeds attached to the normal. The two extra chelae are mirror images of one another, but the one nearer the primary claw is not mirrored from the latter, but is of the same side. Furthermore, the primary claw is a ‘nipper,’ while the supernumeraries are both of the ‘crusher’ type, so that the case proves to be likewise an exception to Prai- bram’s rule (’11), according to which, in heterochelous forms, the extra appen- dages are of the same type as the primary. The case described by Cole (’10), also in a lobster, is an almost diagrammatic example of Bateson’s rule, if allow- ance is made for the effects of torsion. SYMMETRY IN TRANSPLANTED LIMBS 95 Likewise, the well-known experiments of Tornier (05) upon ‘tadpoles of Pelobates, in which the hind-limb bud was divided in an early stage, some of the cases of Lissitzky (10), and Della Valle’s case of reversed regeneration conform closely to Bate- son’srules. Although the end results of the experiments of Tor- nier and of Della Valle are analogous, there is, however a sharp difference of opinion regarding the exact mode of origin of Tor- nier’s double supernumerary hind legs, Tornier maintaining that they both arise from the dorsal part of the pelvis, which was split off by the operation, while Della Valle holds them to be analogous to his own case. Della Valle has laid particular stress upon the supposed identity of change of asymmetry and reversal of polarity, and has sought to make the various cases of superregeneration which have been reported fit into his scheme of ‘doppia rigenerazione inversa.’ The case on which Della Valle bases his discussion of these ques- tions is that of a newt (Triton) in which the left anterior limb was fractured in the region of the brachium and cicatrization pre- vented by tying a silk thread around the limb at that level. Twenty days later the same limb was amputated a short distance below the point of fracture. There régenerated three perfect limbs, one from the distal end of the stump and two from the region of the fracture. Of the latter, one was from the proximal end of the small portion beyond the ligature and the other was apparently a continuation of the stump proximal to the fracture. The first and last of the three were left limbs, i.e., of the same side as the original, while the one which regenerated in a distoproxi- mal direction was reversed. The end result was a triple append- age in which the three members were placed in accordance with Bateson’s rule. Della Valle seeks to make the eases of Tornier (’05) and Lis- sitzky (10) conform to this scheme, and falls into line with Przibram (’09) who had previously given a schematic represen- tation of the same phenomena, which he termed ‘Bruchdreifach- bilding.’ He also interprets in the same way the reduplications obtained by Braus (’04, ’09) and myself (’07) in the transplanta- tion of embryonic limb buds. He suggests that when the limb THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, NO. 1 96 ROSS G. HARRISON bud is implanted in normal location, triplicate appendages could be accounted for in the following way: one member derived — directly from the grafted bud; one member, of mirror symmetry, by an inverse regeneration from the base of the bud, and’ the third member, of original asymmetry, from the wound surface of the host. This view is, however, not borne out by the present experiments. !° When the whole of the evidence bearing on the question is taken into consideration, one cannot but think that too much weight has been placed by Bateson and his followers on the double supernumerary. The other class of cases, where the single supernumerary is symmetrical with the normal appendage with which it is associated, while neither so numerous nor so spectacular, is nevertheless of wide occurrence. Cases reported by Tornier (’97), Przibram (’02), Reed (’04), Zeleny (’05), and Megusar (07) show that truly double appendages in mirror symmetry with respect to each other may be formed by constric- 106 Se dunque noi considerassimo uno di questi innesti praticati invece che in una regione lontana (come p. es. nell’orbita), nell’immediata vicinanza della regione donde fu tolto l’innesto, noi osserveremmo I|’uno presso dell’altro lo svil- uppo oltre che dell’arto normale, anche dell’arto rigenerato dalla superficie di sezione della regione prossimale del corpo, nonché dell’arto sviluppatosi dalla superficie di sezione della regione periferica, identico all’arto che lo ha prodotto, ma con simmetria speculare. La identita anche di questo fenomeno con la dop- pla rigenerazione inversa dalle due superficie di una ferita risulta in questo modo evidente. Della Valle: op. cit. p. 125. There is opportunity to test this hypothesis by comparing the experiments in which the wound-bed was cleaned with those where it was not. In the former, regeneration from the host is precluded (p. 6), and triplicate limbs could only arise by a second reduplication from the base of the graft; whereas in the latter, regeneration from the host should occur in a large number of cases, if at all, and thus yield a large proportion of triplicate appendages. An examination of the results shows that this is not the case. In the first place, as shown in table 2, the total number of reduplications in the series with cleaned wounds is fifty-three, which is 56 per cent of the total number of positive experiments, while there are but sixteen cases (33.3 per cent) in the group with non-cleaned wounds. The disproportion is much greater when the number of triplicate appendages in each group is compared. Out of a total of eighty-seven cases old enough to be deter- mined, there are twenty-five triplicate limbs (28.7 per cent) in the clean-wound experiments and only three in forty-eight cases (6.25 per cent) in the others. It is quite clear, then, that leaving in the wound-bed cells that are capable of giving rise to a new limb reduces greatly, instead of increasing, the chance of formation of supernumerary limbs, so that Della Valle’s suggestion is untenable. SYMMETRY IN TRANSPLANTED LIMBS 97 tion of a simple regenerating bud. This harmonizes with Driesch’s (06) observations on double Echinus embryos. In the present work, the reduplicated extremities are nearly all found to be in minor symmetry, and many of those in which three members are present, if seen only in the fully developed condition, would appear to be cases of paired supernumeraries, conforming, though with some aberration, to Bateson’s rules, The individual histories show, however, that they are mostly simple duplicities in which the supernumerary mirrors the orig- inal, and this seems to be the case in Braus’s experiments, too. Two reduplicating limbs often do develop, but usually each grows as a bud from the original instead of the two arising as a pair in themselves. Each of them mirrors the original limb, so that the two supernumeraries are both of the same side. In other cases the supernumeraries are themselves double, in which event there is strict conformity to Bateson’s rule, but the former constitute a large majority, and conformity there is only superficial, for the original limb is the middle member and not one of the extremes. In view of these facts, there is probably no very fundamental difference between the two classes of reduplications, i.e., between the double supernumeraries symmetrical with each other and the single supernumerary symmetrical with the original; had Bate- son had the developmental stages at his disposal, he himself might not have drawn so sharp a distinction. In accordance with the above, Bateson’s rules might be stated in more general form, so as to include both simple duplicities and symmetrical pairs, as follows: 1. The long axes of duplex or multiplex appendages lie in one plane. 2. Two adjacent members form in structure and position the image of each other, as reflected from a plane mirror bisecting the angle between the respective axes and perpendicular to the common plane of the two axes (figs. 3 and 4). The present experiments show (tables 2, 3, 5, and 8) that, excepting heterotopic grafts, it is in the disharmonic combina- tions that reduplications are most frequent. What, now, is the nature of the disturbance that causes the doubling of transplanted 98 ROSS G. HARRISON limb buds and of regenerating limbs, which, when it occurs, is_ always combined with reversal of one member? ‘The first visible sign of reduplication both in the embryonic limbs and in the re- generating blastema is the presence of two growth centers for the limb in place of one; each becomes an apex of growth, with a resulting bifurcation of the appendage as a whole. The question arises whether the doubling of the growth center is antecedent to or resultant from the reversal of the asymmetry. From the fact that mere mechanical division of a simple regenerating cen- ter??? may bring about doubling, it would seem to be more prob- able, if not certain, that. the existence of two growth centers within spheres of mutual influence is the factor that produces the reversal in one—the one that is less advantageously placed, or in which differentiation is less advanced. The problem before us thus resolves itself into two phases: that of division or repetition of parts and that of symmetry. This was clearly seen by Bateson, who has emphasized the funda- mental nature of the power to divide.!°? No attempt will be made here to analyze this phase of the question. The symmetric relations of the repeated parts are, however, so definite and of such general recurrence that they, too, are beyond question of a fundamental nature. The phenomenon of reversal of asymmetry has been treated by many investigators as one with that of axial heteromorphosis, and yet this is not strictly correct, for the reversal of asymmetry may be brought about by the interchange of the poles of any one of the three axes to which the object is referred, and not neces- sarily the one along which regeneration and differentiation is taking place. ‘This is true not only when regeneration occurs in a proximodistal direction, as in the cases of Tornier, Zeleny, and others, cited above, but also when it takes place distoproximally, as shown in the two experiments reported by Kurz (’12).!° 107 Cf. Tornier (’97), Przibram (’02), Reed (’04), Zeleny (05), MeguSar (’07). 108 “This power to divide is a fundamental attribute of life and of that power cell division is a special example.’’ (Problems of Genetics, p. 38.) 109 In somewhat similar experiments by Morgan (’08 a) only the bone, not the soft parts, was reversed. Nothing is said regarding the exact character of the limbs regenerated. SYMMETRY IN TRANSPLANTED LIMBS . 99 The fundamental phenomenon, therefore, is not that a particular axis is reversed, but that reversal occurs at all, and how it is brought about. Organic polarity, in general, has been based either on the sup- posed polarization of the organic units themselves or upon a supposed gradient of a functional (Child) or material (Morgan, 05) nature, running from one end of the organism to the other. There is evidence for the occurrence of both factors, and what seems most likely is that both are at play. Under certain cir- cumstances they act in the same direction; under different condi- tions one may antagonize and retard, or even overcome, the other, as seems likely in heteromorphic regeneration where polarity is reversed (earthworm, planarians, amphibian tail, ete.). Prai- bram (713), who advocates a theory combining the two factors, which he calls ‘Richtungspolaritit’ and ‘Schichtungspolaritit,’ respectively, nevertheless regards the reversal of polarity as due to actual rotation of the cells. He (’06, 710 a) cites unpublished work by Hadzi in support of this view, and adopts it in his dia- grams (’09) illustrating the five fundamental varieties of opera- tion leading to regeneration (Biotechnik). The figures are not convincing, however, for just as much rota- tion of the cells is shown at the end where polarity is not reversed as at the other end where it is reversed (Przibram, ’09, pl. XV, 1h-3h), and in fact, as expressed in these diagrams, what turning is shown is nothing more than a wound-healing process. Until it is demonstrated that rotation of the cells as a whole takes place solely in heteromorphic regeneration, it cannot be used to explain reversal of polarity. So long as the elementary units of the limb bud have one plane of symmetry left, and the final asymmetry of the limb remains to be determined by its relation to certain axes of the embryo,'!° it 110 Tn the case of asymmetric organisms, the elementary units, if representing the form of the organism at all, must be postulated as asymmetric themselves. In the case of paired organs, each asymmetric in itself, but symmetrical with respect to its opposite, polarization on the transverse axis may be assumed as due to the position of the parts with respect to the other two axes (ef. Przibram, 13, p. 38) and not as necessarily due to actual differentiation of the elements in the transverse direction. 100 ROSS G. HARRISON will of course be possible to account for its reversal by rotation of the elements about the proper axis. As an alternative to the rotation hypothesis, we might, however, consider reversal as due to an interchange in position of two of the determining groups in the elementary units (p. 89, fig. 186). In case of differentiation on all three of the axes, 1.e., if the units themselves are asymmet- ric, then reversal could take place only in the latter way, unless it occurs altogether independently of the intimate structure. There is an analogy for reversal of this kind in the change of the asymmetry of organic molecules of known composition, as, for instance, in the Walden inversion by means of successive sub- stitutions, or in the conversion of dextrotartaric acid into racemic acid, by which transformation half of the dextrorotating groups are changed into the laevo form. Of course, these examples are mere analogies. Such questions have been touched upon by many of those who have studied twins and double monsters, but, unfortunately, the evidence both as to the cause and as to the occurrence of reversal of asymmetry is conflicting. In the case of human dupli- cate twins, it is certain that there is no situs inversus viscerum, except very rarely, and apparently even in double monsters the degree of fusion of the two individuals must be considerable for the asymmetry of the internal organs—heart and alimentary canal—to be reversed. On the other hand, it has been shown by Wilder (’04) that in duplicate twins the friction-skin patterns of the two mates may show mirror imaging, particularly those on the index fingers. ele ae ieydin ris fy mati: Ain pe? Tint gee ee hike Tad castaviiteil ahuit VU if. ‘tein hacia aroha Daisy Oimig ai .ad ime ot = (rs enna ri eh BM, rive vi Plo i } ; : J svgealyreyy re Laie = _ . : 3 a ows oa Fy) rer vee 3 a cy I iat mise Pere. 3 wie hee ee Al temtey he, 138 ; JOSEPH HALL BODINE special interest are: changes occurring with age, differences due to species and sex, the effects of starvation and hibernation, as well as problems dealing with the various phases of metabolism. Results of such nature obtained for higher forms have contri- buted much toward the advancement of various theories of growth, senescence, etc. (Child, 1; Mathews, 2; Minot, 3), as well as to our knowledge of eranee: in organisms one are closely related to age, sex, etc. (Hatai, 4). The present investigation deals with such problems as the percentage of water during growth, starvation, and ‘hibernation’ in different species of grasshoppers, and also the rate of meta- bolism as indicated by determinations of the amounts of carbon dioxide given off by the animals. MATERIAL AND METHODS A. Material Grasshoppers were used in all the following experiments. These animals have been found to be excellent material because of the ease with which they can be obtained, kept under usual laboratory conditions, and handled in experiments. They are sufficiently large to be used individually, and this is of great importance because most of the physiological work heretofore done on insects has been concerned with masses 3 rather than with individual animals. The following species were used: Melanoplus femur rubrum, Melanoplus differentialis, Dichromorpha viridis, and Chorto- phaga viridifasciata, and they will be Beate in the order given. Melanoplus femur rubrum DeGeer, the red-legged locust, is perhaps the most common grasshopper found throughout the entire United States. Its general life-history is practically typical; eggs are laid in the late summer and early fall and remain over winter; nymphs hatch in early summer, and by the last of July and early part of August, in the vicinity of Philadelphia, adults are found. It occurs in rather large numbers throughout the entire summer. The average length of the body of adult WATER CONTENT AND RATE OF METABOLISM 139 males is 23.5 mm., and of females, 24.5 mm.; average weights are: adult males, 0.20 to 0.40 gram; females, 0.25 to 0.65 gram. Nymphs range in weight up to a maximum of 0.35 gram.! Melanoplus differentialis Uhler, the largest grasshopper found in this vicinity, closely parallels Melanoplus f. rubrum in life- history. In length adult males measure 39 mm., and females, 41 mm. Adult males weigh 0.7 to 1.3 grams, and females, 1.3 to 2.8 grams. | Dichromorpha viridis Scudder has a general life-cycle similar to the above-described species. However, it is not as active an animal and occurs in open wet places. Differences in size between adult males and females are marked. Adult males measure 18.75 mm., and females,’ 27.0 mm. In weight adult males range from 0.15 to 0.20 gram, and females, from 0.15 to 0.55 gram. Chortophaga viridifasciata DeGeer is quite different in life- cycle from the above-mentioned species. Eggs are laid in late spring and early summer; these hatch in later summer and early fall; the nymphs live throughout the winter, and in spring grow rapidly, and become adults by early summer. ‘Two-thirds of their active life, in contrast with other species, is spent as nymphs and approximately one-third as adults. Two well-marked varieties oc¢éur, a green form (virginaria Fab.) and a brown form (infuseata Harris). Most females are green and males brown, but some are found of each sex in either color, and as a matter of fact, when green animals are put at a constant temperature of 38°C. they turn brown in a very short time. Adult. males measure 25 mm. and weigh 0.10 to 0.20 gram; females measure 30 mm. and weigh 0.15 to 0.45 gram. For further descriptions of the above species reference is made to standard text-books on entomology and to the works of Morse (6) and Lugger (4). 1 Average dimensions of animals used are taken from Lugger (5), while body weights have been determined by the author. 140 JOSEPH HALL BODINE B. Methods The following general description of methods applies to all experiments and any further details will be given in describing individual cases. All animals were caught in the vicinity of Philadelphia during the summer and fall of 1919-1920, brought into the laboratory, where they were kept in large screened insect cages, designated as stock cages. They remained in these cages for at least a day under the usual laboratory conditions, and were fed during this time on grass. Inasmuch as grasshoppers normally consume a great deal, those kept in the laboratory for a day ate large amounts of the grass, and upon examination the alimentary canal was found to be filled, thus insuring uniformity as to initial amounts of food. General laboratory conditions remained constant throughout the experiments, and any slight temperature changes, usually occurring at this particular season of the year, are noted in data following. Animals were separately weighed in a small covered beaker on a rather delicate balance, determinations being made to four places of decimals. After weighing they were marked by gluing a small numbered tag on the pronotum, which mark could easily be removed and again attached, thus avoiding confusion in keeping accurate records of individuals. After initial weighing they were kept, in groups of five to ten, in small wire insect cages. In determining water content, individuals were killed with chloroform, opened by a longitudinal slit through the abdomen, and then put in an oven at 95° to 97°C. and left there for a period of one week. This was found to be more than ample time for complete desiccation. Carbon-dioxide determinations were made by the barium hydrate titration method of Lund (7), using single animals, and each determination extending over a period of thirty minutes to one hour. In suspending individuals in the respiration bottle they were carefully tied around the prothorax by means of a fine silk thread. This was found to cause them little incon- WATER CONTENT AND RATE OF METABOLISM 141 venience if properly adjusted, and if it was kept sufficiently short, they did not move about to any appreciable extent but rested upon the sides of the bottle. Experiments in which the animal was confined in a small wire cage, just large enough to accom- modate it and not allowing body movements, gave results quite similar to those in which the animal was suspended by the thread. In any such experiments, however, it is quite impossible to entirely eliminate movements by the individual, but by careful handling and manipulation they can be greatly lessened, and comparable, if not accurate, results obtained. In the following experiments emphasis is laid upon relative rather than upon absolute amounts of carbon dioxide given off. Acknowledgment is made to Prof. C. E. McClung for the original suggestion of work on the physiology of the grasshopper, and to Prof. M. H. Jacobs I am deeply indebted for constant advice and criticism during the course of the work. To the members of the Zoological Department of the University of Pennsylvania I am also greatly indebted for generous help and criticisms. OBSERVATIONS A. Water content The physiological importance of water to organisms is too well known to require special discussion. In recent years, many determinations of water content have been made on differ- ent organisms, and on the same organisms under different con- ditions, and the results have thrown much light on such questions of biological interest as the cause of certain tropic responses (Breitenbecher, 8), the nature of the process of senescence (Hatai, 4), the question of the influence of different foods (Bab- cock, 9), ete. Determinations of this sort have been made mostly on higher forms, and no such observations appear to be available for grasshoppers, which I have, therefore, studied rather extensively. a. Relation between body weight and water content. Table 1 gives the body weights and water contents of 981 individuals, 142 JOSEPH HALL BODINE comprising nymphs and adults of Melanoplus f. rubrum and adults of Dichromorpha viridis and Melanoplus differentialis. It is evident from an examination of this table that considerable variation in the body weights for the different species exists. _ These can be explained by a consideration of the differences due to species, age, and random variations. It will be noted, for instance, that of the species studied, Melanoplus differentialis is by far the heaviest, reaching a maximum of 2.9 grams. Range in weight for Melanoplus f. rubrum is of interest, since weights for both nymphs and adults are given, and these show that as the animals become adults there is a progressive increase in body weight up to a maximum for the species. Males never reach the same maximum weights as females, and this is not due primarily to development of masses of eggs by the older females, since in nymphs this relation also holds. Consequently any comparison between males and females of the same weights will not necessarily be between those of the same age. Different conditions, such as food supply, development of reproductive elements, etc., modify the maximum weights of animals, causing some variation among individuals of the same age as shown for Melanoplus differentialis, where the animals are of approximately the same age and show rather large variations in body weights. Closely related to these differences in body weights of the animals are the changes occurring in the percentages of water. With increasing body weight and age, a progressive diminution in the relative water content takes place, as shown especially by Melanoplus f. rubrum, where nymphs have an average maxi- mum of 77.6 per cent and adults an average minimum of 72 per cent. That this diminution in water content is related to age, and not to body weight, is shown by a comparison of these results with those for Melanoplus differentialis, where the ani- mals are of the same age, but differ in body weights, and have approximately the same percentage. of water. “ot a Siete wie a > hig) 1 es Ge sey :, é : “ee tae a ie ( ti) re arent i on , pit as, oe doth is a ad oti wi Ging = Sheed byte ae ts La afditiaahe Fs | - b * es. AS Js : rat Ot Te he Rode be ban a ta) i 7 ' ie: Sts: ee ration Ee: ais pays ig 55, Ny ‘ wicca eee y a 4 sf \ in Pinal ie Pa Wig ieee NG es sea seat Te i pes reat az, viata FA Mght iD: het pi 0) ean, RAS ria a chit Rae i" 3 ; spent ee le i wee Pr APs ie { Mee eeriy * airy Resumen por el autor, Charles W. Metz, Estacion de Evolucién Experimental, Carnegie Institution of Washington. Espermatogénesis de la mosca Asilus sericeus Say. Asilus sericeus Say es una especie de Diptero sumamente favorable para los estudios sobre la espermatogénesis a causa del considerable numero de células y de la exacta serie de estados de crecimiento en el testiculo, asi como la ausencia relativa de estados confusos durante el periodo de crecimiento. Existen cinco pares de cromosomas. La asociacién de los cromosomas en parejas se presenta en la espermatogonia y se retiene durante la ultima divisién espermatogonial. En la telofase de esta division, la disposicién de los cromosomas en parejas es tan intima que realmente equivale a la sinapsis, y la unién que tiene lugar en este momento persiste durante el periodo de crecimiento que la sigue. Los autores no han podido encontrar estados leptoténicos o zigoténicos (sindpticos) propiamente dichos. Los cinco ele- mentos dobles (bivalentes) que aparecen en la telofase permane- cen relativamente condensados y son facilmente discernibles durante todo el periodo de crecimiento hasta la primera divisién de los espermatocitos. La sinapsis precoz y la omisién del estado leptoténico ordinario pueden interpretarse como una manifestacién de la fuerza que origina la asociacién por parejas caracteristica de los cromosomas de la espermatogonia, oogonia y células somaticas de los dipteros. Translation by José F. Nonidez, Cornell Medical College, New Yosk AUTHORS’ ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, NOVEMBER 1 SPERMATOGENESIS IN THE FLY, ASILUS SERICEUS SAY! CHARLES W. METZ anv JOSE F. NONIDEZ Station for Experimental Evolution, Carnegie Institution of Washington TWO PLATES (TWENTY-TWO FIGURES) CONTENTS rte OC UT Ora! St Ss |S TSUN OE OU ae Se ceOmCene Mans od LEB OY, Soe he 165 PR SCHIITORUG Pr, 308 ans 212 2s Cepke SAe aa, Ca yd ee ees oe (ERD ble a. al tee 167 POMORLA DLN CAL TEATUTES Eis ao 0 5% sterajureral EO, Seana To aks cclei fs ol dend Reawavers 167 SS OCEELEINEWI{0 00 mr eee gor Re Ck Te ea ae 168 Bimaltspermatozonial telophasess 2.272 0.) tere tetas aioe ew kee ce 169 ME lye Ow bh period :...\..01. 4 Yhd ess Wa ne cae REA Geis ok cena edu eee 170 atemerow tl perrod,. («2d iesuncse as aoe tothoeiee BART ee Ee Peed are oS RTE histoate 174 Wher speEMmabocy ber CiVISIONS Ani.) ee crsee, ale er Ieee cheese eae. Gas 175 ETG: THEOL SOT geen ali a Rea PE He eo ae dn et ice 176 IDWGUSES 0; Ae Sea ee eam ee SRR a ee ee, ch hte eS See 5 eee 177 SLUIACUN Tg cee ao ee ee eee Fn =e ee 179 ther aiaire rented «Avis toe cocks 1 Ban Die Wen ne yet ONE Re eR, AbATy HME ce ch 180 INTRODUCTION The rapid development of genetical studies on Drosophila in the last few years has drawn considerable interest to the subject of gametogenesis in the Diptera. The phenomena of mutation, crossing over, nondisjunction, ete., occur partly or wholly during gametogenesis, making it desirable that our knowledge of the latter be extended as far as possible. Another feature that has added. interest to the subject is the characteristically ‘paired association’ of the somatic chromosomes of flies as distinguished from those of other animals.2. It seemed possible that this pre- existent association of chromosomes in the soma and early germ cells might affect the processes of maturation in such a manner as to throw additional light on their significance. 1 We are indebted to Mr. C. W. Johnson for identifying the species used. * For discussion of this phenomenon, see Metz, ’16. 165 166 CHARLES W. METZ AND JOSE F. NONIDEZ These considerations led the senior author some time ago to begin gathering material for a study of gametogenesis, in con- nection with other studies on Diptera chromosomes, and the first results of this study are presented here. From the genetical standpoint the present paper represents only a short step in the desired direction, but it may serve as a foundation upon which to base further work—particularly studies on odgenesis, which are under way at the present time. Two reviews of the literature on Diptera chromosomes have been given recently (Metz, 716, pp. 213, 214; Whiting, 717). From these it will be seen that most of the work on the subject has dealt primarily with the sex chromosomes or other special features, and that our knowledge of gametogenesis, epee the growth stages, is meager. In her studies on the sex chromosomes, Stevens (’07, ’08, 710, 11) records several observations on other aspects of spermato- genesis, but unfortunately these cannot be combined to make a connected account. The observations of Taylor (’14) and of Lomen (14) on Culex, in addition to being meager as regards details of spermatogenesis, are, we believe (see also Whiting, °17), faulty on account of the poorly fixed material used. Whiting (17), in a more recent paper on Culex, has given a comprehensive account of the maturation divisions, beginning with the first spermato- cyte prophase. His observations on the earlier stages, however, particularly the earlier part of the growth period, are limited, probably owing to the fact that Culex does not afford favorable material for this purpose. As regards odgenesis in the Diptera, practically nothing has been published, so far as we are aware. Unfortunately, the Diptera have long, and justly, been looked upon as unfavorable objects for cytological study—a fact that has undoubtedly been responsible for keeping our knowledge of gametogenesis in this group far behind that of such insects as the Orthoptera, Hemiptera, and Coleoptera. We have found, however, that some groups of Diptera are much more amenable to study than others, and by making selections from these and by careful attention to technique we have been able to obtain SPERMATOGENESIS IN ASILUS SERICEUS 167 favorable material. It is hoped that the results obtained from this will eventually permit of a satisfactory analysis of the pro- cesses in the difficult Drosophila material. Our survey of the Diptera has not yet revealed any single species that is favorable for a study of all stages of spermato- genesis, but the species considered here combines more favorable features than any other. In this form the seriation of stages through the development of the first spermatocyte up to the maturation divisions is clear-cut and practically complete. Details of certain stages are not depicted with as great clearness as they are in some other species (to be considered subsequently), but in most respects the material is unusually favorable. TECHNIQUE Flemming’s strong solution has been found most satisfactory for fixation and has been used almost exclusively. Heidenhain’s iron haematoxylin and safranin have been used for chromatic stains, with or without counter-stains. A more detailed con- sideration of technique has been given in an earlier paper (Metz, 16, p. 219). All of the accompanying figures are from material fixed in Flemming and stained in iron haematoxylin. TOPOGRAPHICAL FEATURES The testes in Asilus, like those in most other asilids, are a pair of long coiled tubes, each containing thousands of cells. The distal end of the testis contains a clearly marked spermato- gonial region with a central core of giant nutritive cells. Then comes a narrow transition zone in which spermatogonial ana- phases and telophases are intermingled with the earliest stages of the first spermatocytes. The nuclei here are very small. Following this is a broad zone, containing cells, nearly all of which are in one stage. From this point the development involves a gradual transformation, which may be followed with comparative ease through the long growth period extending far down the testis. In favorable material all of these stages appear in One preparation, or even in one section, and since there is such 168 CHARLES W. METZ AND JOSE F. NONIDEZ a large number of cells available for study, even the intermediate transition stages, often so difficult to obtain, are nearly all in serial order. The only exceptions to this rule are found in the stages immediately following the last spermatogonial anaphases. Here development proceeds with great rapidity, and the telo- phase and associated stages are more or less intermingled. But in spite of this it is possible to obtain a clear conception of what takes place, for the immediately succeeding stage (6) is perfectly clear, and with its aid the other figures may be pieced together. The details of these processes will be given below. SPERMATOGONIA The spermatogonia are abundantly represented in our material and are of ample size for study. Those of the last generation or two are noticeably smaller than the earlier ones and have the chromosomes more closely aggregated in metaphase, but other- wise are not appreciably different from the rest. In a previous paper (Metz, ’16) the general peculiarities of chromosome behay- ior in spermatogonia and other diploid cells of flies have been described in detail. For this reason the spermatogonial stages will be passed over briefly here. It is important, however, to keep in mind the fact that the close association of homologous chromosomes, especially in the resting stages and prophases, makes the pairs of chromosomes simulate single chromosomes of other animals. In spermatogonial metaphases of A. sericeus there are ten chromosomes, arranged in five pairs (figs. 3 and 4), the smallest of which is probably the sex chromosome pair, although there is no evident dimorphism to distinguish it. Occasionally the arrangement of one or two pairs is disturbed, but normally they all show the paired association just as in other Diptera (Metz, 716). In anaphase the chromosomes pass to the poles in this paired condition (fig. 5). Their behavior during the telophase will be discussed below. During most of the resting stage the chromatin is diffuse and stains so faintly that its behavior cannot be observed satis- factorily. In prophase it becomes aggregated, and each aggre- SPERMATOGENESIS IN ASILUS SERICEUS 169 gate gives rise to a long double thread by a process of attenu- ation or uncoiling (fig. 1). This thread shortens up immediately into a pair of prophase chromosomes (fig. 2).8 FINAL SPERMATOGONIAL TELOPHASES Since the telophase of the last spermatogonial division is a stage of particular interest, it may be considered separately. In the last spermatogonial anaphase, as in preceding anaphases, the chromosomes go to the poles associated in pairs. In late anaphase the pairing becomes more intimate, due partly to the crowding of the chromosomes at the poles. Then in telophase the crowding is relaxed, the cluster loosens up, and the individual chromosomes may be observed. They are now intimately associated in pairs—so intimately, indeed, that the duality is often obscured. In other words, as the cluster loosens, the chromosomes separate out as bivalents instead of single elements. In figures 6 and 7 different degrees of association are represented. Some of the chromosomes show the dual structure clearly, while others show it very little or not at all. These nuclei are entire, or nearly so, and all of the chromatin is represented. Figure 8 is from a slightly later stage in which the paired association is so intimate that all trace of duality is gone, and only five chromosomes can be detected. The staining capacity of the chromatin is greatly decreased at this time and the chromosomes appear less bulky than before. Needless to say, such figures are difficult to analyze, but a careful study has convinced us that the process is as described—that homologous members join in early telophase and effect an inti- mate union side by side. This conclusion is based not only on the duality of the telophase chromosomes, but on the fact that they are haploid in number (5) instead of diploid (10). The cells are small, affording plenty of examples of uncut nuclei, and in no case have we been able to find one in which the chro- matic bodies approached the diploid number. Indeed, we found no clear case in which more than five were present. ® These features, together with other details omitted in the present paper, will be considered more fully in a subsequent publication. 170 CHARLES W. METZ AND JOSE F. NONIDEZ Probably the density of the stain or degree of extraction has a good deal to do with the appearance or non-appearance of the duality in these telophase nuclei, but there can be no doubt that the union is very intimate. In this relation the chromosomes pass (fig. 9) into the succeeding stage in which they lose their staining capacity to a much greater extent, as they enter the growth period. A careful scrutiny of the late telophase nuclei reveals very little indication of a spinning-out process or a net- work formation, except that due to the linin. The chromosomes appear simply to fade out through loss of color, while retaining, approximately, their form and position (fig. 9). It is probable that the above account should not be restricted to the final spermatogonia, but should apply to’all of the sper- matogonial telophases. The evidence points consistently in that direction (Metz, 716), but we have not been able to make sure of the point in the species under consideration. THE ,.EARLY GROWTH PERIOD; STAGES A AND B Following the final spermatogonial telophase there is a very brief period during which relatively little chromatin is visible in the nucleus, as indicated by figure 10. This stage, which may be called stage a, is also characterized by the appearance of a small nucleolus, as shown by the figures. The nuclei of this period, together with those of the telophase just preceding, are the smallest to be found in the testis and cannot be confused with those of any other stage. Apparently our stage a corresponds to Montgomery’s stage a in the Orthoptera and Wilson’s stage a in the Hemiptera (see below). So far as we can determine, it is structurally similar to the early resting stage of the spermatogonia. The stage is so brief that it is only represented by a few scattered groups of cells at the border-between the final spermatogonia and the clearly marked region in which the next stage (stage 6) is represented. Adjacent to the nuclei of stage a (fig. 10) are others only slightly larger in which the chromatin becomes progressively more deeply stained and condensed, revealing the outline of the chromosomes. SPERMATOGENESIS IN ASILUS SERICEUS CE These are double and haploid in number, corresponding to the telophase pairs. The size of the cells and nuclei indicate that the actual growth period has barely begun when these bodies become visible (fig. 11), and there seems to be little doubt that the preceding transition from the telophase stage has not only been very brief, but has involved little change in the chromo- somes other than that involved in the loss of staining capacity. The intimate association in pairs appears to have remained unaltered. The chromatin now becomes further condensed, revealing the size and shape of the bivalents in a more clear-cut manner (figs. 12 and 13). Attached to one of these (apparently the smallest pair) is the nucleolus, the history of which will be considered later. This stage may be designated stage 6. Structurally stage b resembles the late resting stage or early prophase of the spermatogonia in which the chromatin becomes condensed into five bivalent aggregates that give rise to the prophase chromo- somes.* Since stage b forms a definite point of orientation between the brief early stages and the more extended later ones, it may be well to consider events up to this time before describing the subsequent processes. It is apparent that the synaptic condition has been fulfilled at the very beginning of the growth period by the intimate association in telophase of chromosomes that were, for the most part,® already arranged in pairs. Technically, this association should be called synapsis, for, as will be seen, the union effected in telophase persists throughout the growth period and is responsible for the formation of the bivalent chromosomes of the first maturation division. Compared with the corresponding stages in other animals, this behavior seems to be unique, and it seems legitimate to infer that it is associated in some causal manner with the other 4 See footnote 3, page 169. 5 It is not justifiable to assume that the paired association in anaphase is absolute and invariable, for occasionally the two members of a pair may be sep- arated in metaphase (e.g., fig. 4), and consequently in anaphase. It must be assumed, however, that in these exceptional cases the paired arrangement is restored in telophase or soon thereafter. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, NO. 1 ily CHARLES W. METZ AND JOSE F. NONIDEZ peculiarity of the dipteran chromosomes—their characteristically paired association in somatic cells. It is of interest to consider the events up to this point in relation to those found in other insects, such, for instance, as the Hem- iptera, Odonata, and Orthoptera. A marked similarity is at once noticeable in many features, but always with the difference that the chromatic elements of Asilus are double instead of single. Thus in Oncopeltus Wilson (’12) describes the stages immedi- ately following the final spermatogonial divisions as involving a diffusion of the chromatin in telophase followed by a stage in which definite prochromosome-like aggregates arise (compare Wilson’s figures 48 to 51 with our figures 9 to 13.)° These aggre- gates or masses would correspond to those of stage 6 in Asilus, but instead of being haploid in number and bivalent in compo- sition, they are, in Oncopeltus, diploid in number and apparently univalent in composition. In Lygaeus among the Hemiptera (Wilson, 712), Anax among the Odonata (Wilson, 712), Phrynotettix, Dissosteira, and Chortophaga among the Orthoptera (Davis, ’08; McClung, ’02; Wilson, 712), and probably in numerous other forms, phenomena not essentially different from those in Oncopeltus are found, so that the comparison of Asilus with Oncopeltus may be extended to include several species representing a widespread type of spermatogenesis as regards the earliest stages of the growth period. Apparently the Coleoptera may also be put in this class, although there are so many conflicting accounts of coleopteran spermatogenesis that many cases are open to question. The essential features, however, namely, the resting stage followed by the appearance of more or less condensed masses or aggregates in diploid number, seem to be well established in certain instances (Stevens, 05, ’06, 08 a, ’09; Nonidez, 714, 715; Goldsmith, 719, figs. 17, 18, 19). 6 This resemblance is even more strikingly shown by another species of Asilus (A. notatus) in which the prochromosome-like bivalents are more condensed and shorter than in A. sericeus (discussion, page 178). SPERMATOGENESIS IN ASILUS SERICEUS 173 One author (Arnold, ’08) has described in Hydrophilus piceus (Coleoptera) a precocious reduction of the chromosomes at the beginning of the growth period not unlike that found in Asilus. But the brevity of his description together with the fact that no other observer (Stevens, Vom Rath, 92, Goldsmith, ete.) has noted such a phenomenon in this or other Coleoptera makes it seem probable that Arnold is mistaken in his interpretation. It appears, then, that although a superficial similarity exists between the early growth stages of Asilus and those of various other animals, the divergence between the double (bivalent) chromatic bodies on the one hand and the single ones on the other separates the representative of the Diptera from all the other forms.” If we turn to the plants, however, we find a different situation. Here, although the evidence is not as clear as might be desired, some species appear to exhibit a paired association of “‘prochromo- somes,’ in the early growth period immediately after the last diploid telophase, somewhat like that found in Asilus. Overton (05, 09), for instance, records such an association in Thalictrum, Calyeanthus, Campanula, and Helleborus. In these the last diploid division is followed by a resting-stage network in which definite chromatic bodies (prochromosomes) are scattered about. These are diploid in number, but often, or usually, lie in paired association. Their shape and degree of condensation differ in different cases, but their paired association seems to be fairly constant. In these plants the association may persist from the last ‘premiotic’ anaphase through the growth period and up to the metaphase of the reduction division, although Overton does not commit himself as to the behavior in the telophase and earliest stage of the growth period, as indicated by the following: 7 The earlier literature of spermatogenesis contains numerous references to possible or supposed precocious pairing in the last spermatogonial telophases. For instance, Montgomery, ’00, page 297, on Peripatus, notes a few such ‘excep- tional’ cases; Blackman, ’03, 710, page 141, on Scolopendra, describes a pre- cocious telosynapsis; Stevens, ’03, on Sagitta, suspects an early pairing, and Downing, ’05, on Hydra, makes a similar suggestion. Other and more recent examples could be cited also, but in no case have we been able to find clear-cut evidence of such an association as is exhibited by Asilus. 174 CHARLES W. METZ AND JOSE F. NONIDEZ I have attempted to trace the processes of reconstruction of the nucleus of the pollen mother-cells from the last pre-meiotic division, and to compare the structure of these nuclei with that of ordinary somatic ones, but have experienced considerable difficulty in identify- ing with certainty the last pre-meiotic divisions. After the formation of the nuclear membrane and during the period of nuclear enlargement, the chromatic material becomes rather regularly distributed in the nuclear cavity, the greater portion of the staimable substances lying in the prochromosomes, each suggesting by its form and size that it is derived from a chromosome of the preceding telophases. I am not prepared to discuss the problem as to how the chromosomes of the telophases are modified in passing over into the resting nucleus. (Overton, ’09, pp., 21, 22.) In Oncopeltus and the other insects mentioned above, the prochromosome-like bodies of stage b, whether massive (Wilson’s stage b in Hemiptera and Anax) or more thread-like (Davis’s stage b in Orthoptera), give rise, by a process of unraveling, to long, delicate leptotene threads that then undergo synapsis to form the pachytene or diplotene threads. Since, in Asilus, the chromosomes are already double (i.e., bivalent) it is of especial interest to examine their subsequent behavior. LATER GROWTH PERIOD The transition from stage b to later stages involves merely a gradual lengthening out of the five diplotene threads (figs. 13 to 16) and their polarization with reference to the nucleolus. One member (apparently the smallest) is already attached to the nucleolus. The others, or at least two or three of them, soon become attached and extend out like fingers (figs. 16 to 20). Apparently each thread becomes attached at one end only. No eases have been found in which a complete loop was formed. Fortunately, the threads lie close to the nuclear wall and remain well separated from one another throughout almost the entire growth period, so they may be examined readily. They show no indication of dissociation into single (leptotene) threads at any stage, although their duality is evident throughout. As may be noted from the figures, the nucleus decreases somewhat in size instead of enlarging as polarization progresses. SPERMATOGENESIS IN ASILUS SERICEUS 175 The polarized stage persists almost up to the first spermatocyte prophase, and is modified, toward the end, by a definite contrac- tion period (fig. 17) in which the threads draw away from the nuclear wall and lie close together. Apparently no significance attaches to this contraction for the threads undergo no visible changes and soon spread out again into their previous positions near the periphery (figs. 20 and 21) and condense into the five prophase chromosomes, ready to go on the spindle. Although these processes cover about four-fifths or more of the growth period and are represented by many thousands of cells, they are so simple and involve such slight changes in the chromatin, that in essentials the condition found in stage b (fig. 13) may be said to typify all the succeeding stages up to the prophase, and the whole series may be represented by a few figures. The diplotene threads that appeared at the beginning of stage b have persisted unchanged so far as their diplotene structure is concerned. The contraction stage, occurring in the late growth period, if it has any counterpart, outside of the Diptera, would represent the so-called second contraction, taking place long after synapsis. These events seem to resemble those in Thalictrum (Overton, 09) to the extent that the chromatin remains in the form of relatively condensed, bivalent threads. Compared with animals, however (other than Diptera), there is no such resemblance, for, as just mentioned, the leptotene and synaptic stages usually following stage b are not found in Asilus. THE SPERMATOCYTE DIVISIONS Since this paper is concerned primarily with the growth stages, the maturation divisions will be passed over briefly. So far ~ as known, they present no unusual features. Metaphases of both spermatocyte divisions are clear, and each shows five chromosomes. Only the first is represented here (fig. 22). It is the reduction division, apparently, for no tetrad structure is evident. 176 CHARLES W. METZ AND JOSE F. NONIDEZ THE NUCLEOLUS The history of the nucleolar structures has not been studied in detail. but the nucleolus is so prominent during the growth period that a study of the chromosomes must necessarily reveal the main features of the nucleolar behavior. It is probable that the chromatic part of the nucleolar complex persists from the final spermatogonial anaphase in the form of a pair of chromo- somes, but whether the achromatic portion arises from this or originates independently we are unable to state. The two are united from stage b (fig. 11) throughout the remainder of the growth period. The chromatic portion may be followed directly to the first spermatocyte metaphase where it becomes one of the five bivalent chromosomes. During much of the growth period the nucleolus is plainly compound (fig. 14), being composed of a large, oval achromatic portion and a smaller dense chromatic portion to which is attached a chromatic thread or finger-like projection. The latter is very characteristic and persistent throughout the growth period. The achromatic portion seems to diminish gradually during the later stages, and cannot be detected with certainty in late prophase. However, the degree of extraction of the haematoxylin has much to do with the appear- ance of the structure, and it is difficult to say just what becomes of the achromatic portion. The chromatic portion is presumably the sex chromosome pair. At first sight the finger-like process suggests the presence of an unequal XY pair, but this asymmetry seems to disappear in metaphase and we are unable to verify the point. Likewise, the spermatogonial divisions do not reveal any such inequality in any chromosome pair. It seems more probable that the finger-like process is due to a difference in the degree of conden- sation of the two chromosomes, such, e.g., as that shown by the XY chromosomes of Enchenopa binotata (Kornhauser, 714). In another species of Asilus it is practically certain that the chromosomes involved in the nucleolar complex are the sex chromosomes, and it may be inferred that the same is true in A. sericeus. This is in agreement with the observations of Stevens (08 b), who found the sex chromosomes condensed during the growth period in several species of flies. SPERMATOGENESIS IN ASILUS SERICEUS 177 DISCUSSION Asilus sericeus presents the most simple and clear-cut type of spermatogenesis thus far found in the Diptera, owing to the fact that during the growth period the chromatic threads do not spin out and become entangled to such a degree as they do in other forms. When compared with animals other than Diptera, the most outstanding characteristic of the maturation processes in Asilus is the apparently continuous association of corresponding chromo- somes in pairs. Superficially some of the stages bear a marked resemblance to those in various other forms, but on close exami- nation it appears that only the later growth period and succeeding stages are actually similar in essential features. Previous to this there js an underlying difference due to the fact that in Asilus the chromosomes, whether condensed or thread-like, maintain an intimately paired association from the telophase throughout the entire growth period, with the result that the usual leptotene stage and the subsequent synaptic process seem to be omitted entirely. This is discussed more specifically above. Another feature that should be recalled here is the probable parallelism between the peculiarities in chromosome behavior observed in Asilus and those found in certain plants as recorded by Strasburger, Rosenberg, Miller, Overton, and others (see especially Overton, ’09). Apparently the peculiarities during the maturation stages are in each known case correlated with a noticeable paired association of chromosomes in the somatic cells, which would again lead one to conclude that the two phenomena are causally connected and are both manifestations of the same inherent ‘tendency toward pairing.’ As has been remarked previously (Metz, 716, p. 225), the latter seems to be an accentuation of the tendency or force that unites correspond- ing chromosomes during synapsis in most organisms. It seems to differ mainly in that its effects in the cases mentioned are not limited to the final germ cells, but are visible in somatic and early germ cells as well. What this force is, physicochemically, remains as obscure as ever, although there is very strong reason for believ- 178 CHARLES W. METZ AND JOSE F. NONIDEZ ing that it is due to a likeness in constitution of corresponding chromosomes. Regarding the genetical question of ‘crossing over,’ our obser- vations afford only negative evidence. Since no leptotene threads have been observed and nothing like a typical synaptic stage has been identified, there is little indication of any process that might bring about crossing over during the early stages. It is difficult to determine just what takes place in stage a, but it should be recalled that this stage appears to be like the sperma- togonial resting stage, and that as far as cytological evidence goes there is no more reason for expecting it in the former than in the latter. In subsequent stages there is some evidence of chromosome twisting, but more often the threads lie side by side without twisting, and when they do overlap there is no evidence of break- ing. On the whole, then, what evidence there is would argue against the probability of crossing over in the males of Asilus, which agrees with the genetical results in Drosophila, where crossing over is found only in the female. But this question, from the cytological standpoint, is only in the speculative stage, and will probably remain there at least until further studies are completed, particularly studies on o6genesis. In this connection a word should be said regarding the degree to which the above description may be considered typical of the Diptera. One other species of Asilus (A. notatus) has been studied fully and shows certain noteworthy deviations from the above account. ‘These may be summarized briefly as follows: In stage a following the final spermatogonial telophase, the chromatin stains more deeply than in A. sericeus and gives even clearer evidence of remaining relatively condensed, i.e., not spinning out into threads. Stage a is very brief and is succeeded immediately by stage b, in which the chromatin is likewise more dense than that in the corresponding stage of A. sericeus. It is in the form of short, thick, bivalent prochromosome-like bodies, the dual nature of which is very plain. These show a more marked superficial resemblance to the bodies of stage 6 in the Hemiptera than do those of A. sericeus. SPERMATOGENESIS IN ASILUS SERICEUS 179 The most noticeable difference between sericeus and notatus, however, appears in the stage immediately following stage ). At this time the bivalents in A. notatus, instead of lengthening only slightly and remaining well separated from one another, as they do in sericeus, become greatly attenuated and entangled for a time, making analysis very difficult. Here again the super- ficial resemblance to phenomena in the Hemiptera is more marked than in sericeus, although the actual structural characteristics (persistence of the diplotene condition) appear to agree with those of sericeus. When an attempt is made to compare spermatogenesis in Asilus with that in other genera of flies, confusion enters at once. Other members of the Asilidae show definite resemblances, but outside of the family superficial differences are so great that comparisons cannot be made safely without very careful study. As a case in point we may mention the genus Drosophila. Super- ficially spermatogenesis in this group is exceedingly different in appearance from that in Asilus. Further study and possibly detailed examination of intermediate forms will be necessary before the relationships can be determined. Perhaps much of the apparent divergence between Drosophila and Asilus is due to difference in the cytoplasm, rate of growth of the spermato- cytes, degree of staining of the different nuclear elements, and other secondary features, but there is as yet no certainty that it may not also include fundamental differences in the chromo- somal behavior. SUMMARY 1. The spermatogonial chromosomes of A. sericeus are ten in number, arranged in five pairs. The sex chromosomes have not been identified. 2. In the last spermatogonial anaphase, as in preceding ana- phases, the chromosomes go to the poles associated in pairs. 3. The paired association becomes more intimate in telophase, giving rise to bivalent chromosomes in haploid number. 4, A brief diffuse stage (stage a) ensues, in which the chromatin stains only slightly. 180 CHARLES W. METZ AND JOSE F. NONIDEZ 5. Then the double chromosomes reappear, apparently in the same form and relative position as before, and condense into bivalent prochromosome-like bodies (stage 6). 6. The ordinary leptotene condition seems to be omitted entirely. 7. The bivalent bodies of stage b elongate into diplotene threads that remain relatively condensed and clearly separate throughout the entire growth period, giving rise to the bivalent prophase chromosomes. 8. In another species, A. notatus, the process appears to be essentially the same, but is somewhat confused by a spinning out and intertwining of the threads in the stage following stage b. 9. The usual synaptic process is entirely wanting. Synapsis is effected in telophase at the beginning of the growth period by an intimate association of chromosomes that were already paired in anaphase. 10. Superficially the early growth stages are not unlike those in the Hemiptera and other forms, but the chromatic structures are bivalent instead of univalent. 11. Tetrad structures are not visible. 12. The first division appears to be reductional for all of the chromosomes. LITERATURE CITED ArnNotp, G. A. 1908 The nucleolus and microchromosomes in the spermato- genesis of Hydrophilus piceus. Arch. f. Zellfor., Bd. 2, No. 1. BriackMaNn, M.W. 1903 The spermatogenesis of the myriapods II. Biol. Bull., vol. 4; pp. 187-218. 1910 The spermatogenesis of the myriapods VI. Biol. Bull., vol. 19; pp. 138-160. Davis, H. 8. 1908 Spermatogenesis in Acrididae and Locustidae.. Bull. Mus. Comp. Zool. Harvard, vol. 52, no. 2. Downinea, E. R. 1905 The spermatogenesis of Hydra. Zool. Jahrb., Bd. 21; s. 379. GoutpsmitH, W. M. 1919 A comparative study of the chromosomes of the tiger beetles. Jour. Morph, vol. 32, pp. 487-488. Kornuauser, 8S. I. 1914 A comparative study of the chromosomes in the spermatogenesis of Enchenopa binotata, ete. Arch. f. Zellf., Bd. 12 s. 241-298. LomeN, Franz 1914 Der Hoden von Culex pipiens L. Zeits. Naturwiss., B. 52. SPERMATOGENESIS IN ASILUS SERICEUS 181 McCuune, C. E. 1902 The spermatocyte divisions of the Locustidae. Kans. Univ. Sci. Bull., vol. 1, pp. 185-240. Merz, C. W. 1916 Chromosome studies on the Diptera II]. Jour. Exp. Zodl., vol. 21, pp. 213-279. Montreomery, T. H. 1900 The spermatogenesis of Peripatus. Zool. Jahrb., Bd. 14, s. 277-368. Nonipez, José 1914 Los cromosomas en la espermatogenesis del ‘Blaps lusi- tanica’ Herbst. Trab. Mus. N. Cienc. Nat., Madrid, Ser. Zool., num. 18. 1915 Estudios sobre las celulas sexuales. I. Los cromosomas goni- ales y las mitosis de maduracion en Blaps lusitanica y B. Waltli. Mem. Soc. Esp. Hist. Nat., T. 10. Overton, J. B. 1905 Ueber Reduktionsteilung in den Pollenmutterzellen ein- iger Dikotylen. Jahrb. f. wiss. Bot., Bd. 42, s. 121-153. 1909 On the organization of the nuclei in the pollen mother cells of certain plants. Ann. Bot., vol. 23, pp. 19-62. Srevens, N. M. 1903 On the odgenesis and spermatogenesis of Sagitta bipunc- tata. Zool. Jahrb., Bd., 18, s. 227-240. 1905, 1906 Studies in spermatogenesis. Parts I and II. Carnegie Institution of Washington, Pub. 36. 1907 The chromosomes of Drosophila ampelophila. Proc. VII Inter- nat. Zool. Cong. ; 1908 a The chromosomes in Diabrotica. . . . . Jour. Exp. Zodl., vol. 5. 1908 b PLATE 1 PLATE 2 EXPLANATION OF FIGURES Figures 14-22, Asilus sericeus, middle and late growth stages. 14 Late stage b, four of the bivalents drawing out into diplotene threads, the other attached to the plasmosome, forming the nucleolar complex. Three of the threads are, respectively, at the top, the extreme left, and the extreme right of the figure; the fourth is at a low focus, indicated by its light color, and passes underneath the nucleolus. The nucleus is entire. 15 and 16 Stages progressively later than the preceding, showing the conden- sation of the threads and their orientation with respect to the nucleolus; nuclei entire. 17 Contraction stage, nucleus entire. 18 to 20 Successive stages following the contraction. The diplotene threads extend out like fingers from the nucleolus; nuclei entire. 21 Prophase of the first spermatocyte division; nucleus entire. The four long threads have broken loose from the nucleolus; the latter has become smaller and is mostly chromatic. 22 First spermatocyte metaphase showing the five bivalents. 184 SPERMATOGENESIS IN ASILUS SERICEUS CHARLES W. METZ AND JOSE F. NONIDEZ PLATE 2 alee j it ee \ ee. ult ; ry j f ih . st ¥ ; A ’ ‘ em i‘ ” i D iy J] me ‘Sp : he tea oy ee i L; URNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, NO. 2, iV FEBRUARY, 1921 cpt v ne oe . whi 7 i A | e -~ + Resumen por el autor, Harold H. Plough, Amherst College. Nuevos estudios sobre el efecto de la temperatura en el crossing over.' Los datos presentados en este trabajo constituyen un suple- mento a un trabajo precedente del autor sobre el efecto de la temperatura sobre el crossing over del segundo cromosoma de Drosophila melanogaster. Empleando el mismo método del trabajo mencionado, el autor ha estudiado los efectos de dicho agente sobre prdcticamente el total de la longitud conocida de los cromosomas segundo y tercero, sometidos a una temperatura de 31.5°C. Los resultados obtenidos indican que ni-la temper- atura ni la edad de la hembra madre de una generacion, producen una variaciOn significativa en el crossing over de parte alguna del cromosoma sexual. Solamente la regién media del tercer eromosoma presenta un aumento marcado en el crossing over como resultado de la exposicién a una temperatura elevada y una variacion con la edad. Las regiones de los cromosomas que son ‘‘sensitivas”’ a los cambios del medio ambiente presentan también una proporcién elevada de crossing over sencillo y doble. Es probable que donde el crossing over es menos libre, se pueden observar los efectos del medio ambiente. 1Con este nombre se designa el entrecruzamiento de los cromosomas en las células sexuales de la hembra. Nos parece mas conveniente conservar la palabra inglesa, puesto que ha adquirido un significado preciso que se pierde al tradu- cirla. (N. del T.) Translation by José F. Nonidez Cornell Medical College, New York AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, NOVEMBER 15 FURTHER STUDIES ON THE EFFECT OF TEMPERATURE ON CROSSING OVER HAROLD H. PLOUGH Department of Biology, Amherst College THREE FIGURES CONTENTS LiNGTG is (Gia pole ee Nh ee hay Se epee Rael Eee ew eae SIM 187 LOSSES Ten Str Nes AR AC ee ee PR CO a PL 73 AG CM pec eee 188 Interpretation of the curves of crossing over... .......6.-ceees-eeeeessan 189 Reaction to temperature and high coincidence......................--000- 194 WIS CUSSION Amines. > os oe AT iese MRAP Sica revs = OE One ae SPE Pooh s SANG. coat ntetye Pare 198 Age wnd: temperature effects compared . ..))./..\.'8oh. hak She apardea ce tioete. ¢ 199 STEUER Toy iter ea hr Sid eae ys Meee. | ASO Wei Colley chy gla 201 INTRODUCTORY In an earlier study of the effect of temperature on crossing over in Drosophila melanogaster I showed that temperature both above and below the optimum (22°C.) caused a significant increase in the amount of crossing over between certain genes located in the second chromosome (Plough, ’17). Preliminary work on the first and+third chromosomes indicated, however, that crossing over in these groups was not visibly affected by temperature. No reason for this unexpected result could be assigned, and it seemed worth while to test the first and third chromosomes by the same accurate methods which had been used with chromosome IT. Such data would also give an accurate basis for checking the fact reported by Bridges (’15) that in chromosome J, unlike the second chromosome, there is no signifi- cant variation in crossing over due to the age of the female parent. The large amount of breeding work with Drosophila has resulted—especially through the work of Bridges—in making available a large number of easily workable mutant characters 187 188 HAROLD H. PLOUGH with excellent viability. The most valuable mutants of each chromosome have been assembled into multiple stocks, the use of which has made it possible to determine the effect of environ- mental changes on linkage relations over approximately the whole known lengths of each of these chromosomes in a single experiment. At the same time in these multiples the distances between the different genes are generally not sufficiently great to cause complications due to unobserved double crossing over. My present more accurate data establish the truth of the earlier observation that crossing over in chromosome I is not influ- enced by temperature, but show that there is a section of chromo- some III in which crossing over is increased in the same way as in chromosome II. EXPERIMENTAL The mutant stocks used for the tests were the sex (or first) chromosome multiple stock, scute-echinus-cut-vermilion-garnet- forked, and the third chromosome multiple stock, sepia-spineless- sooty-rough. Scute shows an absence of scutellar bristles, and echinus, a roughened condition of the facets of the eyes. The other mutations have been described. Dichete, a dominant character, was introduced in a small number of preliminary third chromosome tests based on ten-day brood counts. A glance at the chromosome maps in figure 3 will show that the genes used cover both chromosomes at fairly even intervals throughout a large portion of their known lengths. The method of making the tests was essentially the same as that used in my earlier work with the second chromosome. Virgin sister females of the normal wild stock were mated to males of the multiple mutant stock to be tested, and allowed to lay in one set of bottles for about three days. This first set of bot- tles was kept at the control temperature. The P, pairs were next transferred to another set of bottles which was kept con- tinuously at the high temperature. Virgin female offspring . from each set were then isolated and back crossed to males of the original mutant stock used. These back crossed pairs were placed in quarter-pint milk bottles containing banana agar, and EFFECT OF TEMPERATURE ON CROSSING OVER 189 . kept at the control temperature. With the exception of the preliminary ten-day brood test, the pairs were changed from one set of such bottles to another at the end of successive three- day periods throughout the life of the females. (The first change was made in all cases at the end of the fourth day.) The counts of the successive sets of offspring of these pairs furnished the data for determining the effect of the high temperature on crossing over in the developing eggs of the heterozygous females. The control temperature was approximately 24°C. maintained in a wooden stock cabinet controlled by an electric heater with a thermostat. It varied between 22°C. and 25°C. throughout the experiments. The high temperature was 31.5°C. maintained in a Freas electric incubator. This varied as much as 1° above and below, though the normal variation was about 0.5° either way. | The results given by the tests of the first chromosome regions are given in table 1. The bottle counts for each three-day period are added and the percentages of crossing over for each of the five regions calculated and listed in the columns at the right. The successive percentages of crossing over for each of the five regions are plotted as curves in figure 1, the dotted line in each case being the experimental value and the full line the control. The results of the tests of the multiple third chromosome stock are given in tables 2 and 3, with the percentages of crossing over in the columns headed per cent 1, etc. Table 2 summarizes the results of a ten-day brood count made with the sepia-Dichete- spineless-sooty-rough stock, and table 3 shows the three-day- interval results from the same stock without Dichete. 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No section of the first chromosome tested, therefore, shows any effect of high temperature on the amount of crossing over. This is a complete confirmation of my earlier conclusion, made on less exact data, and for a much shorter scute-echinus echinus-cut 31 Fig. 1 Curves of crossing over for different regions of chromosome I. The control values are the solid lines; the values from the heat-treated lines are dotted. The abscissas are days after mating, the ordinates percentages of crossing over. section of the chromosome. Second, a comparison of the differ- ent control lines with each other indicates no significant variation as the female grows older. The control line for second chromo- some regions dropped steadily up to about the tenth day, and gradually rose up to about the twenty-second. The sex chromo- some control values show no significant nor uniform changes, and confirm the conclusion of Bridges (’15) that in this chromo- some the age of the female has no influence on the amount of crossing over. 192 HAROLD H. PLOUGH TABLE 2 Preliminary test of chromosome III, ten-day broods se ss es ro D CENT 4 266 | 30| 48| 49| 7o| 10 | 6 | 12] 7} 17] 6 | 0/2) 0]2 525/11.4|16.4|13.3)20.8 2-3-4 TOTAL PER 1-4 Control—22°C. continuously Coincidence... ....|1.22 0.78 0.55 | Hatched at 31.5°C., mated at 22°C. 103 | 15| 63| 19| 31] 9 | 8 | 9] 13) 15] 6 | 3 | 8] 1 | 2 (305/17.3/33.814.9123.6 Coincidence....../1.02 0.99 0.78 TABLE 3 Summaries of third chromosome series se ss e& ro : DAYS AFTER MAT- ING NON CROSS- OVER 1-3 Control—24°C. continuously 1-4 337 | 129| 47 | 96] 26 | 40 Aa 493 | 180| 97 | 154] 32 | 49 7-10| 507 | 183] 82 | 136] 25 | 45 10-13 | 455 | 157| 72 | 126] 30 | 27 13-16 | 506 | 198] 95 | 123] 24 | 53 16-19 | 380 | 146] 71 | 93] 11 | 28 19-26 | 185 74| 25 | 66] 11 | 18 0 | 677|28.7\11.0/18.9|1.20/1.09/0. 14 2 |1008)26.1)/13.1)/20.4/0.93)0.90/0.04 0 | 979/25.7|10.9)18.5/0.89|0.95|0.05 2 | 872/24.7/12.1|18.0/1.14)0.69/0.14 0 |1004/27.4|12.6)18.3/0.69/1.08)0.47 3 | 732/25.6)/11.6|16.9/0.55|0.87|0.30 0 | 379/27.1/09.5|22.1)1.13/0.79 oo OW Fe tb Heat treated—hatched at 31.5°C., mated at 24°C. 1-4 195 97| 34 | 54) 20] 31 | 3 AG 370 | 228} 67 | 140| 37 | 86 | 11 7-10 2570 |alsO)rady) 76|29)) 37) 6 10-13 257 96} 45 | 78) 15 | 23 | 4 13-16 212 92) 36 | 55) 16 | 22] 2 1 2 435/34 .2/13 .3/20.4|1.01/1.02/0.25 948/39 .0/13.1/26.6|0.76|0.94/0.31 587/33 .5|14.8/20.4|0.99)0.92/0.33 518/25 .8/12 .4|20.2/0.90/0.84/0.32 436|30.0)12.6/18 .3]0.98/0.91)0.21 446/28 .8/11.8/19.9}0.65/0.78}0.09 887 |26 .2|11.1|16.9/0.52/0.79/0.27 = BREE OrFOrF 16-19 208 98) 41 | 67} 10 | 20 19-26 200 81| 34 | 49) 6 | 14 EFFECT OF TEMPERATURE ON CROSSING OVER 193 An examination of tables 2 and 3 and of figure 2 discloses an interesting situation in the third chromosome. It seems clear from table 2 that high temperature causes a definite increase in crossing over between sepia and Dichete and a very marked one between Dichete and spineless, but little if any change in the remainder of the chromosome. ‘Table 3 and a comparison of the full and dotted lines in figure 2 bring out this fact even more definitely, but without separating sepia and spineless by the sepia-spineless spineless-sooty sooty-rough Fig. 2 Curves of crossing over for different regions of chromosome III. The control values are the solid lines; the values for the heat-treated lines are dotted. The abscissas are days after mating, the ordinates percentages of crossing over. Dichete factor. The dotted line for the sepia spineless region begins at a point about 6 units higher than the control, rises to a difference of 18 units, and drops sharply to about the same point at about the tenth day. This indicates, as in the second chromosome, that the eggs which go through the critical period at the high temperature show a much increased crossover ratio, but that those which pass through that period subsequently (i.e., after the females are replaced at the control temperature) are not affected. The dotted line for the spineless sooty region, on the other hand, shows no significant difference. That for 194 HAROLD H. PLOUGH sooty rough shows a rise of nearly six units in the four-to-seven- day period, but since no difference appears either before or after this time, it is probable that this has no significance. The data indicate, therefore, that the percentage of crossing over is increased by exposure to high temperature at one end of chromo- some III, but not throughout the remainder of its length. It is interesting to note that the control line for the sepia spineless region shows the age variation observed in the second chromosome. The value gradually drops to the tenth day and then rises. The rise apparently takes place somewhat earlier than in chromosome II. The other two regions show no signifi- cant difference as the female grows older. The results of the tests may be summarized as follows: a) the sex chromosome shows no significant increase in the percentage of crossing over as a result of the exposure of the developing eggs to high temperature; b) the third chromosome shows an increase in crossing over in the sepia spineless region, but nowhere else; c) a variation in crossing over with the age of the female occurs in those regions which show a reaction to temperature only. REACTION TO TEMPERATURE AND HIGH COINCIDENCE In figure 3 I have drawn to the same scale comparative maps of the principal chromosome regions whose reactions to high temperature have been tested. The percentages of crossing over in chromosome I have been calculated from the ten-day brood counts summarized at the end of table 1, and those for chromosome III from table 2. The map of chromosome II is taken (for the points indicated) directly from the very accurate one given by Bridges and Morgan (p. 302). The regions for which a rise in the percentage of crossing over as a result of exposure to a temperature of 31.5°C. has been recorded—either in this or my former paper—are indicated by diagonal lines, while those which are not changed are solid black. As noted previously, it may develop that one or both of the long regions at either end of chromosome II will show a reaction to temper- ature if they can be broken into short blocks. A rise in total crossing over may be obscured by a compensating rise in double EFFECT OF TEMPERATURE ON CROSSING OVER 195 crossing over, so that no result appears in the count. With the exception of these two regions, in which Bridges (715), Plough (17), and Bridges and Morgan (’19) have recorded an age differ- ence, the diagonal lines also indicate the regions which show a variation in crossing over with age. 0.0 Star 0.Ogrscute 00° sepia 8. echinus 11.4 Dichete 40 102 122 26 24 cut 8 783 spineless 46.5 22 black -4 4 vermilion 2.7 4 purple 6 55 41.1 sooty 96 ap uck: pa waic 65.04 vestigial 3.5 curved 1. forked , 1.9 rough 48 Chromosome I Chromosome III 185.5 speck Chromosome II Fig. 3 Chromosome maps showing all the important regions whose reaction to high temperature has been tested. The regions which show a significant in- crease in crossing over after exposure to high temperature are ruled with diag- onal lines those not affected are solid black. The coincidence values are given outside of brackets enclosing the different pairs of adjacent regions. I have recently laid some emphasis on the fact that in the sensitive section of chromosome II the percentage of increase in crossing over due to high temperature was roughly in inverse proportion to the length of the region involved (Plough, 719). The results in chromosome III demonstrate very plainly, however, 196 HAROLD H. PLOUGH that the masking effect of increased double crossing over is not the only reason why certain regions remain unchanged after exposure to high temperature. Table 2 shows that in chromo- some III the Dichete spineless region (16.4 units) shows an increase of more than 100 per cent, while sepia Dichete (11.4 units) is increased only about 50 per cent, and spineless sooty (13.3 units) is practically unchanged. It is apparent that there are other factors than the mere length of the region responsible _ for the difference in reaction to high temperature. Not only are different chromosomes unlike, but within each chromosome cer- tain regions show distinct differences in behavior from certain other regions. This fact has been apparent to several Drosophila workers in connection with the investigation of the coincidence values, and has been the subject of a special study by Bridges, the results of which are not yet published. It is of some interest to compare the differences in reaction to temperature with the coincidence results. Figure 3 shows also the coincidence of double crossing over for each pair of adjacent regions as a percentage value outside a bracket enclosing the two regions for which it has been calcu- lated. The significance of the value for coincidence has been discussed in detail by Bridges (15), Muller (’16), Weinstein (18), and Bridges and Morgan (19). It represents the per- centage of the expected number of double crossovers actually observed. The size of the coincidence value has been shown by a number of observers to be in proportion, up to a certain point at least, to the distance apart of the opposite boundaries of the regions tested (i.e., to the lack of interference). For instance, a glance at the ten-day counts in table 1 shows that the coincidence value is high when the scute-echinus and vermil- ion-garnet regions (138 per cent) or the scute-echinus and garnet- forked regions (86 per cent) are figured, but low when the regions are closer together. A comparison of the coincidence values given for approximately equal lengths of chromosome shows that they do not correspond. Double crossing over per unit of distance is apparently much EFFECT OF TEMPERATURE ON CROSSING OVER 197 more frequent in certain regions than in others, and this is true not only when the regions compared are in different chromo- somes, but when they are in different portions of the same chromosome. For instance, for the black-purple-vestigial region of chromosome II we get a coincidence of 66 per cent,! while for distances slightly longer in chromosome I-scute to cut or cut to garnet—the values are 40 per cent and 22 per cent, respec- tively. The black-purple-curved region in chromosome II gives a coincidence value of 96 per cent (Plough, 717, table 8), while a similar length in chromosome I—-echinus-cut-vermilion— gives 26 per cent. Even more striking, however, is the fact that the region at the upper end of the third chromosome on the map—sepia-Dichete-spineless, a distance of 28 units—gives a coincidence value of 122 per cent, yet the longer lower region— spineless-sooty-rough (33 units) gives only 55 per cent coinci- dence. This indicates that double crossing over is at least as frequent as though there were no interference at the sepia end of chromosome II, but interference is almost as high at the rough end as in chromosome I. A difference of the same order is apparent in chromosomes I and II. It will now be obvious that those sections of the chromosomes mapped in figure 3 which show a relatively high coincidence per unit of distance are the same ones which show a change in the amount of crossing over as a result of high temperature and of the age of the female. In no case where the coincidence value for a continuous region of 30 units or less is below about 60 per cent do we find an increase in crossing over due to high temper- ature, or, with the possible exceptions noted in the second chromo- some, a change due to age. The chromosomal regions which are ‘sensitive’ to environmental effects all show a minimum of influ- ence of one crossover on another simultaneous crossover in the same region. 1 Bridges and Morgan, ’19, table 42—not 61 per cent, as they give it. 198 HAROLD H. PLOUGH DISCUSSION The fact that high coincidence and sensitiveness to environ- mental effects are found in the same chromosomal regions sug- gests that certain structural features of the crossing over process determine each. Bridges and Morgan (p. 188) suggest that the difference in the amount of interference for short regions between the first and the second chromosomes may be inter- preted in two ways. We may assume that the average length of loop between simultaneous crossovers is the same in each, which means that a region having a given coincidence value in chromosome II is actually the same length as one having the same coincidence in chromosome I. On the other hand, we may hold that the length of loop between simultaneous crossovers is relatively shorter’in chromosome II than in chromosome I, - which means that equal amounts of crossing over then indicate equal lengths of chromosome. Either of these alternatives holds also for the different sections of chromosome III. According to the former interpretation, crossing over takes place relatively less freely in the regions ruled with the diagonal lines and they are actually much longer than the map length indicates. Ac- cording to the latter view, crossing over takes place relatively more freely, and the map lengths are accurate. Bridges (’19, and from subsequent unpublished data) distinctly favors the former interpretation. The effect of high temperature in causing an increase in these regions does not give any clear evidence for either view, though it would seem to support Bridges’ inter- pretation. It is hardly possible that temperature does not act on the whole chromosome equally. Any observed differences between different regions would seem to be due to the fact that slight effects are registered in certain regions and not in others. It is reasonable to suppose that the regions in which a change is observable should be those in which crossing over is less free. It is of some interest to consider what structural conditions in the chromosomes could result in regions of decreased freedom of crossing over. Bridges and Morgan (p. 198) and Bridges (719) suggest that the reason for the difference in behavior of the black curved region in chromosome II may lie in the fact that this EFFECT OF TEMPERATURE ON CROSSING OVER 199 region is near the middle of the chromosome, “with the spindle fiber attachment, and that this middle region is the last part to undergo synapsis.”” Bridges has subsequently applied this same idea to chromosome III and decided its middle point is close to the locus for Dichete. In the latter case the conclusion as to the midpoint of the chromosome has been definitely confirmed with the finding by Strong (’20) of the locus for roughoid at 24.9 units beyond sepia. If, as Bridges suggests, crossing over is less in this middle region because synapsis fails or is slight, the decreased freedom of crossing over might be consistently explained. On the other hand, it should be definitely borne in mind that such behavior is an observable phenomenon, which is susceptible of cytological demonstration. The demonstration that the process of crossing over is accomplished by a simple twisting separation, and reunion of chromosome strands is still incomplete, and we have no cytological data which indicate that in Drosophila the middle region is the last part to undergo synapsis. At the early stage in the growth period of the egg at which crossing over apparently takes place it seems altogether unlikely that the spindle fiber suggested by Bridges is present at all. Until we know more of the actual cytological features of the crossing-over process and of the spindle fiber attachments in Drosophila, such suggestions must be regarded as highly speculative. AGE AND TEMPERATURE EFFECTS COMPARED It has been demonstrated above that in general both age and temperature affect the amount- of crossing over in the same chromosome regions—those probably in which there is a mini- mum of crossing over. It is to be expected, therefore, that the freedom of crossing over is modified by both agents. It is of some interest to note that Bridges and Morgan (p. 199) and also Bridges (19) in identical language conclude that the age variation is probably due ‘‘to a lengthening of the average length of the section of chromosome between simultaneous crossovers,”’ while temperature causes an increase in the freedom of crossing over with no difference in the length of loop. The clearest evidence 200 HAROLD H. PLOUGH for this conclusion is stated to be found in a calculation of the coincidence values for my two-day-interval experiment for the black-purple-curved region reported in my former paper. They state (p. 199): In this experiment the curve of variation in coincidence was the mirror image of the curve of variation (in crossing over) for age. The curve of coincidence corresponding to the curve of temperature varia- tion found by Plough seems to be a straight line cutting the rises and falls of the temperature curve and independent of them. TABLE 4 Coincidence values for control and heat-treated lines in two-day-interval experiment. (For actual counts cf. Plough, ’17, table 14) b pr c PARENTS HATCHED AT 22°C., EXPOSED CONTROL—22°C. CONTINUOUSLY To 31.5°C. FROM 3RD TO 11TH Day NUMBER DAYS AFTER MATING AFTER MATING Per cent of crossing Coincidence Coincidence Per cent of crossing over—b, pr region b, pr-pr, e b, pr-pr, c over—hb, pr region 3 8.3 0.91 Ot Teil 15) 4.9 0.94 1.31 4.8 7 6.8 Ids 0.53 3.8 9 5.8 1.03 1.37 3.8 11 4.2 1.06 0.63 8.8 13 5.1 0.80 0.94 13.9 15 5.3 1.40 0.99 19.2 17 4.2 0.92 0.95 20.0 19 7.3 0.93 0.73 17.5 21 8.2 1.04 IL 5i7/ 6.8 23 7.9 0.63 0.27 4.9 25 7.0 0.98 1.4 I have calculated the coincidence values for the experiment cited and the results are given in table 4. A comparison of my coincidence values with the crossover percentages for the black- purple region shows that the coincidence value varies within rather wide limits in both the control and experimental lines. A smoothed curve gives some suggestion of the relation claimed by the writers quoted, but its significance is doubtful. The same comparison may be made between the similar lines in chromo- some III (table 3). The coincidence values for the different pairs of regions are given in the last three columns. Here there EFFECT OF TEMPERATURE ON CROSSING OVER 201 is surely no mirror-image relation in the control series. In addition, the coincidence is subject to so high a probable error that it would take very marked and constant differences to establish such a conclusion as the one stated. It seems clear that much weightier evidence than that quoted must be given before it can be established that age and temperature act in different ways on the crossing-over process. It is more con- sistent with the results here given that each causes a variation in the actual freedom of crossing over and that the changes ‘in coincidence recorded are without significance. \ SUMMARY 1. It has been shown that a temperature of 31.5°C. causes little or no effect on crossing over in any part of the sex chromo- some, nor is there any significant variation with the age of the female. 2. Crossing over in the sepia-spineless region of chromosome III is increased by a temperature of 31.5°C., the effect being most marked between Dichete and spineless. 3. The same region shows a variation in crossing over with the age of the female parent. — 4. Crossing over in the remainder of chromosome III is influ- enced neither by temperature nor age. 5. The chromosomal regions which are ‘sensitive’ to temper- ature and to age all give a very high ratio of double to single crossing over. 6. This is interpreted as indicating that the effects of environy ment cause observable differences in crossing over only where crossing over occurs least freely. 7. It is shown that the view that temperature and age act op crossing over in different ways is not established. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, NO. 2 202 HAROLD H. PLOUGH LITERATURE CITED Bringces, C. B. 1915 A linkage variation in Drosophila. Jour. Exp. Zodl., vol. 19, no. 1, July. 1919 The genetics of purple eye color in Drosophila. Jour. Exp. Zoél., vol. 28, no. 2, May. Bripaes, C. B., anp Morcan, T. H. 1919 The second chromosome group of mutant factors. Publ. no. 278, Carnegie Inst. Wash. Mouuuer, H. J. 1916 The mechanism of crossing over. Amer. Nat., vol. 50. Pioucu, H. H. 1917 The effect of temperature on crossing over. Jour. Exp. Zool., vol. 24, no. 2, November. 1919 Linear arrangement of genes and double crossing over. Proc. Nat. Acad. Sci. U. 8., vol. 5, May. ; Srronec, L. C. 1920 Roughoid, a mutant located to the left of sepia. Biol. Bull., vol. 38, no. 1, January. WEINSTEIN, A. 1918 Coincidence of crossing over in Drosophila. Genetics, 3, March. . Resumen por los autores, William EK. Burge y Emma Longfellow Burge, Universidad de Illinois.. Una explicacién de la variacién en la intensidad de la oxidacién durante el ciclo vital. Es un hecho conocido que la oxidacién o metabolismo es muy baja en el 6vulo no fecundado, mientras que aumenta notable- mente a raiz del proceso de la fecundacion; que el metabolismo del nifio recién nacido es también muy bajo, pero que aumenta ripidamente llegando a ser muy elevado durante la ninez, dis- minuyendo después gradualmente desde la edad adulta hasta la vejez. Los autores han observado que 0.5 gramos de los huevos no fecundados de Leptinotarsa, macerados, desprenden 18 cc. de oxigeno en diez minutes cuando se tratan con perdxido de hidrégeno, y que 0.5 gramos de huevos fecundados desprenden 35 ec. durante el mismo tiempo. 0.5 gramos de larvas recién salidas del huevo, durante la cuarta parte, mitad, tres cuartas partes del desarrollo y larvas completamente desarrolladas desprenden 280, 800, 1250, 1725 y 1750 cc., respectivamente, y que las ninfas, adultos e individuos viejos desprenden 1800, 1750 y 900 ec. de oxigeno, respectivamente. Comparando estas figuras puede comprobarse que el huevo no fecundado contiene mucha menos catalasa que el fecundado; que el contenido de catalasa en las larvas recién salidas del huevo es menor que el de las larvas mds avanzadas y que el contenido de catalasa en el individuo viejo es menor que el del adulto mas j6ven. La reducida cantidad de oxidacién en el huevo no fecundado se debe probablemente a su escaso contenido de catalasa. La oxidacién aumentada del huevo fecundado y su desarrollo consi- guiente se atribuyen a un aumento de catalasa introducida por la estimulacién del huevo para la produccién mayor de esta enzima por parte.del espermatozoide. Del mismo modo el aumento del metabolismo respiratorio u oxidacién en el jéven y su disminucién con la edad avanzada, se atribuyen a un au- mento de catalasa en el j6ven y a su disminuci6n en el de mas edad. Translation by José F. Nonidez Cornell Medical College, New York AUTHORS’ ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 6 AN EXPLANATION FOR THE VARIATIONS IN THE INTENSITY OF OXIDATION IN THE LIFE-CYCLE W. E. BURGE anv E. L. BURGE Physiology Laboratory, University of Illinois ONE FIGURE As a result of the work of a great number of observers, particu- larly of Hasselbalch (1), Magnus-Levy and Falk (2), and of Warburg (3), it is now known that oxidation or metabolism is very low in the unfertilized ovum, while it increases greatly following the process of fertilization; that the metabolism of the newly born infant also is very low, but increases rapidly, becoming very high during childhood and then gradually decreas- ing from maturity to old age. The present investigation is an attempt to find an explanation for the variation in the intensity of oxidation under the conditions named. Since we (4) had found that whatever increased oxidation in the body, the ingestion of food, for example, produced an increase in catalase, an enzyme possessing the property of liberating oxygen from hydrogen peroxide, by stimulating the alimentary glands, particularly the liver, to an increased output of this enzyme, and that whatever decreased oxidation, narcotics, for example, diminished catalase by decreasing its output from the liver and by direct destruction, we naturally turned to catalase in seeking an explanation for the variations in the intensity of oxidation at different periods in the life-cycle. The Colorado potato beetle (Leptinotarsa decemlineata) was used in this investigation. Catalase determinations were made of the following materials gound up in a mortar: unfertilized and fertilized eggs, quarter, Analf, three-quarter, and full-grown larvae, as well as pupae, adult, and very old beetles. Five- tenths gram of the ground material were added to neutral hydro- 203 204 W. E. BURGE AND E. L. BURGE gen peroxide in a bottle and the amount of oxygen liberated in ten minutes was taken as a measure of the catalase content of the material. . AMOUNTS OF CATAL ASE MERSUREL (NV CC. OF OXYGEN. UNFERTILIZEDEGG ———- =~ —— /6 FERTILIZEDLGG ——- ~ —— JS Newty Harcueo LARVA —— = —— 280 QUARTER Grown LARVA —— ete — 800 tt FS he ake Fur. GRownlakVa— ame Pupa —— & Aputt BEETLE — OL0 BEETLE — Fig. 1 The figures in the chart indicate amounts of oxygen liberated from hydrogen peroxide in ten minutes by 0.5 gram of the material ground in a mortar. The results of the determinations as well as photographs’ of the beetles, pupae, larvae, and eggs are shown in figure 1. It may be seen that 0.5 gram of the unfertilized eggs liberated 18 cc. of oxygen in ten minutes from hydrogen peroxide and 0.5 - gram of the fertilized eggs, 35 cc.; that 0.5 gram of the newly’ hatched, quarter, half, three-quarter, and full-grown larvae INTENSITY OF OXIDATION IN LIFE-CYCLE 205 liberated 280, 800, 1250, 1725, and 1750 ec., and that the pupae, adult, and old bugs liberated 1800, 1750, and 900 cc. of oxygen, respectively. By comparing these figures it may be seen that the unfertilized egg contains much less catalase than the fertilized. This is in keeping with the fact that the oxidative processes are much less intense in the unfertilized egg than in the fertilized one, as observed by Warburg. It may be seen further that the catalase content of a newly hatched larva is less than that of the older larvae in keeping with the fact that in the newly born, and presumably in the newly hatched, oxidation is very low and that it increases very rapidly shortly after birth. It may also be seen that the catalase content of the old bug is less than that of the younger adult in accordance with the fact. that oxidation or metabolism is less in a person of advanced age than in one in middle life. Tt should be mentioned in this connection that our observation of the low catalase content of the unfertilized potato-beetle egg and the high catalase content of the fertilized egg is in keeping with the observation of Winternitz (5), who found that the unfertilized hen’s egg showed no catalytic activity even after prolonged incubation, whereas the incubated fertilized egg rapidly acquired the power of decomposing hydrogen peroxide. They agree also with the observations of Battelli and Stearn (6), who found that the catalase content of most of the tissues, and particularly of the liver, of newly born pigs is lower than the corresponding tissues of the mother, but that the catalase activity rapidly increased, until at the end of the seventh or eighth day it was as high as that of the adult. J. Loeb (7) attributes the development of the fertilized sea- urchin egg to the increase in oxidation, and the increase in oxidation to a change in the cortex of the egg which makes the entrance of oxygen, and hence oxidation, possible, while R. Lillie (S) holds that the cortical layer of the unfertilized egg prevents the diffusion of CO, from the egg and that this CO, prevents oxidation, and hence development. A more plausible explanation for the increased oxidation or metabolism in the 206 W. E. BURGE AND E. L. BURGE fertilized egg, and hence for the development of the egg, would seem to be that the spermatozoon furnishes a substance which stimulates the egg to an increased formation of catalase. Fur- ther evidence that might be presented in support of this view is afforded by the fact that the very same chemicals : (amines, alkalies, acetates, butyric acid, ete.) which Loeb found would bring about increased oxidation and artificial parthenogenetic development of the egg, we found, when introduced into the alimentary tract of animals, stimulated the alimentary glands, particularly the liver, to an increased output of catalase with resulting increase in oxidation. SUMMARY The low rate of oxidation in the unfertilized ovum is attributed to its low catalase content. The increased oxidation in the fertilized ovum, with resulting development, is attributed to an increase in catalase brought about by the stimulation of the egg to an augumented production of this enzyme by the spermatozoon. Similarly, the increase in the respiratory metabolism or oxi- dation in youth and decrease in old age is attributed to the increase in catalase in the young and its decrease in the aged. BIBLIOGRAPHY HassetBpatcH 1904 Bibliotek for laeger, Copenhagen, vol. 8, p. 219. Maanus-Levy unp Fark 1899 Arch. f. Anat. u. Physiol., Suppl. 315. WarsurGa 1908 Zeitschr. f. physiol. Chem., Bd. 57, No. 6. Buree 1918 Am. Jour. Physiol., vol. 45, no. 4; vol. 47, nos. 1 and 3. WINTERNITZ AND Rocers 1910 Jour. Exper. Med., vol. 12, no. 12. BATTELLI E STEARN 1995 Arch. di Fisiol., T. 2, p. 471. Lors 1913 Artificial parthenogenesis and fertilization. University of Chi- cago Press. 8 Lititme 1910 Am. Jour. Physiol., vol. 27, p. 289. IOooa fh WN ‘ hy os fe, bt M i mbaie oe "7 rs Mt 2 las Hk tm Sue a . a if a) nn yi meer Tver im: bi anys ah wigilose My): Hi hiantr i a i ef oe Lasers ts, vith aie bn uf bt ean “foe ‘ ye | miei nats Pye} crit itn, DN aL hie Mt Pr is aoe nod MEO. 1M y ' ; " 7 i sett ae | Pues yay e th stp, iia. Ce a | a pee hs) d a eHKnf i Ny PP MD ; Bi ; ie beat Ai , a at ne c rey wee el | rT] Datura ar en ‘s nt vo ‘ Se : ? ; ha ‘ mM, ie | shy ase loltwamne: | | * f hi c Nic ‘ tah ences ‘ oc heu tia as (a4 F ity ‘2 i iy } bi Lavy x nt pic woe i cag ay aay tru t : f r i 1 , ay ry eh nae’ ae 4 7h rid Th ete ean one ‘6 py “ u A Ps Ls pays a ie j ; . ' cli es 7 40 ig i rs ae ae basen vo f " ated) od mh ay th. 4 yon 3 ieee. © me tee } > ers Y ES fl mids i Wy mee, Cnet He yd 4 pits ah ‘a / i a i Ts sei j yg me i) NG ree Fi 7 ; Ta ain 0 2 7 ni A d ; " vey 64 a ' iu? We ; tay alien me i t 7 m A r as al ith uit Pi ae “a Cet (Oe A wi ' rave to | wk wie ins Phech Matty, Phat . 4 oe. LA wod HAP yh eee 1 7 4 t : | ' é 3 geet ’ i i i i 4 } ’ \ j (oho a” im ¥ } i 1 " é con os ; A ty Wh ~* y ~ i a i, 3 ‘ i - 1 i . l Ae - ods ' Resumen por los autores, Henry Laurens y 8. R. Detwiler, Universidad Yale, New Haven. Estudios sobre la retina. | La estructura de la retina de Alligator mississipiensis y sus cambios fotomecdnicos. El ojo de Alligator posee un tapetum retinal bien desarrollado, formado por la inclusién de guanina en las células epiteliales de las poreciones dorsal y posterior, a una distancia de 1.5 mm. de la entrada del nervio 6ptico. El pecten consiste en una especie de copa pigmentada ligeramente elevada, que cubre la entrada del nervio 6ptico. En toda la retina se encuentran conos y bastones, pero la proporcidn de ambos es diferente en distintas regiones. Los bastones son todos del mismo tipo, los conos de dos tipos: grueso y delgado. Los primeros son mas numerosos, presentdndose especialmente en las regiones posterior y ventral de la retina. Los conos del segundo tipo se encuentran solamente en la porcién ventral y no son numerosos. ‘También existen conos dobles. Ninguno de los conos y bastones contiene gotas de grasa. Los nticleos de los bastones son de forma oval y la mayor parte de ellos se proyectan a través de la membrana lmitante externa en una extensién variable. Los niicleos de los conos son piriformes y ocupan un nivel mds profundo que el de los nticleos de los bastones, formando una segunda fila. Los bastones presentan un cambio de longitud media de unas 4 micras, y son mas largos a la luz. Los conos sencillos presentan un cambio medio de 2.1 micras y son mds cortos.a la luz. Los miembros mas pequefios de los conos dobles presentan un cambio medio de longitud de 3.5 micras, y los mayores de 2.7 micras. La emigracién del pigmento es ligera, y su media es 1.6 micras, pero cuando se combina con el cambio de longitud de las células visuales produce una emigracién efecti va igual a su suma. El trabajo termina con consideraciones tedricas sobre los cambios fotomecdnicos y la teorfa de la duplicidad bajo un punto de vista comparado. Translation by José F. Nonidez Cornell Medical College, New York AUTHORS’ ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 6 STUDIES ON THE RETINA THE STRUCTURE OF THE RETINA OF ALLIGATOR MISSISSIPPIENSIS AND ITS PHOTOMECHANICAL CHANGES HENRY LAURENS AND S. R. DETWILER Osborn Zoological Laboratory, and the Anatomical Laboratory, School of Medicine,’ Yale University THIRTEEN FIGURES INTRODUCTION Owing to the variation in kind and distribution of the visual cells, the reptilian retina offers an interesting field of investi- gation in structure and function, with particular reference to the probable functions of the rods and cones under diurnal and nocturnal conditions (duplicity theory). Detwiler (16) studied the structure and photomechanical changes in the retina of a number of turtles and of lizards, and with the idea of making another contribution to our knowledge of the conditions holding in the reptiles the present investigation was undertaken. The work was started in the spring of 1917, but was necessarily abandoned, and only recently has it been possible to resume it. The literature on the crocodilian eye is small and rather unsatisfactory. Heinemann (’77), in a study of the eyes of Mexican reptiles, described the visual cells of Crocodilus rhom- bifer cuv. as consisting of characteristic rods alternating with considerably fewer and shorter cones. In addition to the typical rods he found a much less numerous kind, with very long outer segments of platelet structure. The cones are described as being also of two kinds, thick and slender, neither of which contain colored oil drops, and which occur singly and together to form double cones. There is an ellipsoid with a small central body in the peripheral portion of the inner segment of both and 207 208 HENRY LAURENS AND S. R. DETWILER a paraboloid in the slender cones. He could distinguish the cone nuclei from the rod nuclei by their more spherical form and larger size, but describes both as occupying a single layer. Tafani (’83) studied the retina of the crocodile, Champsia lucius, and found rods to be the predominant type of visual cell. In the anterior part of the retina there are practically no cones, but as the fovea is approached they become gradually more numerous than the rods. He found, unlike Heinemann, only one kind of rod. He describes the cones as being short, with a barrel-shaped inner segment, but with an outer segment similar to that of the rods, although his figures do not bear out his description. The differences between the nuclei of the cones and of the rods, which occur in a single layer, he considers too slight and inconstant to be considered as of any significance. Chievitz (89) described in some detail the pigmented epi- thelium and the tapetum. In the eye of Alligator mississip- piensis the tapetum extends through the entire upper half of the retina in the form of a bright band, reaching nearly to the ora, while its lower margin lies about 2 mm. above the entrance of the optic nerve in a 32-cm. specimen. In this bright band he found a fovea in the form of a very superficial, narrow furrow with thickened edges, and running horizontally across the entire tapetum about 1 mm. from its lower edge. He could not decide whether rods as well as cones occurred. In a vertical section of the eye of Crocodilus intermedius the tapetum is seen as a longitudinal bright stripe in the middle of the pigmented epi- thelium. In this region the middle part of the epithelial cells contain a number of fine, whitish, opaque granules of guanin, which when removed leave the middle portion of the cells color- less, while the choroidal and the vitreal portions contain melanin. The nucleus lies in the guanin-containing portion, directly against the basal pigment. At the margin of the tapetum black pigment is present in almost the entire cell; toward the middle, the vitreal pigment is gradually reduced and eventually exists only in the form of isolated, iregularly distributed small masses, between which the guanin comes to the edge of the cells. In the alligator the pigment in the choroidal portion of the cells is sparse and RETINA OF ALLIGATOR MISSISSIPPIENSIS 209 the nuclei are very close to the basal cell boundary. The pig- ment processes reach as far as the inner segments of the visual cells, the outer segments being deeply imbedded in the guanin- containing portion of the epithelium. Krause (’93) described the visual cells in the retina of Alligator mississippiensis and quotes from Hofmann’s description of the retina of Crocodilus vulgaris. In the alligator, Krause considers that the rods could be taken for small cones, because the slender inner segments are slightly tapering, while the outer segments are almost cylindrical. The inner segments of the cones, on the other hand, are thick and the outer segments short and pointed. Hofmann describes the rods of the crocodile as numerous except in the fovea and the surrounding regions of the retina. They are very similar to the red rods of the frog, and Krause reproduces a figure from Hofmann of a rod and cone. ‘The cones are single and double. Krause reproduces (again from Hofmann) cones with very long, pointed outer segments from the fovea. There are no rods in the area and only single cones, the inner segments of which become narrower as the fovea is approached. Accord- ing to Krause, the nuclei of the visual cells in Alligator mississip- piensis are all in one row, the cone nuclei being rounder than those of the rods. In the crocodile, Hofmann says that they occupy two rows, with the rod nuclei next to the external limiting membrane. Abelsdorff (98) considers that very strong support is given by the conditions in the reptilian retina to the view first put ‘forth by Max Schultze, that the rods serve for the reception of weak and colorless light stimuli. He ealls attention to the fact that most reptiles have practically only cones, the exceptions being the geckos (in some of which the cones seem to be entirely lacking), the crocodiles, and the boa. These are all nocturnal animals. The crocodile, he says, on account of its rod-rich retina, is not only capable of seeing in a very weak light, but can find its way about in pitch darkness, this property being enhanced by the light reflecting tapetum in the upper portion of the eye, the rods being thereby doubly stimulated. Abelsdorff points out that it is particularly in water that the upper part of the eye 210 HENRY LAURENS AND 8. R. DETWILER needs an increase in intensity of the light impression more than does the lower part of the eye, because the upper portion receives only what little light may be reflected from the bottom. He figures the tapetum in a sagittal section where it can be seen in the upper portion of the eye between the choroid and retina proper, going over in the lower portion of the eye, with a gradually increasingly thick black border, into the guanin-free, melanin-containing portion of the pigmented epithelium. At- tempts to demonstrate a change in position of this pigment in light and darkness were unsuccessful. It is interesting to note in this connection that Garten (’07, p. 89) considered it worth while to have this experiment repeated, which he did, with results (p. 108) substantiating those of Abelsdorff. Abelsdorft describes the rods of the alligator as being similar to those of frogs, but of narrower diameter. He found that the visual purple, investigated by direct observation of the opened eye as well as ophthalmologically (assisted therein by the presence of the tapetum), was not confined to the upper portion of the eye, but, by turning the retina over and looking at it from the visual cell side, could be seen as well in the lower portion. He investigated the bleaching of the purple in daylight as well as the relative amount of bleaching and the time relations upon exposure to light of various wave length. The fact that the purple was seen throughout the eye would seem to indicate that the rods occurred throughout the retina, although, if one chose to follow Edridge-Green, it might be assumed that the purple diffused into the regions where the rods were not present. Finally, Garten (’07, p. 109) describes the visual cells in Alligator lucius. In the upper part of the retina (guanin portion) there are large cylindrical rods only, which are surrounded in the light as well as in the dark eye by a mantle of guanin. This part of the retina is absolutely cone free. In the lower portion of the retina the visual cells are relatively small, possessing a very thin tapering outer segment, which in light as well as in dark eyes is buried in pigment. Garten considers these to be all cones. He refers to Abelsdorff as having described visual purple in the lower portion of the retina, and thinks, since he RETINA OF ALLIGATOR MISSISSIPPIENSIS Oat (Garten) has shown that there are only cones in this region, that this matter should be reinvestigated. The conditions in the alligator eye Garten uses in substanti- ation of his conception of the functional value of the migration of pigment in connection with the movement of the visual cells. Since he finds the two parts of the retina containing exclusively cones or rods, he argues that pigment migration therefore should not take place, because the perceiving power of the eye would not thereby be in anyway enhanced. Garten calls attention to the importance of the fact that the conical visual cells go over very gradually into those of rod form, and points out that it is exactly at this transition place that Chievitz localized the fovea. In two general reviews by Piitter (12) and Franz (13) the conditions found in the crocodilian retina are summarily given, but no new matter contributed. It should be recalled that Piitter is of the opinion that, although some of the elements in the reptilian retina may be cylindrical in form (that is rod-like), they are all nevertheless to be regarded functionally as cones, on account of their dendritic mode of connection with the bipolar . cells. METHODS The alligators, which were between 45 and 55 cm. long, were treated as follows. Two animals were placed in a dark room for twenty-four hours, at the expiration of which time one of them was removed to bright diffuse daylight for seven hours. ‘At the end of this time both were killed. The upper jaw, with the eyes, was removed with a pair of large bone forceps, bisected, and dropped into a large dish containing an abundance of Per- enyi’s fluid. The time required for this operation did not exceed thirty seconds, and in the dark was carried out in the weak light from a photographic lamp. The pupil of the allgator is vertical, and when the animals are placed in light. the aperture remaining after a few seconds’ exposure is but a mere slit. The pupillomotor reaction is so decisive, characteristic, and easily measurable, that experiments have been begun on the relative efficiency of spectral lights upon it. These will be reported later. | es HENRY LAURENS AND S. R. DETWILER The halves of the upper jaw containing the eyes were allowed to remain in the fixing fluid for an hour, in light or darkness, respectively, without being disturbed. After the expiration of this time, they were carefully dissected out and dropped into Fig. 1 Diagrammatic drawing of sagittal section of the epithelial pigment layer. The guanin is indicated by the lightly stippled, the melanin by the heavily stippled area; the choroid is shown by horizontal lines. X 11. fresh fixative, where they remained from four to five hours longer. Sagittal and horizontal sections 104 thick were made, stained in eosin and toluidin blue, iron haematoxylin and eosin, or in Ehrlich’s haematoxylin and eosin. All methods yielded good results. RETINA OF ALLIGATOR MISSISSIPPIENSIS PALL ANATOMICAL Epithelial pigment layer. A retinal tapetum occurs in the dorsal and posterior portions of the retina to within 1.5 mm. of the entrance of the optic nerve. It is formed by the inclusion of guanin in the cells of the epithelial layer. The relative amount and distribution of the guanin and the ordinary melanin is shown in figure 1. In the region designated by B the epithelium is relatively devoid of melanin, which forms a narrow border of a Fig. 2 A portion of the tapetum designated by the letter B (fig. 1), showing broad zone of guanin, a narrow vitreal border of melanin, and a few scattered needles of pigment near the choroidal margin. 665. Fig. 3 A portion of the epithelial layer designated by letter C (fig. 1), show- ing choroidal guanin-containing portion and vitreal melanin-containing portion. X 665. Fig. 4 665. few needles along the vitreal margin and occurs also as scattered needles here and there in the choroidal portion of the cells (fig. 2). As the optic nerve is approached, the amount of melanin grad- ually increases as the guanin decreases, until within about 1.5 mm. above the entrance of the optic nerve guanin is no longer found. The gradual increase in the amount of melanin as the optic nerve is approached is seen in figures 2, 3, and 4, which show in detail the condition as found at the levels B, C, and D in figure 1. Above the level B in figure 1 the guanin again gradually decreases in amount and the melanin shows a corre- sponding increase (level A, fig. 1). THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, NO. 2 214 HENRY LAURENS AND S. R. DETWILER The guanin is light grayish-brown in color (stained sections), finely granular and fairly uniformly distributed through the cell (fig. 2). In the tapetum (level B, fig. 1), the guanin-con- taining protoplasm, although covering over the outer segments of the visual cells, does not show the finger-like projections which so typify the melanin-containing portions of the epithelium (figs. 2, 3, and 4). In the lower portion of the retina the epithelial layer is entirely devoid of guanin. Here the melanin, in the form of delicate brownish-black needles, occupies the entire cell body and finger- like processes of the cells which project over and embrace the outer segments of the visual cells. The nuclei of the epithelial layer are spherical and occupy the choroidal portion of the cell body. Light and darkness have no effect on their shape and position. Visual cells. The retina of Alligator mississippiensis contains both rods and cones, differing in this respect from the retinae of turtles and lizards (Detwiler, ’16). The two kinds of visual cells in the alligator are not uniformly distributed, the cone-rod ratio changing in different parts of the retina. Histological examination of the retina has yielded the significant fact that no portion is rod or cone free and that there is no gradual transi- tion from the conical elements into rod-like forms, as Garten (07) claims. There are, however, areas which predominate in rods as well as areas which contain only a few rods. Viewing the retina as a whole, it can be said with justice that it is char- acteristically a red-retina. Rods. The structure of the rod is uniform throughout the retina. It consists of an inner segment composed of a cylindrical myoid and an ellipsoid and a cylindrical outer segment (fig. 5). No rods with conical outer segments could be found. The rod nuclei are typically oval in shape and le just beneath the external limiting membrane projecting above it for variable distances in both dark and light eyes, and thus form the outer part of the external nuclear layer. Cones. There are two kinds of cones, of which the predomi- nating type is a large thick visual element very similar to that RETINA OF ALLIGATOR MISSISSIPPIENSIS 215 found in the turtle retina. The inner segment consists of a short broad myoid, a broader refractive paraboloid, and an ellipsoid, while the outer segment is relatively short and conical (fig. 5). This type of cone is found particularly in the posterior and ventral portion of the retina. The second type of single cone (not very numerous), which is found only in the ventral portion of the retina, has a considerably longer myoid and a Fig. 5 A portion of the visual layer showing rods and large single cones. < 935. Drawing made at about 2.5 mm. above the entrance of the optic nerve (level C, fig. 1). Fig. 6 Elongated single cone and a neighboring rod taken from the ventral portion of the retina. X 935. Animal in darkness for twenty-four hours. Fig. 7 A double cone taken from the lower portion of the retina. Animal in darkness for twenty-four hours. X 935. Fig. 8 A double cone taken from the tapetal portion of the retina. Animal in diffuse light for seven hours. X 935. narrower paraboloid. The conical outer segment is long, slender, and pointed (fig. 6). The double cones consist of a larger and a smaller member. The former are very similar in shape to the large single cones. The latter is characterized by a long slender myoid and the absence of a paraboloid and has not been observed to occur singly (figs. 7 and 8). All cones lack oil drops, differing in this respect from those of the turtle and lizard. 216 HENRY LAURENS AND S. R. DETWILER Relative distribution of rods and cones. In the upper peripheral portion of the retina there are very few cones. In the region designated by A (fig. 1), the ratio of rods to cones is about 95:5. The cones in this region, mostly single and of the large type, are irregularly distributed. In the typical tapetal portion (fig. 1, B) they are shghtly more numerous (about 15 per cent). The majority of the cones, however, in this region are double, unlike those from the more peripheral portion of the retina. As the optic nerve is approached the number of cones shows a corresponding increase. In the region designated by C (fig. 1) TABLE 1!) DISTANCE FROM NUCLEUS TO OUTER SEGMENT IN DISTANCE FRO} Rents He DISTANCE FROM NUCLEUS |___ x TO PARABOLOID IN |DISTANCE FROM ELLIPSOID IN ROD NUCLEUS TO NEAREST PIGMENT Sieein Double cones NEEDLE IN Cones | Rods | Cones | Rods Paes =e Large Small IDEN. cols oanmee ISH We || Webs | Woes || OLS | Go) USO TLRS Tightens ecetes WD | eee) || WAIL | aly |) abe | eh | O55 9.9 IDISREAG. Ses aesl| Ze 20 | Beth | 2G | 2) oth |) Bee 1G 1 Total number of rod measurements, 280. Total number of cone measurements, 190. Total number of pigment measurements, 60. Measurements made at about 2 to 3 mm. distance from the entrance of the optic nerve, in the region designated by C in figure 1, with the exception of the measurements of double cones which were taken from region B. approximately 40 per cent of the visual cells are cones. Here the double cones are still more numerous than the single cones. In the guanin-free portion above the optic nerve (fig. 1, D) cones and rods are about equal in number, the single cones again exceeding in number the double. The cones outnumber the rods in the lower portion of the retina, the number increasing from the region of the optic nerve toward the ora serrata, and including all types. In this region only a few scattered rods are present. About 75 per cent of the cones in this region are single, the majority of which are like that shown in figure 5. The small type (fig. 6) is relatively scarce. RETINA OF ALLIGATOR MISSISSIPPIENSIS PALet The cone nuclei are easily distinguished from the rod nuclei; the former being somewhat pear shaped, the latter more or less oval or elliptical. Furthermore, the cone nuclei occupy a deeper level than the rod nuclei and constitute a second row. EXPERIMENTAL Effects of light: Rods and cones. When sections of eyes of animals which have been exposed to diffuse light are compared with sections of eyes of animals kept in darkness, it is seen that there is an average difference in the lengths of the rod myoid of 4u (table 1). The relative lengths of the rod myoids in the dark and light conditions are shown in figures 9 and 10, as well as TABLE 2 DISTANCE FROM ROD NUCLEUS TO ELLIPSOID (MYOID) IN Region 3mm from optic Tapetum (region B, nerve fio) IPT TSS st depeche cists tenacers eievores aston Gos oketien st S1oKs 11.5 8.9 1D aire ea A Go ree ee ae ees el de oe C25 6.9 DIET ETE COA Ee chs c cleus oiacusie apekelearteneteers 4.0 2.0 diagrammatically in figure 11. The change in the length of the rods is found to be less extensive in the tapetal area (fig. 1, B), where rods predominate, than in the region close to the optic nerve, where the cones are considerably more numerous (table 2). The effect of light on the cones is not so easily demonstrable. The results, however, of several series of measurements (table 1) show that the cone myoids of light eyes are slightly shorter (2.1u) than those of dark eyes (figs. 11, 12, 13). The contraction is found to occur in the double as well as in the single cones. The measurements of the double cones show that the myoid of the smaller member has shortened more than that of the larger (figs. 7 and 8). Further evidence of changes in the length of the visual cells in light and darkness is afforded by a study of the relative positions of the cone and rod ellipsoids. In the dark 218 HENRY LAURENS AND S. R. DETWILER condition (fig. 9) the ellipsoids of the single cones are usually found to be on the same level as the rod ellipsoids. On the other hand, in the light condition (fig. 10) the cone myoid is seen to occupy a level closer to the external limiting membrane than that of the rod ellipsoid. This change in relative position is the result of the combined effect of rod elongation and cone contraction, ad i > ae =o eae: Mack, 5S Eo ere eer) — Se HE 4 roe Fig. 9 A portion of the retina 3 mm. from the entrance of the optic nerve (region C). Animal in darkness for twenty-four hours. XX 9365. Fig. 10 935. Fig. 13 Single cones. Animal in darkness for twenty-four hours. XX 935. ellipsoid and a portion of the myoid is ensheathed. The amount. of cone contraction in the light, being only slightly more than the extent of the pigment migration (table 1), the relation between the position of the pigment and the cone ellipsoid is practically the same in both dark and light eyes, the choroidal portion of the cone ellipsoid being in both covered by pigment (figs. 9, 10, and 11). The description of the position of the pigment in relation to the ellipsoids of the visual cells in both dark and light eyes pertains only to conditions found in the posterior part of the retina (about 2 to 3 mm. from the entrance of the optic nerve). Changes in the position of the pigment in. 220 HENRY LAURENS AND 8S. R. DETWILER the more peripheral region of the retina could not be demon- strated. Near the margin of the retina the visual cells are greatly shortened and the pigment is found to extend down almost to the external limiting membrane. DISCUSSION Photomechanical changes of the retina. The question of the functional significance of pigment migration and the changes in position of the visual rods and cones in light and dark adaptation is one about which much has been written (Garten, ’07, and Helmholtz, ’11). It may therefore appear redundant to add anything in the way of a theoretical consideration of this function. But there are a few points which still lack clarity. In the eyes of those animals in which these changes take place they represent a mechanism for the adaptation of the eye to day and to twilight vision (Herzog, 705; Exner and Januschke, ’06). In dim light (twilight vision), when the rods alone are capable of being stimulated to any degree, or in complete darkness, the pigment moves back and leaves free the spaces between the rods, resulting in a less complete insulation of these elements. Under these conditions, with the entrance of a small amount of light the part played by the individual rods in the reception of the light is greater, owing to refraction and diffusion, than if the rods were covered over by a thick mantle of pigment, in which case only the light which passes through the retina in the direction of the long axis of the rods could enter them. ‘The presence of a reflecting tapetum further enhances the favorable conditions. The cones in twilight vision are not functional, on account of their high threshold, and they elongate and thus move out of the way. The rods contract and thereby optimum conditions are presented for their stimulation. In bright light (day vision) the pigment by migrating forward protects the rods, which have a low threshold and which have been made particularly sensitive by the accumulation of visual purple in the dark, from too strong stimulation by absorbing the direct and scattered light. The rods elongate, while the less sensitive cones are drawn out of the pigment by the con- RETINA OF ALLIGATOR MISSISSIPPIENSIS Pega traction of their myoids and are thereby made freely accessible to the stronger light stimulus, thus presenting optimum con- ditions for their stimulation. The fact that. these photomechanical changes have not been demonstrated in the eyes of man and mammals does not con- stitute a denial of their taking place in the eyes of other animals, and there is no ground against explaining as above the phenomena in such animals. Adaptation-of the mammalian eye is brought about by different means, viz., the formation and bleaching of visual purple. The process of light and dark adaptation is not dependent upon the phototropic movements of the elements of the retina, but these movements may take part in the process of adaptation, that is in the formation and bleaching of visual purple. That the pigment, as such, has anything to do with the formation of visual purple is not probable, because visual purple occurs in the eyes of albinos and in the pigment-free portions of the retina of many animals, for example, the cat. The significance of the pigment is probably a purely optical one concerned with the absorption of scattered light. In connection with the function of the retinal epithelium in the formation of visual purple, the paper by Kolmer (’09) is to be noted. Kolmer finds numerous droplets and granules on and between the rods in the retina of various vertebrates. These he regards as secretion products of the pigment epithelium. In the retina of frogs kept in darkness the droplets and granules are larger and more numerous than in the illuminated retina, and after illumination of the eye with direct sunlight are not to be seen at all. Since they are lacking in the eye of lizards and snakes, Kolmer assumes that they have something to do with the visual functions of the rods, the organs of twilight vision, and perhaps with the appearance of visual purple. It is interesting to note that pigment migration is still assumed by some to take place in the human eye. Ramon y Cajal (’11, p. 363) believes that the function of the pigment is to prevent the impression of halo, and that the dazzling sensation which one experiences on going from a dimly lighted place into a bright one Dao HENRY LAURENS AND S. R. DETWILER is due to the fact that the pigment, which has moved back into the body of the epithelial cells as an effect of darkness, requires some time to be brought out again into the prolongations of the cells to ensheath the individual visual elements. Bard (719), in a highly theoretical paper, in many respects offering views widely divergent from those usually held concern- ing vision of form and of color, explains many things by the assumption that both pigment migration and cone contraction take place in the human eye. Cobb (19, p. 444) also states that pigment migration takes place in the human retina. He says: aside from the changes in the size of the pupil there are two anatomical factors undoubtedly concerned in dark and bright adaptation: the exhaustion and regeneration of visual purple (or possibly other photo- chemical substance); and the migration of the pigment of the hexagonal cells. This last may be a protecting device that acts fairly promptly, and has the effect of enclosing the retinal rods, and by its own light- absorbing qualities reducing the amount of light absorbed by the indi- vidual rods. It is conceivable that asudden flash of light might antici- pate this action and produce a strong destruction of the photochemical material in a short time, before the pigment cells have had time to react, while with gradual onset of hight the time is adequate for the pigment cells effectively to assume this protective function. And on page 445: Some of the curves strongly suggest two factors playing a part in dark adaptation . . . . allowing the interpretation that the results are arising from two more or less independent mechanisms one of which overtakes the other in effect, at the end of about four minutes. We believe that in the migration of pigment, the contraction of the cones, and the elongation of the rods there is exemplified the response of irritable protoplasm to a definite, adequate stimulus. In some cases the response is very marked, though of varying degree (fish, frog, bird); in others it is not demon- strable (man and mammals). In some it may serve an easily comprehended purpose; in others, in terms of the theory explain- ing it, it may seem to be useless. Nevertheless, if it can be demonstrated (as in the turtle, lizard, and alligator) it cannot be explained away or ignored because it seems to serve no useful purpose. RETINA OF ALLIGATOR MISSISSIPPIENSIS 228 In the eye of the alligator the migration of the pigment and the change in position of the visual cells seem to be correlated with the relative distribution of rods and cones. The rods show the greatest difference in position in light and dark eyes, in the regions designated by C in figure 1 (rod-cone ratio 60 to 40), and by D (where the rods and cones are about equal in number), much less in the region B (rod-cone ratio 85 to 15), and not demon- strably at all in the region designated by A, or in the region (E and F) below the optic nerve, where the rods represent only about 5 per cent of the total number of visual cells. The pig- ment can be demonstrated to move forward only in the posterior portion of the retina (regions C and D), thus corresponding to the regions where the maximum change in position of the rods takes place. The cones throughout the retina show the same (slight) degree of shortening in the light, except that the double cones, which are most numerous in the regions B and C, show a slightly greater amount of change in length. Garten (’07, p. 38) weakened the general application of the suggestion put forth by Herzog (’05) and Exner and Januschke (06) by observations which seemed to show an extremely high sensitivity to stimulation by weak light, so that the light con- dition of the visual cells was considered as assumed in dim light. Arey (’19) has recently brought forward evidence indicating that the sensitivity of the retinal pigment and of the rods and cones is nowhere nearly so high as is generally believed, and the con- ception that the changes observed in those eyes where marked effects of light are obtained are adaptive has been thereby placed on much surer ground. We should not, however, a priori, deny that light effects similar, if less marked, changes in retinae so constituted that there can be no, or little, question of any advantage to be gained by a correlative shifting of the position of the visual elements. Garten cites the facts that in the selachians, which presumably have pure-rod retinae, although the literature on the subject is not without disagreement,! there is little, if any, pigment, and that 1 Schultze, ’66; Krause, ’76 and ’95; Neumayer, 97; Schaper, 99; Hesse, ’04; Franz, 705; Retzius, ’05; Garten, ’07; Cajal, 711, and Piitter, 712. 224 HENRY LAURENS AND S. R. DETWILER in pure-cone retinae, where the pigment, as assumed, is necessary for the absorption of the ight scattered by the highly refractive cones, there is practically no pigment migration. But Detwiler (16) found a demonstrable pigment migration and cone con- traction in the eyes of both turtles and lizards. Garten further argues (p. 109) that photomechanical changes should not take place in the crocodilian eye, because of the exclusive presence of rods or cones in the various portions of the retina. But, as we have demonstrated above, the structural conditions, at least in Alligator mississippiensis, differ from his description, and changes in the position of pigment and of visual cells do take place in light and darkness. The duplicity theory. The duplicity theory of von Kries, or the theory of the double retina of Parinaud, based on the findings of Max Schultze (’66), is of the greatest importance in compara- tive work on vision. The hypothesis is generally regarded as well substantiated, particularly by the facts of twilight and day vision. For brief accounts and references to the literature of the theory and its development the reader is referred to Nagel (05), Helmholtz (11), and Parsons (’15). Briefly stated, the theory holds that the rods are sensitive only to ight and darkness, and by virtue of their power of adaptation in the dark through the regeneration of visual purple they form the apparatus for vision in dim light. The cones, on the other hand, are the apparatus subserving bright vision as well as the perception of color. But in another way, the rods are the apparatus for achromatic scotopic vision (twilight vision), the cones the apparatus for photopic vision (day vision). The cones are not necessarily assumed to be utterly useless at night, but only rela- tively so, being quickly fatigued, on account of their high threshold. The presence and relative distribution of rods and cones is therefore a matter of the first importance. But without prej- udice it can be said that this is a very unsatisfactory matter as far as the comparative literature is concerned. Early contri- butions to the histology of the visual neuro-epithelium either have not been reinvestigated, but assumed to be correct, or * RETINA OF ALLIGATOR MISSISSIPPIENSIS 225 when investigated a divergence in the opinions of later investi- gators is the rule rather than the exception. The question as to what constitutes a rod and what a cone would seem to be a simple matter, but the great variety in the forms of the cells in different animals makes difficult a gener- alized classification. The contentions of Pitter (12) for a functional classification based on the type of connection of the visual cell with the bipolar, rather than a structural one, are good if kept within limits, but it seems to us that they are carried too far. As everyone knows, the foveal cones of man and mam- mals are cylindrical in shape, and are therefore much more like rods in general appearance than cones. But their known func- tion fits in with the general conception of the physiology of the apparatus for color and bright-light vision. ‘roland (’17) has pointed out that the shape of the foveal cones suggests that the function of the cone figure is structural rigidity rather than differentiation of response. Whether a visual cell is a rod or a cone is determined by the presence of one or more of three structural factors, viz., 1) the shape of the outer segment; 2) the shape of the inner segment, and, 3) the mode of connection between the visual cell and the bipolar cell. When we find visual cells which, from their general form (outer and inner segments), would be called rods, possessing terminal connections typical of cones (e.g., frogs, diurnal birds, see Ramon y Cajal, 94, pp. 31, 164, and ’11, pp. 340, 327), there is no, or very little, reason why they should be called cones simply because they terminate in dendrites, and considerable reason for continuing to designate them as rods on functional as well as structrual grounds. The conditions in the geckos may be cited as another example. From description and illustra- tion it would seem as if no more typical rods could be found. Coupled with their structure there are functional characteristics, which will be referred to later, and which, from all that we know about rod vision, indicate that the visual cells are as functionally typical rods as can be found. Our work on the retina of the alligator shows that rods as well as cones occur there, structurally as well as functionally, as exemplified in the inverse changes in 226 HENRY LAURENS AND S. R. DETWILER light and darkness. But Piitter would call them all cones because their centripetal termination is similar to that found in the cones of man. The designating a visual cell as a rod or as a cone on morpho- logical grounds is not therefore useless, as Pitter claims, but, as he also points out the structural basis (form of inner and outer segments), is brought into line with the functional by what we know of the respective functions of the visual cells in man, viz., threshold, visual acuity, ability to see movement, and vision of color. The rods are visual elements with a low threshold, but with possibilities of summated conduction, due to the connection of more than one of them with a single bipolar cell; the cones are visual elements with a high threshold and isolated conduc- tion, based on the histologically found type of connection. Pitter, in speaking of the conditions in the nocturnal birds, admits that the visual elements have knob-like endings, and that the visual cells are morphologically typical cones, although they have assumed what he regards as the most distinctive character- istic of rods. Pitter reverses himself here and is, as well, incom- plete, because, as Ramon y Cajal (’94, p. 104) points out, in these retinae there are rods ending like those of mammals, while the cones which have almost entirely similar endings, reach deeper and come into connection with a different set of bipolars, so that there is a further morphological differentiation here between rods and cones. It does not seem at all certain to us that Hess ('10 and ’13), by his work on the turtle and on birds, has disproved or weakened the general truth of the duplicity theory. He claims to have demonstrated an adaptation in the turtle retina, where there are cones only. The claim that he and Katz and Révész (13) make, that adaptation in diurnal birds is a function of the cones, does not seem warranted, owing to the fact that rods are present in considerable numbers, as Hess himself describes, particularly in connection with the presence of visual purple. ‘The phenom- enon, similar to the Purkinje phenomenon, which they state to have observed, may therefore, and most naturally, be a function of the rods and not of the cones. Katz and Révész RETINA OF ALLIGATOR MISSISSIPPIENSIS apa (13) also state that the rods of nocturnal birds (owls) in bright light approach, or are similar, in function to cones. But this is without anatomical foundation for the simple reason that the retinae of such birds contain numerous cones (Garten, 07; Hess, 713, p. 581) which show, with the pigment, photomechanical changes. In this connection the view of Parsons (15, p. 204) may be quoted: If we regard the rods as the more primitive type of visual neuro- epithelium, as we are probably justified in doing, the persistence of recognizable rod attributes in the cones, even if modified, differentiated, and rendered more complex, might well be expected. Apart therefore from the difficulties of isolating the physiological results of excitation of the rods from those of excitation of the cones it may be anticipated that the latter cells will retain some measure of the functions which are in the highest degree characteristic of their prototypes. Hence, if it should ever be conclusively proved that the rod-like foveal cones of the human eye possess some trace of visual purple and are endowed with some slight degree of light-adaptation it would not be surprising; neither, on the other hand, would it militate seriously against the view that the rods and cones have become essentially diverse in function. Troland (’16) has demonstrated by careful experiments, cor- roborating the earlier work of v. Kries and Nagel, that the phe- nomenon of Purkinje does not take place in the rod-free portion of the human retina. And Watson (715) and Lashley (16) show that Hess’ contention that the spectrum is shortened for the bird’s eye as compared with the range of wave lengths seen by the human eye, is not correct. It is not out of place to add that in the condition known as night-blindness, in which the rods are insensitive, or practically so, dark adaptation is almost abolished or is much slower than normal, and that Purkinje’s phenomenon is much less marked than in the normal eye or absent altogether. One further remark concerning Hess’ work. He (10) claims that many turtles are nocturnal and cites authorities supporting his contention. Ramon y Cajal (11, p. 361) says that reptiles in general (in which of course he is incorrect, witness the alli- gator and the gecko) do not see in darkness. Rochon-Duvig- neaud (717) does not believe that turtles can be called nocturnal because they are incapable of flight from an enemy or of pursuing 228 HENRY LAURENS AND S. R. DETWILER prey, and he thinks that they detect the plants and insects upon which they feed by the sense of smell. Rochon-Duvigneaud asks the very apt question in reference to Hess’ claim of adapta- tion in the fowl, why it is, if they possess the power of adaptation almost as well-developed as that of man, that they go to roost long before man ceases to enjoy good vision. With reference to the duplicity theory and the distribution of rods and cones, the work of Abney (16 and 717) is most interesting and important. By determining the minimum intensity of ight of various wave length which can be perceived at the fovea and up to ten degrees from its center Abney and Watson (716) obtained results indicating that in some cases the fovea of man is free from rods, which increase rapidly as the fovea is left, while in others there is a plentiful supply of rods at the fovea, their distribution, at any rate up to ten degrees, being very nearly uniform, and, if anything, in excess at the fovea. In the first group the light appears colored as long as it is visible at all, particularly in the green. In the second group the light loses color a considerable time before it is extinguished, except in the red. In other words, there is an achromatic inter- val. Abney (717) later examined persons suffering from night- blindness, in which the rods are generally believed to be non- functional, for extinction of color from the red to the blue. The light was found to vanish when its color was extinguished, so that the same reduction in intensity of the light was the threshold for both light and color, similarly to the cases men- tioned above where there are no rods at the fovea, indicating that there is an absence of sensitive rods in the whole retina of the night-blind. If dark adaptation is directly associated with visual purple the vision of an animal possessing rods only as compared with that of an animal with cones only, both in respect to ability to see in light and darkness, after longer or shorter adaptation to the one or the other condition, and as well the relative stimulating value of spectral lights of equal energy, should be expected to be markedly different, quantitatively as well as qualitatively. Now we have in the reptiles admirable subjects for investigating this RETINA OF ALLIGATOR MISSISSIPPIENSIS 229 very question, especially in the lizards, for example, the geckos as compared with other lizards and particularly the horned toad which has a retina, from all structural indications, peculiarly adapted for day vision only. By investigating in the gecko and the horned toad the relative visibility of wave lengths of equal energy and the relative powers of adaptation, we will obtain information concerning the question of the selective response of rods to different wave lengths as compared with that of cones. Visual purple absorbs all wave lengths except a little red and violet. The rods therefore are presumably sensitive to all wave lengths except the extreme red and violet. Since rods are in general ‘color-blind,’ there is opportunity here of differentiating between wave length and stimulating value. In connection with the question as to which kind of visual cell represents the more primitive condition, the histogenesis of the retina is worthy of investigation. But the histogenesis of the neuro-epithelium is a subject about which our knowledge is most imperfect. In general, the rods are regarded as the more primitive type of visual cell (Graham Kerr, 719), while the cones are considered as specialized rods. According to Leboucq (’09), the two kinds of visual elements develop simultaneously and are distinguishable only by the fact that the axis of the diplosome is perpendicular to the surface in the case of the rod and parallel to it in the case of the cone. Cajal (11, p. 356) says that the cones and rods evolve in the same way and that it is difficult to distinguish between them at the beginning (also Fiirst, ’04). Cameron (711) reiterates a view earlier championed by him- self (705) as well as by Bernard (’03) to the effect that the cones represent early stages in the formation of rods. We mention this here because of the fact that in looking over some slides of early amphibian embryos, the eyes do seem to contain nothing but cones or conically shaped elements. Graham Kerr (p. 137) finds that the visual elements (rods) in the retina of Lepidosiren shorten in the light and elongate in the dark, which is similar to to the usual behavior of cones and contrary to that of rods. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, NO. 2 230 HENRY LAURENS AND S. R. DETWILER The shape of the pupil is a subject of interest in a paper dealing primarily with the eye of the alligator because of the vertical slit form of the pupil in this animal. In two recent articles reference is made to this subject. Rochon-Duvigneaud (17), in listing the characteristics of the eye of the geckos which make them adapted to nocturnal vision, includes the form of the pupil—a vertical slit—which shows a rapidity of movement surpassing that of the human pupil and approaching that of birds. In dim light the pupil is a large oval, or even round, as in the cat. In bright light it is closed completely. According to Rochon-Duvigneaud, a round pupil can dilate as well as an oval one, but it cannot be entirely closed, and he believes that it is in the way of a protection against an excess of light in an animal adapted to twilight vision that an oval pupil finds its chief function. It is possible to imagine that a pupil in the form of a vertical slit can be opened wider than if it were round (for example, the cat). Hartridge (’19) views the function of a vertical slit pupil (as seen in the cat) from another angle, viz., the function of the lens and the aberrations caused thereby, and the habits of life of the cat family in the nature of their being tree-climbing and tree- dwelling animals which hunt their prey chiefly at night. An oval pupil in which the long axis is vertical will cause the lens system to form images in which the aberrations of horizontal contours are greater than those belonging to vertical contours. The contours of trees and their branches are principally vertical, therefore if the illumination of the image formed on the retina could be increased by sacrificing the definition of horizontal contours it would be an advantage. ‘This is effected by the use of the oval pupil since the aberration of vertical contours is little greater than that of a circular pupil of the same horizontal diameter, while the intensity of the image formed on the retina is as much the greater as the vertical diameter of the oval is greater than that of the circular pupil. It seems more likely to us that the function of a vertical slit- shaped pupil is for protection; that is, to permit of its being almost, if not entirely, closed. In thinking of animals that have RETINA OF ALLIGATOR MISSISSIPPIENSIS 231 this type of pupil, we find that many of them are essentially nocturnal in habits—the gecko, the alligator, the cat—animals in which the rod functions are predominant over those of the cones. At night or in dim light the pupil of the cat is wide open and round. Furthermore, in the daytime, when the pupil is a vertical slit or oval, cats hunt and catch a great deal on the ground, for example, birds and squirrels, as well as chase the rapidly swirling leaves. The cat is furthermore said to have very defec- tive daylight vision and to be colorblind (DeVoss and Ganson, 15). The alligator hunts along horizontal contours, and yet one finds that the shape of the pupil is a vertical slit. SUMMARY 1. The eye of the alligator possesses a well-developed retinal tapetum in the dorsal and posterior portions of the retina to within 1.5 mm. of the entrance of the optic nerve. It is formed by the inclusion of guanin in the cells of the epithelial layer (figs. 1, 2, 3, 4). The pecten consists of a slightly raised pig- mented cap covering the entrance of the optic nerve. 2. Typical rods and cones occur throughout the retina, the cone-rod ratio being different for different regions, but character- istic for particular regions (p. 216). 3. The rods are all of one type (fig. 5), the cones of two, thick and thin, of which the first is by far the more numerous, occurring particularly in the posterior and ventral portions of the retina. Those of the second type are found only in the ventral portion and are not numerous (fig. 6). Double cones also occur (figs. 7 and 8). None of the cones contain oil drops. The rod nuclei are oval or elliptical in shape, and the majority of them project through the external limiting membrane for a variable extent, the rest of them being just under it. The cone nuclei are pear shaped and, occupying a deeper level than the rod nuclei, con- stitute a second row (figs. 5, 6, 9, 10). 4, The rods show a change in length of their myoids averaging 4u, being longer in the light and shorter in the dark (figs. 9, 10, 11 and table 1). The single cones (thick and thin) show an average change of 2.lu (figs. 11, 12, 13 and table 1). The Dae HENRY LAURENS AND ‘S. R. DETWILER smaller member of the double cones shows an average change in length of 3.5u, the larger member of 2.7u (figs. 7, 8 and table 1). 5. The actual change in position of the pigment between light and dark eyes is slight, averaging but 1.64; but when combined with the change in length of the visual cells, gives an. effective migration equal to the sum of the two (figs. 9, 10, 11). 6. A theoretical consideration of photomechanical changes and of the duplicity theory from a comparative point of view is appended. BIBLIOGRAPHY ABELSDORFF, G. 1898 Physiologische Beobachtungen am Auge der Krokodile. Arch. f. Anat. (u. Physiol»), 8. 155-167. ABNEY, Sir W. DE W., anpD Watson, W. 1916 The threshold of vision for dif- ferent colored lights. Philosoph. Trans. Roy. Soc., vol. 216A, pp. 91- 142. ABNEY, Sir W. pe W. 1917 Two cases of congenital nightblindness. Proc. 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CG ey eet wor hahha Fuh: eg sia it : & me “seal seas sant Baas eos eee is Riso IL 2) ais algal | hari ; lone Oye Ha) ry) has Ue A ti Mey abe soe | Ai daar sbi Py 4 Ry Vethuges a ; a a ph tin nis Pee. | wis ah eeate ihe sani Disa Spiny Lyte Fey “wi eag plane etiviny ois eolat anh ices 7 “tebiberih ey cone ne aM LG ee ae wh Ne: eat —- aly + he oben! i Ob id Hoe di : dogs, ye a y: Bp aA is babisauemany i thiths anna agheb 9-0 ee nae armeione lye by. eared ab rT ati] any igs ay Le ie d ia Shang. aa dtp 3 crt HA’ Leisy aah oe . ie: Arn pig a host “A se ide evi oe Dipys ae ai “bp ante adh semis tha fay: oth sii By .ar Fs | Me yi inn ey ret eed 2 pine he oh sta 0 Pe ve Bey POA a th ang HEO) a9 | ab 2 a yg Nad a Net ; oT 4 onus as aieanciiis, t a Ns she natit itil ay sane HY, Gly ip hes wt ang tanual’ Tomi hye irs lie: 78 is 3 ith gy: elatio” bah sl daly Se nse ae oh 0 ‘ . y, Be Po | THE SELECTED Cc B & rs PAIR GAVE e |g : . | ee al a g 4 Smulrae F, | 28 TU) TY) Ay a Gh e3] hl a ay 4271 ,575,27 .11| 56.3,21: 98=21.43 F. x 97| 381/25 .46 1a || Ae 18) a 5| 8] 4] 5] 1] 2} |1 ,087|8 ,760)27 .58/139 .3 34:189=17 .99 F, x 206) 787|26.18 F; | 56) 1 1 3| 7| 9} 5| 9/11} 4] 2} 2} 2/2 ,188/8 660/25 .27|154 .6)44:210=20.95 F x 197} 920,21.41 F, | 45/10 if LG Aa 7) Ay 2) 2 905/4 ,234/21 .37| 94.1) 9:104= 8.65 Fs x 125} 612)/20.42 Fy | 438/74) 1) 2 3] 5} 4) 7| 3) 4 478/2 ,899 16.49) 67.4) 0:131= 0.00 Fio x 0; 53) 0.00 Fi 5| 6 0} 87} 0.00} 17.4) 0: 12= 0.00 Fie x 2| 148) 1235 Per cent of crossovers e) 28 26 24 22 20 18 16 14 12 Series B 10 8 6 4 2 0 1 3 5) 7 g ata 13 15 Ati 19 21 23 P43) 27 2 Generations Fig. 1 Series A, A’, and B, low selection 340 J. A DETLEFSEN AND E. ROBERTS recourse to at least two methods of dealing with such unpro- ductive pairs. We can either include in our frequency distri- bution only those females on which we have ample data to give a somewhat reliable crossover value and ignore all pairs giving less offspring than a fixed minimum (fifty individuals for, example), or we can simply include all females and thus withhold no data. The latter course seemed preferable and we have followed it. There were five pairs showing lower crossover values than the one we selected, as follows: 10.0; 12.5; 16.0; 16.6; 20.7. We did not always select the lowest absolute value, for in many cases this was based upon an insufficient number of offspring. It was also necessary to keep fertility in mind, in order to insure the perpetuation of our selected line. This explanation will make clear why we could not always choose the lowest absolute crossover value in the frequency distribution of any given generation. In table 1 the italicized frequency in each distribution shows the relative position and value of the selected pair. No dispersion can be given for the Fs, Fu, Fe, etc., since these represent en-masse matings. An x represents the point to which the progeny of the pair selected in the preced- ing generation regressed. The average number of offspring per pair shows how reliable the crossover values usually were in this experiment. The crossovers, total, and the crossover value for each selected pair are also given in the last column. Those generations which have any number of pairs entered under that heading are generations in which all matings consisted of pairs, while the other alternating generations were en-masse matings. Since the crossover value of a female may be based upon a small number of offspring in some cases, and thus give an apparently wide deviation which has little significance, we have not calcu- lated the variability of each generation in this paper. For example, a female showing a crossover value of 10 per cent based upon twenty offspring might well show 30 per cent if one hundied and fifty offspring had been secured, since age and changing temperature affect crossing over; or she might even show 10 per cent based upon twenty offspring as a sheer fluctuation of sampling. EFFECT OF SELECTION ON CROSSOVER VALUES 341 The first two selections seemed to show little or no effect. Although the values of the selected pairs were low, their progeny regressed practically to the parental average. Possibly this means that all wide deviations were not necessarily due to genetic causes and that we had difficulty in distinguishing between wide environmental variates and wide variations due to genetic causes. Selection was thus effective only when by chance we chose a wide variate due to the latter set of causes. For example, in the F;, we chose a female showing 17.99 per cent cross- overs, but her progeny gave an average of 26.18 per cent. After the F;, progress was very rapid. The F, gave 16.49 per cent, and the Fi, to Fi; gave about 0 per cent. These last generations in this series were based upon small totals, because the excessive heat (90° to 100°F., day and night) for long-continued periods reduced fertility to a minimum and eventually annihilated our stock in this one. However, series A’, which was derived directly from series A, gave just as low crossover values with larger numbers and under better conditions. We may be quite sure that temperature was not the cause of low crossing over; for, if we may anticipate, series B showed effects of selection under normal temperature conditions. Series A’, low selection; derived from series A In the F; generation of series A, two selections were made. One female (2 14) gave 9:104 = 8.65 per cent, and a second female (2 10) gave 1:91 = 1.10 per cent. The former was used to continue series A, while the latter was used to begin a new series, A’. Table 2 and text-figure 1 give the main facts pertaining to series, A’. We began this series to insure keeping alive some of the low crossover material of series A during con- tinuously hot weather. Our facilities did not permit controlling temperature, and the whole experiment was in a precarious situation during the early summer months of 1916. We found that mating a number of females en-masse assured more progeny than the same number of females mated in individual bottles— evidently because the larger number of larvae carried the yeast through the culture and kept molds down. Hence, during the 342 J. A DETLEFSEN AND E. ROBERTS summer months of 1916, we made numerous en-masse matings in this series to insure keeping the stock alive. Beginning with a single F; pair of series A showing 1:91 = 1.10 per cent, the new series A’ was run for nine generations. All generations were en-masse matings except F, and Fy, in which paired mat- ings were made to ascertain what the crossover values of the individual females might be in this line. In the F, the average crossover value for the total population was 8:397 = 2.02 per cent. The wide dispersion in this generation does not carry TABLE 2 Series A’: derived from series A THE DISTRIBUTION OF CROSSOVER GENERA- NUMBEE) VALUES IN EACH GENERATION cet ere || Are CROSSOVER Paes 4.5 @.5 | 16-5 | 13:5) 16:5 | 19:5 I, 1 1 91 1.10 Fs x 1 86 1.16 Fo 18 iA | 1 1 8 397 2.02 Fio x 0) 61 0.00 Fi x 0 133 0 00 Fis eel 4 373 1.07 Fi3 x 9 1,473 0.61 Fis 25) 25 | 10 2p25e 0.44 Fi; xX 0 289 0.00 EIR te Fee Rs Sots a ae od Sere IE AR Sin 33 5 ,156 0).64 1See text. much weight because cultural conditions were poor and fer- tility was low. Pair no. 4, for example, gave 3:15 = 20 per cent, but such a pair might well give a much lower crossover value with a larger number of offspring. The Fi; gave 10:2253 = 0.44 per cent, and the numbers are large enough to be significant. This generation included twenty-five pairs which gave a total of 2:977 = 0.20 per cent, and an en-masse mating which gave 8:1276 = 0.63 per cent. There can be no doubt but that an original crossover value of 33 per cent has been changed by selection, at least, that a marked change has followed selection. For nine generations the stock bred true to about 0 per cent crossover. The totals for series A’ were 33:5156 = 0.64 per cent. EFFECT OF SELECTION ON CROSSOVER VALUES 343 Series A’, like series A, was eventually lost in the latter part of the summer of 1916 because of an unavoidable succession of events. We regretted the loss of this stock because we had hoped to make a genetic analysis of the last generations in an attempt to learn what was taking place during selection. How- ever, the data as they stand indicate that crossing over is not a very stable phenomenon and that it can be rather easily modi- fied. We surely cannot concur in Morgan’s (’19) view that crossing over ‘‘gives numerical results of extraordinary con- stancy.” We immediately began a new selection experiment, hoping that we could duplicate the results of series A and A’. Series B; low selection Series B, like the preceding series A and A’, began with the mating of a single white miniature female and a wild red long male. In fact, as a prelude to series B, we made eighty such paired matings, for we had found some non-disjunction in our original stocks and in series A and A’. Since non-disjunction theoretically lowers the percentage of crossing over (Bridges, ’16), we wished to assure ourselves, if possible, that this cause might not be operative in producing low crossover values in our selection experiment. Of the eighty white miniature females tested we found eleven giving either matriclinous daughters or patri- clinous sons or both. This must mean secondary non-disjunc- tion in the white miniature stock, for the exceptions were -too numerous to be considered primary. We chose white miniature 2 53 mated to a wild male as the foundation pair for our experi- ment, because this pair gave fifty-two wild-type daughters and seventy-eight miniature white sons. While they showed no exceptions, it does not prove that 2 53 may not have been non- disjunctional (XXY), for a ratio of 0: (52 + 78) might well occur as a chance ratio where an average of 4.3 per cent of excep- tions is expected from XXY females (Bridges, ’16). However, in the present paper we are concerned only with the question whether selection based on variable crossover ratios can be effec- THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, NO. 2 344 J. A DETLEFSEN AND E. ROBERTS tive. Whether non-disjunction has any necessary relationship to our result will be discussed in a subsequent paper. TABLE 3 Series B: low selection nm | THE DISTRIBUTION OF CROSSOVER VALUES 5 IN EACH GENERATION z 3 2D |UD | ad | 2D | Ad | 1D] 29 | 29 | ag | 29 | 19 | 20 | ap | 19 ° | at ES) Sah Sh Se Pa ee ess Sai) iS) F, 34 3] 5} 3/10) 6} 3} 2| 2'2 ,056 F, 1 129 F3 | 47 HUE 1) 9] 5} 8} 7] 6} 6} 2) 1/2 ,204 F, 1 330 1B Hokey al) al at Al19/17| 5/19}11) 2 5,798 F, itil al 379 Bie | ola: 1} 7) 4) 4/18/18) 9) 8)-1) 1) |5,490 Fs iM Ulett) I 650 Fy | 88 1|12/12|13|17|13} 9| 3] 2 1/3 ,230 Fio i) SAP PAN AL 595 Fu | 84 8) 6)13/24/14/10)10) 3) 1 | {1,896 Fie 8) 3) 1) 1 815 Fi3 | 63} 3} 9/13|16)10} 6) 5 759 Fiy Lies 1 1 149 Fis 1 68 Fig | 79} 5) 7/22)29)12) 2) 1) 1 1,416 Fy7 6| 1 642 Fig | 74) 6} 5/28/22) 9) 1) 2) 1 1,195 Fi5 3] 9] 4| 2 975 Feo | 52] 4} 5)12|16) 9} 5} 1 641 Fo | * 1) 5) 6 1,108 Foo | 67) 3) 6)15/22/12) 7) 2 1232 Fo3 il Ap 4 1 392 Fes | 47/10)13)16) 5) 3 431 Fo; TNT e/a aL 223 Foe | 38} 5)13)12) 6} 1 1 280 Foz Le 65 Fes | 46/10) 8/12] 7] 6) 3 215 Fag Q| 10 ATION Totals 7,189 » 530 8 ,089 1,141 23 ,618 1,411 21,974 2,858 17 ,550 3,502 11 937 4558 7,439 790 609 14 ,765 6 027 12 166 8 ,786 6 533 11,407 1 717 3,649 6,801 3,119 3,903 1,152 2 650 158 THE TOTAL POPULA- TION IN EACH GENER- Crossover values 28 .60 24.34 25 .02 28 .92 24.55 26.86 24.98 22 74 18.40 16.99 15.88 17.88 10.20 18.86 7 9.59 10.65 9.82 11.10 9.81 9.71 10.51 10.74 6.34 7.15 rds 5.64 8.11 AVERAGE NUMBER OF OFFSPRING PER PAIR L749) Os 144.7) 0: 39: OPAs7/\) “We 43: 6.33 211.4) 61: THE SELECTED PAIR OR SELECTED EN MASSE GAVE 219 2a 129: N74. Na br Aaye 330:1141 =28 .92 288.0} 48: 296 :1130=26.19 360.0) 36: 217:1185=18 .31 Pea! ities 191:1179=16.20 LADEN eo: a i tala PLSE A abe Do: 68: 186.9} 12: 122:1009=12 .09 164.4] 4: 47: 125. 6|) 20: 199:2027= 9.82 580= 24 .34 645 = 27.13 230= 20.87 251= 14.34 276= 6.16 96= 5.20 512=12.89 197= 7.61 207 = 12.08 609=11.17 169= 7.10 77= 5.19 464=10.13 179=11.17 63= 1.59 768= 6.64 107= 0.00 454= 8.59 141= 4.26 873= 4.93 39= 0.00 : 129= 6.20 The F,; sibs from @ 53 were mated in thirty-four pairs and gave as a whole a crossover value of 28.60 per cent (table 3). EFFECT OF SELECTION ON CROSSOVER VALUES 345 In order to further test our foundation stock, the F, offspring of 2 25 (one of the eighty Pi ? 9, and similar to 2 53) were tested en-masse and gave 1142: 3553 = 32.14 per cent. The F; offspring of several other F; 2 2 were mated en-masse and gave 830: 2923 = 28.40 per cent. All of these facts indicate that our foundation stock was quite normal with respect to crossing over and gave crossover values of the same general magnitude as those ordinarily used in plotting maps of the sex chromosome. . The main facts pertaining to series B are given in text figures 1 and 2 and in tables 3 and 4. Table 3 was constructed in the Per cent of crossovers 14 12 10 § Series B 6 ae 2 0 SU aliSdow So RST DGD ere Al SaqreeSinh “40s 249 Generations Fig. 2. Series B, continued, low selection same way as table 1, with the following exception: in series B, records of the en-masse matings of the offspring from several promising pairs were kept and the crossover values of all these are put in the form of a frequency distribution, but the italicized frequency shows the position of the en-masse mating which was derived from the pair eventually selected to continue the experi- ment. The italicized frequency in the distribution of the pairs likewise shows what the value of the chosen pair was. The first three columns at the right of table 3 give the data for the total population in each generation.’ The average number of offspring per pair shows that the fertility was high and selection was based upon what seemed to be adequate numbers. © The last column gives the number of crossovers total, and crossover value 346 J. A DETLEFSEN AND E. ROBERTS of the selected pair in each generation and the same data for en-masse matings from these selected pairs. Text figures 1 and 2 give a graphic representation of the progress made in series B. The graphs are based upon the crossover values in the selected line; i.e., ali en-masse matings except the selected one have been neglected in plotting the graph. In other words, the graph relates only to the actual line of selection, and all side lines have no weight in determining the coordinates. It will be clear that those generations in table 3 which have any number of pairs entered under that heading were generations made up entirely of paired matings, while all other generations were en-masse matings. ; The first three selections had little or no effect, but it cannot be said that selection was very rigid during these generations. In F, we selected a pair giving 36:251 = 14.34 per cent and made some progress, for the next seven generations (F;—Fy,) fluctuated between 10 per cent and 23 per éent. The subse- quent nine generations (I'j;—F.3) fluctuated around 10 per cent. Selection was carried on up to Fs, and the last six generations (F..-F2,) varied around 6 per cent. After that we simply carried the stock without selection, and have found it to breed quite true to low crossover for twenty-two generations. The F.5—-F 59 have given values around 6 per cent. These last twenty- one generations are shown in table 4. There are some features of tables 3 and 4 which require com- ment for the sake of clearness. Temperature conditions made it necessary to breed the offspring of the selected pair in the Fi; for two generations by the use of en-masse matings. Hence, the matings in the F,, and F,; show no pairs and selection was interrupted. This was the only case in which the usual sequence of selecting in alternate generations was not followed. The Fs; showed a rather abrupt rise in crossover value (12.50 per cent), 4 An independent mutation of gray to yellow which occurred in the F2; should perhaps be put on record. One female (9 no. 30) proved to be heterozygous for yellow, and this gene was linked to white and miniature. Hence the mutating gene came through the spermatozoon from the gray white miniature father of 2 30. This new gene for yellow proved to be identical with the original yellow mutation found by Wallace in 1911 (Morgan and Bridges, 716). EFFECT OF SELECTION ON CROSSOVER VALUES 347 which was without doubt due to high temperature, as our records indicate. The fertility was low and we obtained with much effort from en-masse matings in the F3, and F3; only forty-eight and eighty individuals, respectively, while under ordinary con- ditions several thousand would have been possible. As soon as normal conditions were restored, the usual low crossover values were again found. The Fu showed a rather unexpected rise TABLE 4 Series B—Continued GENERATION CROSSOVERS TOTALS CROSSOVER VALUES Big ; 6 144 4.17 Fa Vf 171 4.09 Fis 4 48 8 33 ie 10 80 12.50 Lora 52 ‘ 643 8.09 Ris 48 1,147 4.18 Fe 55 1,032 5.33 By; 46 814 5.65 Bye 39 697 5 60 F 39 55 954 5 77 By, 72 1,074 6.70 Bi 94 1,015 9 26 Be 463 8 ,564 5.41 we AT 901 5 22 Ba 103." 1,312 7 85 Fis 43 661 6 51 By 59 992 5 95 Faz 69 1,021 6 76 ie 45 734 6.13 Ba 81 1,081 7 49 Fo 96 1,375 6.98 (9.26 per cent), but since there were no unusual temperature conditions, we must regard this somewhat higher value as with- out peculiar significance. The subsequent generations dropped to about 6 per cent again. ? 348 J. A DETLEFSEN AND E. ROBERTS Series C; high selection In the F, generation of series A, pair number 21, was chosen to begin a high-selection series, series C. While this series was carried for only eight generations, and then discarded in order to devote time to the other series, nevertheless brief mention should be made because the results may aid us in interpreting series A, A’, and B. We were not able to make progress in selecting upward, as the averages of table 5 show. (Table 5 was constructed in the same way as tables 1, 2, and 3.) On the contrary, we were much surprised to find that in the F, generation a number of pairs suddenly dropped to very low TABLE 5 Series C: high selection GENERATION NUMBER OF PAIRS CROSSOVERS TOTALS CROSSOVER VALUES Fi 28 427 1,575 27.11 F, 162 512 31.64 F; 35 1,407 4,842 29 .06 F, : 436° 1,355 Some ia 90 6 465 21,071 30.68 Fe 684 2 267 30.17 F, 72 3,893 13 ,705 28.41 Fs 296 1,298 22 .80 crossover values; in fact, much lower ratios than one would find in any ordinary population such as our original stocks or our F, of table 1. The distribution of the F, in series C is given in table 6. It will be noted that nine pairs gave values lower than 6 per cent. Their values were as follows: . 4:72 = 5.56 5:279 = 1.79 9:164 = 5.49 1:142 = 0.70 1:82 = 1.22 0:123 = 0.00 4:104 = 3:85 2:40 = 5.00 0:49 = 0.00 Total, 26:1055 = 2.46 EFFECT OF SELECTION ON CROSSOVER VALUES 349 There can be no doubt that these crossover values are signifi- cantly different from any ratio in the F; in table 1, or from the usual ratios shown by random stock females. Furtherrnore, there is an interval of about 10 per cent between the lower and higher groups of table 6, in which we found no crossover values. The natural inference is that any attempt to increase the amount of crossing over leads to double crossing over, and thus to very low crossover values (practically zero). That is, these nine females showed a marked decrease in crossover values, despite high selection, because they gave almost nothing but double crossovers. In other words, their low crossover values are, after all, the result of:effective high selection. -Mr. L. E. Thorne, who had this series under observation, was called into military service and we did not make any further tests on this material. TABLE 6 The distribution of crossover values in the F; generation of series C THE DISTRIBUTION OF CROSSOVER VALUES SSHOEAEGSTE: » | ec DM ie a SS ER ae ae OF PAIRS | 19/19] 15 CROSSOVERS TOTAL ai[ale CROSSOVER VALUE 72 5| 4 1 1| 6| 8/15/16} 8) 6} 1) 1) 3,893 13 ,705 28.41 We hope, however, to repeat the high-selection experiment and test out the region between white and miniature in such females which apparently give uniform double crossing over in a region in which single crossing over is the rule. DISCUSSION AND SUMMARY As far as we are aware, there is only one record of a similar selection experiment. Gowen (719) selected for high and low’ crossover values, but his results and conclusions are diametri- cally opposed to ours, since: he found selection ineffective, and concluded there were no differences in modifying factors for crossing over in his experiment. He continued selection for only five generations in the low series and six in the high, using the region of the third chromosome between sepia and rough. 350 J. A DETLEFSEN AND E. ROBERTS While it is possible that this chromosomal region may fail to show the same phenomenon which we found in the sex chromo- some, we are rather inclined to believe that the difference between our results and Gowen’s is more likely due to differences in the method of procedure, for Gowen states that his ‘“‘chief difficulty lies in the few individuals that it was possible to include in a given generation.”’ Gowen gives only the mean total crossing over in each generation, and we do not know how rigid his selec- tion may have been, for he does not state how many pairs were included in each generation nor does he give the frequency dis- tribution for crossover values. We suspect that he found the same impediments in using strict brothet-and-sister matings which we found and which prompted us to use en-masse matings in alternate generations to increase our numbers. We are carry- ing on selection experiments in other regions of the sex chromo- some and in the autosomes, which should decide whether other regions and chromosomes are similarly affected by selection. We have no reason to suppose that they will not be. The effects of selection upon crossover values may be due to one or a number of causes, some of which suggest themselves almost immediately. It would hardly be profitable to expatiate on these, since we are making tests, which we hope may indicate what has really happened in the course of selection. Briefly stated, we think of the following possible causes which may have been operative in modifying our crossover values: 1. We may perhaps have dropped out a large part of the chromosome between white and miniature, thus bringing these two genes closer together. We can probably disregard this as a cause, for although ‘deficiency’ reduces crossing over (Bridges, 17) nevertheless the lethal action of deficiency would be seen in a disturbed sex ratio. We found no such disturbance. 2. Is it possible that we may have moved the locus of the genes on the chromosome? This would mean that the locus of a gene is not permanently fixed, but that a given gene is found in a characteristic position in the majority of cases. If we have done this, and at the same time have not moved other genes, then linkage tests should disclose this fact, for the order of the genes would be changed. EFFECT OF SELECTION ON CROSSOVER VALUES 351 3. In series A and A’ we found much evidence of non-disjunc- tion. Bridges (’16) stated that XXY females should logically show a decrease in crossing over, because heterosynapsis takes place in about 16.5 per cent of the cases and precludes crossing over in these cases. However, Bridges also showed that the experimental results disagree with such an expectation, for cross- ing over was not decreased among the regular sons of XXY females, but as far as the evidence goes it was slightly increased. For some time we labored under the impression that much, if not all, of our decreased crossing over was associated with the presence of non-disjunction (Detlefsen and Roberts, ’20). We are now rather inclined to believe we were in error. It should not be a difficult matter to free our low crossover stock in series B from non-disjunction and thus dissociate this possible cause from the others. We could in this way demonstrate that non- disjunction was only accidentally present in our experimental material and that our results are quite independent of non-dis- junction. 4. Have we reduced the frequency of the usual single ‘chromo- some twist’ between white and miniature to a minimum? Wein- stein’s (718) results indicate that crossing over takes place in the sex chromosome with about forty-six units as the modal distance between successive crossovers. Similarly, Gowen (719) found twenty-five to thirty units in the case of the third chromosome. We began with two genes which were about thirty-three units apart, and which should therefore show a-single crossover as the characteristic or modal number. This would mean that in series A, A’, and B we have practically eliminated the usual single crossover in this region, while in series C we were on the way to increasing it to two crossovers (i.e., a double crossover), which would give us no crossing over as far as these two genes were concerned. Does this mean that we can decrease or increase the amount of ‘twisting’ which members of an homo- logous pair of chromosomes may show, and which is supposed to be responsible for crossing over according to the chiasmatiype theory? If selection can accomplish this, then we may reason- ably suppose that numerous hereditary modifying factors are 352 J. A DETLEFSEN AND E. ROBERTS present in a general population and are the basis and cause of this variable chiasmatype relationship. If this explanation is correct (and we are inclined to believe it the most plausible one of those we have suggested here), then we cannot escape a marked change in our view-point on crossing over and related phenomena. If, for example, all of the difference between prac- tically no crossing over in our series A and A’ and normal cross- ing over (33 per cent) is due to numerous modifying factors, then we naturally begin to wonder just what part ‘distance between two genes’ on a chromosome may play in determining linkage values. Our current view is that ‘‘the percentage of cases in which two linked genes separate (amount of crossing over between them) is necessarily proportional, other things being equal, to the distance between the genes,” (quoted directly from Weinstein (’18)). » But evidently the percentage of crossing over is a vari- able which is the expression of different possible combinations of multiple modifying factors; hence the percentage of crossing over cannot be proportional to the distance if the distance remains uniform. For example, in series B we find 6 per cent crossing over, and so we should conclude that the distance in this stock is 2/11 or 18 per cent of what it was when we began selection! Thus, to maintain our original position, we must conclude that the percentage of crossing over and distance are correlated variables, if the proportion between the two is to remain reasonably constant. We then naturally begin to wonder what has happened to all of the distance (and the genes) between 0 and 33 in series A and A’ where crossing over has been prac- tically eliminated. In view of these considerations, it would perhaps be simpler to conclude that the percentage of crossing -over is not necessarily proportional to the distance. ‘The dis- tance may remain fairly constant, but the amount of crossing over (i.e., twisting of the chromosomes) will depend upon numer- ous hereditary factors. One recalls in this connection Goldschmidt’s (17) suggestive paper in which he postulated variable forces that hold genes to their customary loci on the chromosome and which allow an exchange between allelomorphs in a certain average percentage EFFECT OF SELECTION ON CROSSOVER VALUES 353 of cases. While we cannot subscribe fully to this theory for cogent reasons advanced by Sturtevant (717), Bridges (’17), and Jennings (718), nevertheless Goldschmidt’s proposed theory would not appear entirely. supererogatory, for a crossover value is apparently a variable and the variation is due to or controlled by multiple hereditary factors. A cross between low crossover stocks and the original population, and testing out a large num- ber of F, segregates should throw the desired light on this ques- tion. Unpublished data indicate that segregation in crossover values does take place as one would expect on the basis of the multiple-factor explanation. 5. May we suppose that we have been taking advantage of small mutations in the nature of modifying factors arising during the course of selection? While this is possible we are inclined to doubt it, for favorable mutations evidently do not take place in the direction of selection as readily as this view would imply (ef. Muller and Altenburg, 719). The following conclusions may be drawn from the data of this paper: 1. Crossover values are very variable and part of this vari- ability is due to genetic causes. 2. Low selection has been effective in two entirely independ- ent series, A and B. 3. The low crossover stock bred true to about 0.6 per cent (almost zero) for nine generations in series A’ (derived from series A). 4. The low crossover stock bred true to about 6 per cent for twenty-two generations in series B. 5. High selection probably induces double crossing over, as shown by series C. 6. Crossing over in the various regions of the sex chromosome (and other chromosomes?) is probably controlled by multiple incompletely dominant factors. 354 J. A DETLEFSEN AND E. ROBERTS LITERATURE CITED Brivces, C. B. 1915 A linkage variation in Drosophila. Jour. Exp. Zodl., vol. 19, pp. 1-21. 1916 Non-disjunction as proof of the chromosome theory of heredity. Genetics, vol. 1, pp. 1-52, 107-163. 1917 An intrinsic difficulty for the variable force hypothesis of cross- ing over. Am. Nat., vol. 51, pp. 370-373. 1917 Deficiency. Genetics, vol. 2, pp. 445465. DetLersEN, J. A. AND Roperts, E. 1920 Variation in the percentage of cross- overs and selection in Drosophila melanogaster. Anat. Rec., vol. 17, p. 336. : GoupscuMipT, R. 1917 Crossing over ohne Chiasmatypie? Genetics, vol. 2, pp. 82-95. Gowen, J. W. 1919 A biometrical study of crossing over. On the mechanism of crossing over in the third chromosome of Drosophila melanogaster. Genetics, vol. 4, pp. 205-250. JENNINGS, H. S. 1918 Disproof of a certain type of theories of crossing over between chromosomes. Am. Nat., vol. 52, pp. 247-261. Moraean, T. H., AND Bripaes, C. B. 1916 Sex-linked inheritance in Droso- phila. Publ. Carnegie Inst. Wash. D. C., no. 237, pp. 1-87, 8 fig., 2 pl. Moraean, T. H. 1919 The physical basis of heredity. J. B. Lippincott Co., Philadelphia and London. 305 pp., 117 fig. Mutuer; H. J., anp ALtTENBURG, EX. 1919 The rate of change of hereditary factors in Drosophila. Proc. Soe. Exp. Biol. and Med., vol. 17, pp. 10-14. PuioueH, H. H. 1917 The effect of temperature on crossing over in Droso- phila. Journ. Exp. Zool., vol. 24, pp. 147-210. Sturtevant, A. H. 1917 Crossing over without chiasmatype. Genetics, vol. 3, pp. 301-304. 1919 Inherited linkage variations in the second chromosome. Pub. Carnegie Inst. Wash. D. C., no. 278, pp. 305-341. WernsTEIN, A. 1918 Coincidence of crossing over in Drosophila melanogaster (ampelophila). Genetics, vol. 3, pp. 135-172. Me a’ : -) ' ‘ > ~ hs 4 « ate M f : = as THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, NO. 3, APRIL, 1921 ; d i \ Resumen por la autora, Ruth B. Howland, Osborn Zoological Laboratory, Yale University. Experimentos sobre el efecto de la extirpacién del pronefros de Amblystoma punctatum. Las condiciones que siguen a la extirpacion bilateral del pro- nefros de Amblystoma punctatum demuestran claramente que estos O6rganos son necesarios para la vida del embrién. Todos los embriones desprovistos de rifones cefdlicos presentan debili- dad cardiaca y edema, muriendo al cabo de doce dias. La doble extirpacién de los tibulos pronéfricos no afecta al desa- rrollo normal de los glomérulos. La presencia de un solo pro- nefros es suficiente para mantener la vida del animal. Después de las operaciones unilaterales el resto del pronefros presenta una marcada hipertrofia, aumentando un 100 por ciento el Area de la superficie secretora; el contenido ctbico de la masa de células 63 por ciento, y la longitud de los tubulos 21 por ciento sobre lo normal. El conducto segmentario procedente del é6rgano hipertrofiado posee un dimetro medio mucho mayor que el de cualquiera de los dos conductos del animal normal. El] riién hipertrofiado también presenta indicios de una pequefa cantidad de hiper- plasia, puesto que el nimero de nucleos presente en él es 16 por ciento mayor que el normal. En el lado operado los glomérulos se desarrollan normalmente, y los embudos anterior y posterior pueden regenerar a expensas del epitelio celbmico. La condicién del conducto segmentario varia considerablemente. En _ los casos extremos esta representado solamente por una pequena masa de células degeneradas. En los embriones en que se ha extirpado el rudimento del corazén en un estado muy temprano del desarrollo, el desarrollo inicial de los glomérulos es normal, pero pronto se altera a causa de la enorme distensién de los vasos sanguineos. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, MARCH 14 EXPERIMENTS ON THE EFFECT OF REMOVAL OF THE PRONEPHROS OF AMBLYSTOMA PUNCTATUM! RUTH B. HOWLAND Osborn Zoological Laboratory, Yale University TWENTY-THREE FIGURES CONTENTS PANEL GBC DION Ear re cee 5 2G aco clases Veet oote ers oie Maven, AP erGee neptta eam ra soe A, nl sesoee cia aye 355 Materialemethods, and, normal/deyelopment. 4.2. +25ss66- 0642006650046 oe 361 Bilateralexcision of pronephric rudiments. 2. 2226-65. Ss foc de lees 365 Mod Ckoio PebatlOlen: ah Yosrgs eau see ot a oS elas hit ceca 365 Mieetrorbilateraleremovale. 4-4 elke: kee COE Ee ees Oe oe em eta 366 Unuilatendleexcisionsol pronephricudiments. +. 4 eee cess oe oe eee 371 Postoperative effect on the embryo as a whole....................... 342 Effect of unilateral excision on the remaining Prancohnas: Peony ole Effect of unilateral excision on the glomerulus........ ae Od Effect of unilateral excision on the other components of nics Sy ae hsitky Oud Effect of removal of the heart on the development of the glomerulus. ..... 381 SUM MAryaaind TeONClUISION Sey revises rs seis eat lei os le ee eo ochre a action 382 Wer a tUTeV ChE yori s nara ascrs wn. tro 5 hycuil sicce SoS ERI Aordeco aes eee ss 384 INTRODUCTION The common occurrence, among the lower orders of verte- brates, of a more or less persistent head kidney or pronephros has led to the accumulation of a very comprehensive literature on this subject from the standpoint of pure morphology. Little evidence has been furnished in any group, however, of the role which these organs play in the life of the embryo. Price (710), working on the head kidney of one of the myxi- noids, Bdellostoma stouti, a form in which it persists throughout adult life, follows up his earlier descriptions of the development 1 A preliminary report of the results obtained was published in 1916. (How- land, R. B.,’16. On the Effect of Removal of the Pronephros of the Amphibian Embryo. Proc. Nat. Acad. Sc., vol. 2.) 309 356 RUTH B. HOWLAND of the excretory system with a study of both the structure and the function of this organ in its fully formed state. The head kidney of Bdellostoma is shown to be a composite structure, possessing at its earliest appearance all the characteristics of a pronephros, with the single exception of the typical glomerulus, but later fusing with the anterior end of the mesonephros and losing all connection with the exterior. Structurally, then, the head kidney in at least one of the myxinoids is rendered incap- able of playing the réle of an excretory organ, but since it is connected with both coelomic cavity and the circulatory sys- tem, and since, also, it has been proved possible to transfer certain substances from the coelomic fluid directly into the cir- culation, Price concludes that its probable function is the trans- ference of lymph from the body cavity into the blood-vessels. Since the discovery of the organ by Johannes Miiller (29), the origin, development and morphology of the amphibian pronephros have been described by many investigators, chief among whom are von Wittich (’52), Fiirbringer (’78 a), and Field (91). An excellent review of the early controversies concerning its structure is also given by Field. ‘The presence of a well- developed pronephros in the amphibian embryo, its early appear- ance, and its relatively large size have led to the general assump- tion that it is a functioning organ. Its characteristic structure, consisting in a glomerulus which extends freely into the coelomic * cavity, a coiled tubule furnished with open ciliated funnels for the intake of coelomic fluid, and a simple duct establishing direct connection with the exterior, further points to its function as excretory in nature. Still, from the physiological viewpoint, no experimental evidence as to the extent to which the embryo — is dependent upon it for the elimination of excretory products had been offered at the time of publication of my first note (’16); Since then Swingle (19), working independently upon the embryo of Rana sylvatica, has obtained results which in the main agree with my own. In the series of experiments described in the present paper, the necessity of these organs for the life of the embryo has been proved by the fact that death follows, in time, after the removal REMOVAL OF PRONEPHROS OF AMBLYSTOMA aon of both pronephroi. The uniform occurrence of a pronounced edema after bilateral extirpation, similar to the condition which follows certain pathologic conditions in the permanent kidneys of the higher animals, suggests a further parallel between the larval kidneys on the one hand and the permanent kidneys on the other with respect to their function. Extirpation of the coiled portion of one or both pronephroi has also afforded the opportunity of investigating the question of correlation in develop- ment through a study of the effect of its removal on the other components of the excretory system. ‘The response of one kidney in cases where it has been left functioning alone has further led to the consideration of the factors involved in the restoration of the normal secreting area through the process of compensatory hypertrophy. Although no invariable rule can be formulated as to the type of regulation which may be anticipated as a consequence of the abnormal conditions imposed by extirpation of an embryonic region or organ, a survey of the results obtained in the many instances already investigated shows that, in a large proportion of cases, there occurs a more or less complete regeneration of the excised part. Byrnes (98 b) and Harrison (18) have shown, for instance, that the limbs of the amphibian embryo, if removed at an early age, will soon be replaced through the regenerative capacity of the surrounding tissue. ‘This is also true of the audi- tory and nasal placodes, the lens, and the gills. The possession by the amphibia, of the regenerative power to such a high degree would naturally lead to the presumption that removal of the pronephric rudiment might result in a similar replacement of this organ. This, however, as will be shown later, is not the case in the Amblystoma larva, for the adjustment consequent on removal of the pronephros is not in the nature of a restitution, but is a compensating hypertrophy? of the remaining head kid- 2 A peculiar instance of compensatory hypertrophy in another organ is cited by Kochs (’97) in his work on Triton, where the amputation of the fore leg often resulted in a marked hypertrophy of the tail. Retardation or acceleration in the growth of a fore leg in the larvae of Rana esculenta and Bufo viridis may be induced by removal of a hind leg, according to Kammerer (’05), the hastening or arrest of growth depending on the rapidity of wound healing. 358 RUTH B. HOWLAND ney and of its duct—such compensation as is common in the adult kidney of the higher vertebrates. The degree of com- pensation which has been attained by the single kidney has been estimated in terms of increase in the secreting surface of the pronephric tubules, as well as by measurement of the volume of the cells making up the walls. Although the literature on the subject of hypertrophy in the kidney of the higher forms is too extensive to permit of a dis- cussion in any detail in the present paper, it may be well to mention several of the more important standpoints from which the subject has been treated. The question of direct causal connection between the demand for increased functional activity and the changes in the com- pensating organ has been definitely settled by such experimental studies as those of Sacerdotti (96), in which the kidneys of unoperated dogs were stimulated to compensatory overgrowth by injection of the blood from completely nephrectomized ani- mals. In this instance, as in all the early pathological literature dealing with this subject, the exact nature of the histological changes evoked in the stimulated organ was given only second- ary consideration. Recently, however, with the general accep- tance of the distinction between the terms hypertrophy and hyperplasia, more accurate observations of the condition of the kidney constituents consequent on increased activity have been made. Both forms of regulation may occur in the same organ, each being limited to a definite area. Wolff (00), in his con- tributions on the macroscopic and microscopic conditions of the hypertrophied kidney after resection, draws a sharp distinction between those changes which occur in the region of the lesion, and those occurring in the uninjured portions of a resectioned kidney. In the former location he observes that mitoses appear at the end of two days, resulting in the formation of new epithe- lium. In the uninjured portion of the remaining kidney tissue, however, no new formation of either urinary tubules or of glo- meruli takes place as compensation for those excised, but here a sufficient restitution occurs through increase in size, and the normal balance is restored. From the histological viewpoint, REMOVAL OF PRONEPHROS OF AMBLYSTOMA 359 this process is sure to be almost entirely one of hypertrophy, not a hyperplasia, of the kidney elements. In the glomerulus and urinary tubules the former process occurs exclusively, in the epithelial cells themselves, hypertrophy with a negligible amount of hyperplasia. The clearest and by far the most accurate statement of the exact histological conditions found in the hypertrophied kidney is given, however, by Galeotti and Villa Santa (’02). These authors approached the problem from a widely different view- point, their main object being to determine whether the hyper- trophied kidney of an adult animal would show the same histo- genetic potency as that of an animal which had not attained its full growth. From their study of the kidneys of young and adult dogs and rabbits, careful estimates were made of the num- ber of glomeruli found in sections of normal and hypertrophied kidneys and of the relationship between the average surface area of the glomeruli and the number present. Furthermore, the diameter of the lumen in the tubuli recti was accurately measured and the secreting surfaces in the two kidneys obtained for comparison. The volumes of the cell walls in the two cases were also computed and contrasted. The thoroughness of the methods used in these computations gives added weight to their conclusion that, whereas in the young kidney hyperplasia may occur, the adult kidney has lost its potency for addition of new parts, and can only respond by the enlargement of those elements already present. Kittleson (’20), in his recent report on the effects of inanition and refeeding on the growth of the kidneys in young rats, con- firms the opinion of former workers that starvation inhibits the formation of renal corpuscles, and further concludes that ‘‘refeed- ing after stunting results not only in a hypertrophy of the renal corpuscles but also in.an increase in number, which may even exceed the normal.’”’ This would indicate the possibility of both hypertrophy and hyperplasia of the same kidney element, induced by these abnormal conditions. No instances are on record of experiments dealing with the production of hypertrophy or hyperplasia in the adult am- 360 RUTH B. HOWLAND phibian kidney, although Levi (’05) claims that the anuran mesonephros has, in one species, the power of regeneration after injury. He destroyed both the urogenital anlage and that of the wolffian duct and tubules in Bufo larvae by means of a red-hot needle, and obtained after a certain time complete regeneration of the excretory organs. His method is, however, open to the criticism that no accurate estimate of the actual extent of the injury done to the organs by this type of operation can be made. The introduction of a hot needle may cause only minor displacement or destruction of a few cells of the duct or coils. . Certainty as to the degree of injury can be assured only by complete excision. Removal of a portion of a given organ or system has been found to have a varied effect on the formation of its other constit- uents. ‘The extent to which the growth of one part is influenced during its development by other developing portions of the embryo varies widely, the scale of difference ranging from com- plete interdependence to those extremes in which each con- stituent possesses the potency for self-differentiation without the influence of any formative stimuli. In view of this variation in the degree of correlation exhibited by closely related parts during their early growth, it was of interest to consider, particularly in cases of bilateral extirpation of the pronephros, the effect of the absence of the tubules on the forma- tion of the glomeruli. The operated animals showed without exception a differentiation of glomerular tissue perfectly normal in position and size. The glomerulus therefore possesses the power of self-differentiation, and is entirely independent of the presence of the tubular elements. It gives me great pleasure to acknowledge my indebtedness to Prof. R. G. Harrison, at whose suggestion this investigation was begun, for his helpful and constructive criticism during the course of my work. REMOVAL OF PRONEPHROS OF AMBLYSTOMA 361 MATERIAL, METHODS AND NORMAL DEVELOPMENT Embryos of Amblystoma punctatum were used for all the experiments. The stages chosen for operation varied from the condition in which the first loop of the pronephric tubules appears as a slight, ventrally directed curve of the duct (fig. 1, stage 30)% to that in which the two funnels, together with the first loop, appear as a broadened Y (fig. 2, stage 32). In all cases embryos were used before contraction of the body muscles began, as movement not only hindered the operation, but often tore open the wound after successful removal of a kidney. Figs. 1 and 2 Embryos in the stages used for operating. PR, pronephros located below the third and fourth myotomes. Figure 1, earliest stage (stage 30); figure 2, latest stage (stage 32). Anaesthetics were unnecessary, and the slight motion due to the ciliated epithelium was controlled by holding the animal in the field with an operating needle. The body tissues in these early stages are easily distinguished from each other through slight differences in pigmentation, and, in addition, are so loosely bound together as to allow removal of the pronephric mesoderm without dislocating the cells of contiguous regions. In a few ‘Instances portions of the somatopleural layer ventral to the pronephric rudiment were included in the tissue removed, result- ing in retarded development, in abnormalities, or even in total absence of the limb on this side (Harrison, 718). 3 See Harrison, R. G., 718, Jour. Exp. Zodél., vol. 25, no. 2, p. 417, footnote 9. 362 RUTH B. HOWLAND The general methods employed in operating are so well known that no detailed description of them is necessary here. The special technique required in removal of the pronephros and in the construction. and measurement of the models will be described in later sections. The pronephric swelling is one of the earliest and most clearly defined of the developing organs. Its position may be accu- rately located at a stage not long after the closing over of the neural folds, when the first eight pairs of muscle plates may be seen, and, like these, it differentiates in an anteroposterior direction. It is found in the region immediately underlying the 3 Figs. 3 and 4 Embryos showing the first looping of the tubule, due to rapid growth of the cells just posterior to the nephrostomes. Figure 3, a—a, original axis of the pronephric rudiment. Figure 4, b—b, direction of first bend of the tubule. third and fourth myotomes as a bulbous thickening tapering posteriorly into a short thickened ridge. Operations at this period, although having the advantage of not interfering with developing nerves or blood-vessels, are inadvisable, since the mesoderm is still so compact that excision results almost invari- ably in the removal of more than the pronephric rudiment. In succeeding stages, the delimitation of the segmental duct pro- gresses, and the original bulbous enlargement becomes pitted in in two places on its coelomic border, establishing the nephros-- tomal openings into the anterior and posterior funnels. These two openings lie opposite the midline of somites three and four. At the same time the tubule, which originally lies along the longitudinal axis of the body just below the muscle plates (fig. REMOVAL OF PRONEPHROS OF AMBLYSTOMA 363 3, a—a), lengthens rapidly in the region of the fourth myotome, and bending outward and downward in an acute angle over the upper surface of the yolk (fig. 4, b—b), forms a U-shaped loop. A little later this is bent over anteriorly, and may even come to lie shghtly farther forward than the anterior nephrostome. The pronephros of Amblystoma differs from that of certain of the Anura in the possession of two instead of three nephros- tomal canals,‘ and in the absence of the common chamber or ‘pronephric pouch,’ the funnels, instead, narrowing directly Figs. 5 and 6 Diagrams showing the region where greatest growth occurs in the early and late stages of development of the pronephros. Figure 5, condi- tion before the coiling of the longitudinal tubules connecting the funnels. Fig- ure 6, growth of these tubules in the older kidney. a.f., anterior funnel; p.f., posterior funnel; c.t, longitudinal tubule; s.d. segmental duct; x-y, region of great- est growth during early development resulting in the formation of the ventro- lateral portion of the pronephrie coil, p.c. Shaded areas drawn from wax models, pronephric coil indicated by curved lines. into the U-shaped tube just described. With further multi- plication of the cells just below the funnels, two longitudinal tubules are established (figs. 5 and 6, c.t., and fig. 17, L.T.), separating the anterior and posterior nephrostomes from each other and from their original point of junction with the looped tubule (x), as they grow. This growth is at first a very slow process as compared with that of the U-shaped portion. In the latter region the active proliferation of cells results in a rapid * One instance is on record of the presence of a third funnel on both sides in an Amblystoma larva. See Field (’91). 364 RUTH B. HOWLAND coiling of the tubule, the early increase in size of the kidney being limited mainly to this region, between the connecting tubule and the proximal end of the segmental duct (figs. 5 and 6, x to y). This eventually forms the ventrolateral region of the fully formed organ, which will again be referred to in con- nection with the discussion of edema. In still later stages® the nephrostomal canals and their connect- ing tubule also elongate, and are thrown into loops and folds (fig. 6, l.¢.), retaining their dorsal position and extending slightly laterally over the coils already formed. This portion may then be termed the dorsolateral region, as contrasted with the ventrolateral portion already mentioned. That part of the tubule which is a direct continuation of the segmental duct never becomes strongly convoluted, but retains its original posi- tion along the ventrolateral boundary, slanting obliquely toward the dorsal surface over the kidney from the anterior margin. Minor folds may occasionally occur along its course. Increase in growth is outwardly evidenced by a more and more pro- nounced swelling in the pronephric region. Operated speci- mens may be easily distinguished from normal animals, even after healing is complete, through the absence of this thickening on the operated side. Posterior to the pronephric coils, the segmental duct extends backward along the body just below the ventral surfaces of the muscle plates. The junction between pronephric coils and the proximal end of the segmental duct is always in the immediate region of the posterior funnel. With the subsequent downgrowth of the myotomes the formation of the shoulder-girdle and anterior limb buds, the pronephros becomes partly covered, and comes to lie deeper in the body, and nearer the midline of the embryo. The edges of the myo- tome also extend downward over the segmental duct, making its removal in this stage extremely difficult. ® Excised kidneys in the older stages may be slightly stained, and the capsular nuclei, thus made visible, removed. The tubules may then be easily uncoiled for observation in water or weak alcohol. Oil is an unsatisfactory medium, both for examination and preservation, as it not only increases the brittleness of the tubules, but renders them too transparent for clear definition. REMOVAL OF PRONEPHROS OF AMBLYSTOMA 365 Parallel with this development, although not appearing at so early a stage, the rudiment of the glomerulus is formed. This first appears as a thickening in the opposite or splanchnic wall of the coelom, extending over an area as long as the distance between the anterior and posterior funnels. Vascular cavities soon appear, and at an early stage these become continuous with branches of the dorsal aorta. The arterial supply is derived directly from this source; the venous supply from the postear- dinal vein, which enlarges in the region of the pronephros, form- ing a large venous sinus, into which the fully developed kidney projects. The tubules are thus continually bathed in the blood returning from the posterior part of the body (Field, 791). BILATERAL EXCISION OF PRONEPHRIC RUDIMENTS Mode of operation The first experiments consisted in the removal of the proneph- ric rudiments on both sides, to test the functional necessity of these primary organs in the life of the embryo. In the largest proportion of cases, a period of several hours, or even a day, was allowed to elapse between the removal of the right and left pronephros. However, the two excisions may follow each other immediately without incurring serious results. Sharp-pointed needles, inserted in glass rods, were substituted for the more generally used iridectomy scissors. Controls were kept under identical conditions of light, temperature, water, etc. ‘lwo methods were employed in removing the pronephros. In the first, three straight cuts were made, one beneath and one along each side of the pronephric swelling. The flap of ecto- derm thus defined was loosened from the underlying mesoderm, and the organ removed from below. In the second and more satisfactory method, a single incision was made, dorsal to or immediately over the thickening, the tubule raised upward from below, pulled outward, and excised. In loosening the nephros- tomal surfaces of the funnels, as much of the tissue in a dorso- median direction was removed as seemed possible without disturbing the splanchnopleuric wall, since in this region the glomerulus normally arises. 366 RUTH B. HOWLAND Larvae in which the triple-incision method was used healed much more slowly than those to which the second method was applied, since contraction of the cut edges left a gaping wound much greater in extent and permitted more oozing of the yolk. On the other hand, when only one cut was made, a critical inspection of the excised tubules was necessary to make sure of total removal. In the majority of cases the incision is entirely healed at the end of an hour and a half. : Effect of bilateral removal Most conspicuous of any of the postoperative conditions resulting from bilateral excisions was a pronounced edema, par- ticularly in the anteroventral region. It is well known that edema of the amphibian embryo commonly occurs as the result of a variety of causes. Narcotized embryos reared in a solution of acetone-chloroform (chloretone), though structurally normal in other particulars, show a slight edema and pericardial effusion as a result of weakened heart action (Harrison, 04). More pronounced abnormalities result when early developmental stages are exposed to the heat of direct sunlight, or may be induced experimentally by exposing the embryos to radium rays (O. Hertwig, ’11). McClure (719), in his recent work on edema in anuran larvae, draws attention to the ‘‘less extensive tubular complex which normally occupies a dorsolateral position in the pronephros, and into which the nephrostomal canals directly open,” and the ‘‘tubules which normally constitute the greater portion of the kidney and which occupy a medial and ventral position.”” From a study of the histological conditions existing in edematous frog larvae, he concludes that there is a functional as well as a morphological difference between these two regions, for in all of the embryos in which edema had become apparent, the ventrolateral tubules were either entirely absent or but poorly developed. From this he argues that deficiency in the ventrolateral tubules alone may be the cause of edematous conditions. REMOVAL OF PRONEPHROS OF AMBLYSTOMA 367 In bilaterally operated Amblystoma larvae, the swelling which first appeared in the region of the wounds progressed gradually forward, the pericardial cavity soon becoming enlarged (figs. 7 7 Figs. 7 and 8 Section and entire sketch of embryo, from which both kidneys had been removed, to show pericardial effusion. Figure 7, section through peri- cardial (p.c.) region. Figure 8, camera drawing of embryo, showing swelling in the heart region. Fig. 9 Section through an embryo from which both kidneys had been removed. Both glomeruli (gl) are present, extending out into the enlarged coelomic cavities (c); A, aorta. and 8). Later the fluid caused a pronounced distention of the abdominal cavities (fig. 9), and in extreme cases the gills also became swollen and distorted. Slowing or entire absence of circulation accompanied this condition and sloughing of the 368 RUTH B. HOWLAND ectoderm was not infrequent. Microscopic examination of sections through edematous embryos showed the tissues of the body to be in various stages of degeneration. Pressure of the accumulated fluid often forced the intestine ventrally or to one side and the fibers of the muscle plates were separated by large vacuoles. The muscle fibers themselves also became vacuolated, and in extreme cases the whole region was reduced to a spongy mass of irregular fibers with scattered nuclei. Although the splanchnopleural mesoderm which gives rise to the glomeruli had been left intact during the operations, the question nevertheless arose as to whether normal conditions of development would obtain for these organs in the absence of other parts so closely allied with them. Sections made through the operated region in embryos killed four days and six days after double excision showed capillary tufts extending out into the much-dilated coelomic cavity (fig. 9, gl). The tubular region cannot, therefore, be considered to exert any influence in the nature of a formative stimulus on the development of the glomeruli, since these parts of the system arise quite inde- pendently. Furthermore, the presence of the glomeruli in these ~ operated cases would tend to strengthen the view supported by McClure (19) that the glomerular filtrate, given off directly into the coelomic cavity, collects here in excess, producing the typical edematous condition already described. ‘These embryos also showed well-developed anterior and posterior funnels, extending laterally into the regions from which the tubules had been removed, and ending blindly there. Segmental ducts were present, in some cases the lumina being flattened dorsoven- trally, in other instances the cells of these tubules showing marked signs of atrophy. The condition of the funnels and segmental ducts after extirpation of the kidney will be dealt with more fully in connection with the question of unilateral removal. Efforts were made to bridge over the interval between the operations and the beginning of functional activity of the meso- nephros. The first means applied was that of pricking the body wall as soon as abnormal distention was evidenced. ‘The larvae REMOVAL OF PRONEPHROS OF AMBLYSTOMA 369 were immersed in 0.4 per cent NaCl in an attempt to balance the loss of essential salts through the escape of the glomerular filtrate. Although slightly stimulated heart action resulted, probably due both to the stimulus of operation and to relieved pressure in the pericardial cavity, only temporary benefit was derived in this way, for, with the accumulation of new fluid, the former pathologic condition was restored, and death followed after a short interval. It is quite possible that if a sufficient number of experiments were made, varying the constituents of the solution in which these larvae were kept, a satisfactory medium might be found for prolonging the life of these animals. A determination of the optimum salt percentages of such a solu- tion has not yet been undertaken. The second means employed was the transplantation of the pronephric rudiment to the region of the mesonephros. It was by this means possible to test the capacity for reestablishment of function, through union with the segmental duct. In a series of thirty embryos, the right head kidney, without the ectodermal covering, was placed under the skin farther back on the same side. Twenty-four hours later, the left pronephros was removed from those embryos which had responded well to the first opera- tion and were apparently recovered. The transference and proper orientation of the kidney in these operations was easily accomplished and the wounds healed entirely in the usual short time, but the general edematous condition common to embryos on which only the bilateral excision had been performed sub- sequently developed. With the exception of a few which were preserved and sectioned at the end of a week, all of this series died within twelve days, showing no indication of resumption of function by the transplanted tubule. The transplanted tubule still retained its identity, although as a general rule the cells were pressed together in a solid mass, and only in a few instances a distinct lumen was visible. Removal of the pronephros resulted here in the partial or total atrophy of the segmental duct, to which attention will be called in greater detail below. How- ever, in cases where the transplanted tubule had been placed in the immediate region of the segmental duct, no connection was 370 RUTH B. HOWLAND restored between these two components, nor did the duct, posterior to the transplant, give any evidence of functioning. A slightly different method was applied in a third series of experi- ments. The pronephros was not alone transplanted, but was taken together with the overlying ectoderm, the surrounding mesoderm, and even small portions of the ventral myotomal walls. This transplant was procured from another animal, and was transferred into a previously prepared incision and held in place until healed. On the next two successive days the left and right pronephric rudiments were removed. No appreciable a, a s'p| YY? sD a Fig. 10, Aand B- Diagrams to show the location and extent of operations made in removal of different segments of the embryonic pronephros. In A the seg- ment removed, x, was a part or the whole of the U-shaped tubule anterior to the segmental duct. In B the segment removed, x, consisted of a part or the whole of the rudiment of the funnels. difference was noted in the ensuing condition of this series, and itis safe to conclude that under these circumstances the excised tubule is unable to readjust itself and function in its new location. Interruptions to the development of the pronephros by a less radical operation also go to strengthen the belief that the regen- erative capacity of the kidney tubule is either very limited or very slow in taking place. In a number of embryos from which one pronephros had been extirpated, a small portion of the opposite rudiment was also excised. The segments removed (2) were at two levels, as designated in figure 10, A and B. The funnels were undisturbed in one group (A), a short piece of the ® This does not apply to the coelomic epithelium which lines the nephrostomal opening, as will be shown in a later section. REMOVAL OF PRONEPHROS OF AMBLYSTOMA 371 first loop being removed, while in the second group (B), the funnel rudiment was cut off just anterior to the first bend. Of the twenty specimens used, all exhibited symptoms identical with those induced by bilateral excision. The extent of regeneration which would occur in a defective tubule if the opposite kidney were allowed to remain intact is still a matter for investigation, but it is not improbable that a given portion of a tubule may possess a prospective potency which would insure the restoration of an excised section. How- ever, in dealing with an organ where the demand for functional activity follows so closely upon this disturbance of normal con- dition, the requisite time for readjustment may be the factor lacking. Accumulation of excretory fluid may inhibit the regen- eration which might be the normal consequence, if excretory activity were maintained by an undisturbed kidney. A funda- mental difference thus places excision of the kidney in a category apart from the majority of regeneration or transplantation experiments upon the amphibian embryo which have been reported up to the present, for the effects consequent on extirpa- tion and transfer of limb rudiments, optic vesicles, or nasal pits, though abnormal, are not of a nature to interfere with any of the vital functions of the embryo. UNILATERAL EXCISION OF PRONEPHRIC RUDIMENTS Conclusive evidence having been obtained as to the essential nature of the pronephros in the life of the embryo, a further study of the correlation of the development of this organ with that of the other components of the excretory system was then undertaken.?7 Unilateral excision of the pronephric rudiment served.as a practical means to this end. The technique of operating has already been discussed in the previous section, but a word of explanation is necessary regard- ing the controls used in this series. Since a more or less pro-. nounced retardation in growth was the unavoidable consequence 7 As has been previously stated, the glomerulus was found to develop normally even in the absence of the pronephrie coil. ote RUTH B. HOWLAND of such operations, the controls were always more advanced than the operated embryos, so that, for the comparison of the excretory organs, others had to be selected as described below (p. 373). Postoperative effect on the embryo as a whole Every outward evidence of successful readjustment to the new conditions imposed was shown by the operated larvae. Adverse symptoms, such as edema and general sluggishness, were absent, and, barring the slight retardation already mentioned, normal progress continued, except in those cases in which the limb bud was disturbed or entirely removed. The ectodermal surface which showed a slight concentration of pigment in the - initial stages of wound healing gradually became indistinguish- able from the surrounding regions, and differed from the opposite side only in the absence of the distention caused by the under- lying pronephric coils. Effect of unilateral excision on the remaining pronephros The pronephros remaining after removal of one head kidney obviously takes over the function of excretion usually per- formed by the two organs. Beginning with the fourth day after excision, operated embryos were killed for obser- vation each day for a period of two weeks. Sections showed distinct changes in the several remaining components of the excretory system, particularly in the head kidney functioning alone, the size of which was indicative of a marked compensa- tory hypertrophy. Since the controls taken frcm the same egg mass and carried along under the same conditions as those to which the operated forms were subjected invariably showed on sectioning a more advanced stage of development, the first step in determining the nature and extent of the change in the oper- ated kidney was the establishment of a criterion for comparison of an operated with a normal embryo. An operated individual (PN 7) was chosen as a typical case and a large number of nor- mal larvae of apparently the same age was examined to obtain REMOVAL OF PRONEPHROS OF AMBLYSTOMA ate one in which the stage of development was identical. In many embryos where superficial features, such as length, breadth, and condition of limb and gill rudiments, were the same as those of PN 7, it was found on sectioning that the internal organs varied widely in degree of development. The normal larva finally selected (PN 7 d) tallied* not only in external measurements, but showed the several internal organs (retina, lens of eye, digestive tract, etc.) to be in a stage corresponding to those of BINT As a further check against the possibility of error in the choice of a normal duplicate, a second duplicate was chosen, and the respective volumes of the kidneys of the two roughly compared by the following method:? On drawing-paper of uniform thick- ness the serial sections of the entire kidney of PN 7 d and of the second duplicate (PN 7 d, no. 2) were projected and the lumen of the tubule outlined. The drawings of each kidney were then carefully cut out. No attempt was made to assemble them in the form of a model, but the weight of the paper used for each was taken as a standard for comparison. The weight of PN 7 d was 2.35 grams and that of PN 7 d, no. 2, 2.26 grams, giving a difference of only 0.09 gram, or about 4 per cent—a variation so small as to be considered negligible. The larger normal kidney (PN 7 d) was used for comparison with the hypertrophied one in order to lessen the possibility of exaggerating the difference between the two. After the normal duplicate (PN 7 d) had been selected, several methods were open for the determination of the nature and degree of the hypertrophy of the remaining pronephros in the embryo from which the organ on one side had been removed. 8 The slight variation would tend rather to minimize the contrast than to accentuate it, since, if either, PN 7 d is the more advanced. * In connection with the review of Kittleson’s paper (see previous reference), I find that somewhat the same method was employed by him in his estimation of the relative surface areas and weights of the kidneys of rats. The weight in grams was reduced to square centimeters by estimating the average area in square centimeters of one gram of paper, and from this the total volume of the kidney was estimated. 374 RUTH B. HOWLAND Wax models of the unoperated right pronephros of PN 7 and the corresponding organ in PN 7 d were constructed by means of the Born method, at a magnification of 200 (figs. 18 to 21). Unlike the normal model, to which reference has already been made in an earlier section, these two are reconstructions of the lumen of the tubules without enclosing walls. The model of the kidney of the operated embryo not only showed a consider- able increase in the thickness of the tubule as contrasted with that of the normal, but its length is also appreciably greater. This was determined by taking the average of five measure- ments. - A flexible but inelastic cord was pinned along the sur- face of the wax for its entire course, and its length thus recorded. On each measurement the cord was pinned along a different sur- face, so that the data would be of a representative nature. For the model of the normal kidney the average length was found to be 155 em., at a magnification of 200, with a probable error of +0.181: for PN 7, 188 cm., at a magnification of 200, with a probable error of +0.155, showing an increase of 21 per cent over the normal condition. Microscopic examination of the tubules showed the walls in the normal organ (PN 7 d) to be relatively thick and made up of cuboidal cells. The hypertrophied tubules (PN 7) were thinner walled proportionately, the cells often flattened and elongated, and the lumen strikingly larger than that of the unoperated specimen!® (figs. 22 and 23). Outline drawings were made of the outer and inner boundary of the walls of the hypertrophied and of the normal tubules at a magnification of 600 (figs. 11 and 12). A non-elastic cord was then pinned at frequent intervals along the inner lines, removed and measured, and the circum- ference obtained. Five different sections were used at different levels in each case, and in each section from three to five tubules were measured, making a total of twenty-one measurements for each kidney. The average measurement obtained for PN 7 d 10 Tn a series of experiments reported by Detwiler (’18), the pronephros was often carried along in the transplantation of the limb rudiments. The enlarge- ment of the undisturbed pronephros and its contrast with the normal condition may also be seen on examination of his plates (Jour. Exp. Zo6él., vol. 25, 1918, pl. 3, figs. 18 and 19). REMOVAL OF PRONEPHROS OF AMBLYSTOMA By a was 78 mm. (0.13 mm. in actual size), as contrasted with 130 mm. (0.216 mm. in actual size) for PN 7—a fact suggesting the large percentage of increase in functional capacity of the two kidneys determined and described below. From these projec- tions also the thickness of the walls was estimated by taking the average of sixty measurements in each kidney. The average thickness of the wall of the normal kidney is 10.9 mm. at a mag- oo. oe Se Fig. 11, PN 7d Normal tubules with thick walls, and cells bulging into the lumen. X 240. Fig. 12, PN 7 Hypertrophied tubules with large lumens and thinner walls. nification of 600, or 0.0181 mm. in actual measurement. For the hypertrophied kidney, the average thickness is 7.8 mm. at a magnification of 600, or 0.013 mm. in actual measurement. Having the length of the two kidneys, determined from the models, and considering each kidney as a simple cylinder, with the average measurement just obtained as its circumference and the length as its height, the areas of the inner surfaces in. each were computed. In the normal kidney the area of the secreting surface was found to be 1.007 sq. mm. as contrasted 376 RUTH B. HOWLAND with 2.037 sq. mm. in the compensating organ—an increase of more than 100 per cent (table 1). A comparison of the volumes of the cells making up the walls of the two kidneys is likewise of great importance in determining the nature of the response brought about by a unilateral opera- tion. Although in the calculation of the surface area of the tubules it seemed sufficiently accurate to regard them as cylin- ders with the average circumference as their boundary, it did not seem possible to apply such a geometrical method in estimating the volume of the walls, for the cells (figs. 11 and 12), especially in the normal kidney, often bulge out into the lumen, making this, as seen in projected outline, very irregular. ‘The method already described in the selection of a normal model was again TABLE 1 Showing the area of the inner or secreting surfaces of the normal kidney, PN 7 d, and the hypertrophied kidney, PN 7 INNER OUTER AREA OF ACTUAL SERIES NUMBER eter eee (5¢ 600) eNet AREA OF (X 600) (X 600) S360 1000) ql oe eee cm. cm. cm. sq.cm. sq. mm. PN 7 d (normal). 7.8 13.6 465 3627 1.007 PN 7 (hyper meohied).. 13 18.6 564 7332 2.037 used (p. 373). Paper of uniform weight was obtained, and to insure further accuracy, section number. one of the normal kidney was projected on one half of a sheet, section one of the hypertrophied organ on the same sheet of paper, and so on through the series. The walls of the two kidneys were then cut out, and the aggregate of each weighed separately. The paper representing the walls of PN 7 d weighed 20.39 grams that of PN 7, 33.4 grams, showing an increase of 63 per cent in the weight of the latter. Since the weight in this case is in direct proportion to the volume, the hypertrophied kidney may then be considered as showing an increase of almost two-thirds beyond the normal. A count of the nuclei in each kidney, all the sec- tions of which were cut at 10u, shows a small percentage (16 per cent) of increase in the number of cells found in the larger kidney, REMOVAL OF PRONEPHROS OF AMBLYSTOMA oid there being 1950 nuclei in PN 7 d and 2252 in PN 7. Although this may indicate a certain amount of hyperplasia, that is, increase in actual number of cells present, it is not large enough to account for the great percentage of increase in both secreting surface and in volume. This increase must be mainly attrib- uted to actual hypertrophy or enlargement of the cells already present. Effect of unilateral excision on the glomerulus A study of the condition of the glomeruli subsequent to double excision was necessarily limited by the early death of the embryos. The occurrence of the glomerulus in these cases has already been noted. On examining larvae from which only one pronephros had been removed, both glomeruli were found to be invariably present. The one on the operated side, however, exhibited less uniformity in size and shape than the normally functioning one. In some eases the outer layer of the glomerulus and the epithelial lining of the body wall had coalesced, this being the case not only on the operated, but on the normal side as well. On the operated size, however, the distance to be bridged is very greatly increased, since the absence of the pronephric swelling increases the width of the coelomic cavity there (fig. 13). Since the development of this part of the kidney unit is quite independent of the presence of the tubular portion, its functional activity can be counted on to continue undisturbed. No hyper- trophy in glomerular structure, then, would be anticipated, nor was such found to be the case, for the structural conditions existing allow free passage of the filtrate ventrally from one side to the other through the coelomic cavity. Filtration continues on both sides, the demand for increased physiological activity falling only on the tubules of the unoperated side. Effect of unilateral excision on other components of this system Removal of one head kidney has a widely varied effect on the formation of the segmental duct on the operated side. The process of development of the non-functioning ducts took place 3/8 RUTH B. HOWLAND irregularly, and, in general, only to a limited extent. As shown in table 2, every gradation occurred, ranging from a condition in which the lumen, though small and flattened dorsoventrally, appeared throughout the entire length (A 51, 5 days) to a con- dition where only the occasional presence of a few degenerating cells indicated the location of the atrophied rudiment (A1, 10 days). Fig. 18, PN 2 Showing the increased width of the glomerulus (gl) on the oper= ated side. Description in text. X 40. Increased activity of a single kidney also has a definite effect on the segmental duct of that side. Cross-sections of the duct of an individual with unilateral operation, when compared with either of the ducts of a normal larva of the same age, show a marked increase in diameter. Although in the excision of the pronephric rudiment great care was taken to remove as much of the somatopleural mesoderm as seemed feasible without disturbing the underlying tissues, an examination of the operated embryos showed that a large num- REMOVAL OF PRONEPHROS OF AMBLYSTOMA 379 ber possessed well-developed anterior and posterior nephrostomes ending blindly (figs. 14 and 15 and table 2). This would indi- cate that the adjacent coelomic endothelium possessed the \ } Un Neat / ty, ae? r) 6 /{ } ; NY Lee OZ : S \ ai ah f oi, Tf lif / ss c/ Fig. 14, PN 2. Showing double anterior funnel (d.f.). X 30. Fig. 15, PN 7 A, regenerated anterior funnel (a.f), on the operated side. B, regenerated posterior funnel (p.f) on the operated side. X 30. capacity for regeneration of this portion of the organ, even though, as we have already seen, this property is not shown by the tubules themselves. Of the fifteen embryos tabulated TABLE 2 Showing condition of ducts, nephrostomes, and glomeruli in embryos from which one pronephros has been removed SERIES NUMBER A 54 A 51 A 51 PN 3 PN 4 PN 5 PN 6 PNG 10 11 17 29 15 21 21 9 DUCT ON OPERATED SIDE Canalized at in- tervals, flat- tened Small, canalized, flattened Small, flattened, atrophied pos- teriorly Small, canalized Present anteri- orly atrophied posteriorly Present anteri- orly, atrophied posteriorly Small, shghtly canalized A few degenerat- ing cells Small, irregu- larly canalized Very small but canalized pos- teriorly Small, discontin- uous anteriorly Very small, (whole mount) Small (whole mount) Small anteriorly, central portion discontinuous Small anteriorly, none posteri- orly DUCT ON UNOPERATED SIDE Round, canal- ized, definite Large, round, canalized Large, round Large, round Large, round Large, round Large, round Large, round Very large Large, round Large, but flattened - anteri- orly, Large, (whole mount) Large, (whole mount) Flattened anteri- orly Large, round 1 For PN 7, 9 days, see figs. 18 and 20. 380 NEPHROSTOMES GLOMERULI Anterior present, posterior ab- sent Anterior present, posterior pres- ent No anterior, pos- terior present Anterior present, posterior ab- sent Anterior present, posterior pres- ent Anterior absent, posterior pres- ent Small anterior, no posterior Anterior present, posterior ab- sent Anterior present, posterior pres- ent 2 anterior, 1 pos- terior Anterior present, posterior pres- ent No anterior, pos- terior present No anterior, pos- terior present Anterior present, posterior pres- ent Anterior present, posterior pres- ent Both present Both present Both present Both present Both present Both present Both present Both present Both present Both present Both present Both present Both present Both present Both present REMOVAL OF PRONEPHROS OF AMBLYSTOMA 381 (table 2), twelve had well-formed anterior, and twelve had pos- terior nephrostomes. In one instance (PN 2, 17 days) the anteror nephrostome had doubled, suggesting the three pro- nephric openings normally found in anuran larvae (fig. 14 and table 2). A study of the development of the mesonephros in operated animals will be the subject of further work. EFFECT OF REMOVAL OF THE HEART ON THE DEVELOPMENT OF THE GLOMERULUS In a series of experiments dealing with the effect of removal of the heart on certain other organs of the embryo, Doctor Harri- son removed the rudiment of the heart in larvae of stages 29 to 30. Through his kindness, these embryos were made available to me for a study of the effect produced on the glomeruli. With the incoming of new material (March, 1920), these cases were further augmented by additional experiments. The glomerulus in Amblystoma punctatum, as has been stated in an earlier section, normally begins to differentiate from cells of the splanchnopleural wall below and at each side of the aorta, in stage 36. Within these clusters of cells vacuolated areas soon appear, and in a short time connect with the aorta." In embryos from which the heart has been removed before any contraction of the cardiac muscles occurred, the initial develop- ment of the cell groups is normal. However, as the connection with the aorta is established, the more or less compact nature of the tufts can no longer be maintained, but from pressure of the blood plasma which has collected in and is distending the blood- vessels, the vacuolated centers of the glomeruli are torn apart. As this accumulation of fluid increases, the outer walls of the tufts become more and more flattened, and consequently less easily distinguishable from the wall of the aorta, with which they are still continuous, finally losing their identity as separate organs. It is of interest, however, to note their early formation under these circumstances as additional proof of their independ- ent power of development. 11 A detailed description of this process together with plates is given by Field, 91 (pl. 1, figs. 8, 9, 10; pl. 6, figs. 48, 49, 50, 52, etc.). 382 RUTH B. HOWLAND SUMMARY AND CONCLUSIONS From a study of the results obtained after bilateral and uni- lateral extirpation of the head kidney and of the heart rudiment of Amblystoma larvae, the following conclusions may be drawn: 1. Conditions ensuing on bilateral removal of the pronephros show clearly that this organ is necessary to the life of the embryo, although the presence of one pronephros suffices to keep the organism alive and in a healthy condition. All embryos from which both head kidneys had been extirpated died within from eight to twelve days, evidencing during that interval weakened heart action, edema, and effusion into the pericardial and abdom- inal cavities. Pricking the body wall to relieve the edematous condition proved ineffective. 2. Double extirpation does not affect the normal development of the glomeruli. These appear in embryos killed four days after the operation. 3. The pronephros remaining after the removal of one head kidney takes over the function of excretion usually performed by the two organs, and, concomitant with the increased physiolog- ical activity, presents marked morphological changes. 4. The adjustment consequent on unilateral removal consists not in the regeneration of the lost part, but in compensatory hypertrophy of the remaining organ, a response which has long been known to occur in the adult kidney and in other glandular organs, both paired and unpaired. 5. The area of the secreting surface in the hypertrophied kidney shows an increase of over 100 per cent when contrasted with the normal (2.037 sq. mm.; 1.007 sq. mm.). 6. The cubie content of the mass of cells constituting the hypertrophied kidney as shown by their relative weight is increased 63 per cent above the normal. 7. The length of the tubules shows an increase of 21 per cent. 8. The number of nuclei in the hypertrophied kidney exceeds that of the normal by 16 per cent, due to the occurrence of a small amount of hyperplasia. | 9. In single, as well as in double, extirpations, the glomerulus develops normally in the absence of the pronephric tubules. REMOVAL OF PRONEPHROS OF AMBLYSTOMA 383 10. Anterior and posterior nephrostomal funnels are regen- erated from the coelomic endothelium in a large proportion of operated embryos. 11. The segmenta’ duct on the operated side shows great variation in development, ranging from a condition in which the lumen, though small and flattened dorsoventrally, appears throughout the entire length, to a condition where only the occasional presence of a few degenerating cells indicates the location of the atrophied duct. 12. Increased activity of a single kidney also has a definite effect on the segmental duct of the same side. Cross-sections of the duct of an individual with unilateral operation, when com- pared with either of the ducts of a normal larva of the same age, show a marked increase in diameter. 13. In embryos from which the heart rudiment has been removed in a very early stage, the initial development of the glomeruli is normal. Subsequent distention of the aorta tears the cells apart and they soon lose their identity as lateral capil- lary tufts. — 384 RUTH B. HOWLAND LITERATURE CITED Byrnes, EstHer F. 1898 On the regeneration of limbs in frogs after the extir- pation of limb rudiments. Anat. Anz., Bd. 15. Derwiter, S. R. 1918 Experiments on the development of shoulder-girdle and the anterior limb of Amblystoma punctatum. Jour. Exp. Zod6l., vol. 25; N10. 2: Fietp, H. H. 1891 The development of the pronephros and segmental duct in Amphibia. Bull. Mus. Comp. Zoél. Harvard. Coll. 21. Firprincer, M. 1878 Zur vergleichenden Anatomie und Entwicklungs- geschichte der Exkretionsorgane der Vertebraten. Morph. Jahrb., Bd. 4. GaLrorti, G., UND Vinia-Santa, G. 1902 Uber die kompensatorische Hyper- trophie der Niere. Beitr. z. path. Anat. u. allg. Pathol., S. 121-141. Harrison, R. G. 1904 An experimental study of the relation of the nervous system to the developing musculature in the embryo of the frog. Am. Jour. Anat., vol. 3. 1918 Experiments on the development of the fore limb of Amblystoma, a self-differentiating equipotential system. Jour. Exp. Zodl., vol. 25. Hertwie, O. 1911 Die Radiumkrankheit tierischer Keimzellen. Arch. mikr. Anat., 2te Abt., Bd. 77. How.tanp, Rurx B. 1916 On the effect of removal of the pronephros of the amphibian embryo. Proc. Nat. Acad. Se., vol. 2. KAMMERER, P. 1905 Uber die Abhiingigkeit des Regenerationsvermégens der Amphibienlarven von Alter, Entwicklungsstadium und spezifischer Grosse. Roux’s Archiv, Bd. 19, 2. Heft. KirrLeson, Jonn A. 1920 Effects of inanition and refeeding upon the growth of the kidney of the albino rat. Anatomical Record, vol. 17. Kocus, W. 1897 Versuche iiber Regeneration von Organen bei Amphibien. Arch. mikr. Anat., Bd. 49. Levi, Gruseprpe 1905 Lesioni sperimentali sull’ abbozzo urogenitale di larve di Anfibi e loro effetti sull’ origine delle cellule sessuali. Roux’s Archiv, Bd. 19., McCuovre, C. F. W. 1919 Experimental production of edema in larval and adult anura. Jour. Gen. Physiol., vol. 1, no. 3. Miiier, JOHANNES 1829 Ueber die Wolff’schen Kérper bei den Embryonen der Frésche und Kréten. Meckel’s Arch. f. Anat. u. Physiol., Jahrg. 1829, S. 65-70. Taf. III. Pricz, G. C. 1910 The structure and function of the adult head kidney of Bdellostoma stouti. Jour. Exp. Zodl., vol. 9. SacerpoTTi, C. 1896 Ueber die compensatorische Hypertrophie der Nieren. Virchow’s Archiv, Bd. 146, S. 267. Swinete, W. W. 1919 On the experimental production of edema by nephrec- tomy. Jour. Gen. Phys., vol. 1, no. 5. Wotrr, M. 1900 Die Nierenresektion und ihre Folgen. Berlin. Hirschwald. WirticH, von 1852 Beitrige zur morphologischen und histologischen Entwick- elung der Harn- und Geschlechtswerkzeuge der nackten Amphibien. Zeitschr. f. wiss. Zool., Bd. 4, S. 125-167, Taf. X, XI. PLATES ABBREVIATIONS A, anterior PC, pronephrie coil AF, anterior funnel PF, posterior funnel LT, longitudinal tubule SD, segmental duct P, posterior hr 385 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, NO. 3 16 PLATE 1 EXPLANATION OF FIGURES Model of young normal pronephros, ventrolateral view. Model of young normal pronephros, dorsolateral view. 386 125. x 125. REMOVAL OF PRONEPHROS OF AMBLYSTOMA PLATE RUTH B. HOWLAND PLATE 2 EXPLANATION OF FIGURES 18 Model of hypertrophied pronephros, ventrolateral view (PN 7). 19 Model of normal pronephros, of same age as figure 18, ventrolateral view (PINEZ,"d)3 REMOVAL OF PRONEPHROS OF AMBLYSTOMA PLATE 2 RUTH B. HOWLAND 5389 PLATE 3 EXPLANATION OF FIGURES 20 Model of hypertrophied pronephros, dorsolateral view (PN 7). 21 Model of normal pronephros, of same age as figure 20, dorsolateral view (PN 7, dd; 390 REMOVAL OF PRONEPHROS OF AMBLYSTOMA PLATE 3 RUTH B. HOWLAND era ke Pil eed —s ON OF FIGU a € ATI pronephros ; EXPLA Ss ypertroph ion 22 Sect cathe mona aatie a x = are Fee Eke qe ry hb att ae ot ge ee oar | REMOVAL OF PRONEPHROS OF AMBLYSTOMA PLATE 4 RUTH B. HOWLAND nee . 23 “Section of normal pron wy ” . ' » : t * ua ‘ 9 are . 5 he ~ 4 é a cee Tee ee . # tn - ’ b. = Seay : ried i oe i. 4 >. . ‘ + = y . - PLATE St.) oo EXPLANATION OF FIGURE. ae el ephros (PN 7, d). SNe ie oa ‘ 5 s € ~ o = : = eae . ‘ ‘ ~ ' 5 ‘ . Man t ’ ' i * ra = * . * a a ‘ ‘ ‘ : 394 3 - = ee ; . 4 REMOVAL OF PRONEPHROS OF AMBLYSTOMA PLATE 5 RUTH B. HOWLAND 395 Resumen por los autores, W. A. Kepner y W. Carl Whitlock, Universidad de Virginia. Reacciones alimenticias de Ameba proteus. En este organismo existen dos tipos generales de reaccién en presencia de los alimentos: (a) Cuando la presa no puede escapar la amiba la rodea estrechamente; (b) Cuando puede escapar la amiba corta su retirada envolviéndola con sus pseud6podos, y entonces la presa queda capturada. Estos dos tipos de reaccion alimenticia no son fijos, sino que varian notablemente. Al reacclonar en presencia de un objeto que se mueve general- mente en un plano horizontal, la amiba rodea la presa primero en este plano, y después corta su retirada en un plano vertical. Generalmente una reaccidén tiene lugar mediante cooperacién del ectoplasma y el endoplasma, aunque el primero por si solo puede llevar a cabo una reaccién del segundo tipo. Tanto el ectoplasma como el endoplasma son muy contractiles cuando las condiciones lo exigen. La fragmentaciOn de un animal como Paramoecium en dos pedazos es primariamente un proceso fisico y no quimico, y la digestion comienza después que la presa ha sido fragmentada. El proceso de la ingesti6n del alimento es reversible. Alimento medio ingerido, casi ingerido o completamente ingerido puede ser expulsado. Las reacciones de Ameba difieren de los fené- menos fisicos y quimicos en que son cualitativas mas bien que cuantitativas y se llevan a cabo en interés del organismo. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR’S ABSTRACT OF THIS PAPER {SSUED BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 7 FOOD REACTIONS OF AMEBA PROTEUS WM. A. KEPNER AND W. CARL WHITLOCK University of Virginia SIX PLATES (TWENTY-ONE FIGURES) The observations presented in this paper are selected from many that have been recorded by members of the staff of this laboratory. Some of the observations were taken from speci- mens in Petri dishes, some in uncovered drops, still others under cover-glasses, while many were secured from amebas that had been kept in hanging drops until time was available for making observations. We have found Ameba proteus reacting to two types of food. The first type embraces the following forms: desmids, Mouge- otia, quiet Oscillatoria, encysted Chlamydomonas, and bacterial gleas; while the second group of food bodies comprises flagellates like Chilomonas, Peridinium, and Euglena, ciliates like Parame- cium caudatum, Colpidium, Cyclidium, and rotifers. The first of these groups of food objects is characterized by being non- motile, the second group by being motile. Some of the non- motile objects give off oxygen, while others give off carbon diox- ide. The same may be said of the motile group; it, too, may be subdivided into the forms that give off oxygen and those that yield carbon dioxide to the surrounding medium. Therefore the most conspicuous difference between these two groups of food is the non-motility of the first group and the motility of the sec- ond group. Correlated with this conspicuous difference between the two types of food of Ameba there is a two-fold food reaction on the part of these rhizopods. Ameba’s conduct toward non-motile food is much less complex than its conduct toward motile food. The less complex type of reaction is concerned with ingesting forms that do not set up currents in the surrounding water and 397 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, No. 3 398 WM. A. KEPNER AND W. CARL WHITLOCK that do not present the contingency of escape. ‘The more com- plex type of food reaction of Ameba is concerned with the cap- ture of forms that set up currents in the surrounding water and that do present the contingency of escape. It was interesting to us to find that Leidy (’79) had shown in his figure 5, plate 1, two Urocentra captured according to our second type of food reac- tion, while a green plant cell was ingested apparently by the first type of food reaction. EXAMPLES OF THE FIRST TYPE OF FOOD REACTION a. Objects that yield oxygen to the water On March 15, 1919, we found that in a Petri dish there were many filaments of Oscillatoria that were quite quiet. None of these filaments were to be seen moving as Oscillatoria filaments frequently do. An ameba had ingested one end of one of these quiet filaments (fig. 1). In endeavoring to take this specimen from the Petri dish on to a slide, the capillary canula of the pipette dragged over the free end of the algal filament in such manner as to tear the ameba from the substratum and turn it through about 180 degrees. The ameba was now given time to fix itself again to the bottom of the dish. The free end of the filament was then pushed against with the canula of the pipette. This time instead of the ameba’s being torn from the bottom of the dish, the part of the ameba’s body that surrounded the fila- ment was bent from position a to position 6 (fig. 2.) The ameba was now drawn up into the capillary pipette and transferred to a hanging drop. The compound microscope showed that, de- spite this relatively rough handling, the ameba yet held on to the filament of Oscillatoria. Within two minutes after the cover- glass was placed over the glass ring, the Oscillatoria was egested. Soon after this the ameba again ingested one-third of the length of the filament and then threw it out a little later. The ameba a third time set to ingesting the plant. When about one-fourth of the filament was within the body of the ameba, a paramecium collided sharply with the projecting end of the alga at right FOOD-REACTIONS OF AMEBA PROTEUS 399 angles, and the ameba then gave up its efforts to lay hold of this food. Throughout all of the time that the ameba was working on this rather long filament of Oscillatoria it had within its body a filament of Oscillatoria that was 30u long when first seen. Within the course of our observation, this ingested filament was broken up into three pieces, one 10y, one 5u, and one 1duz. After we had secured several observations showing that the ameba laid hold of quiet Oscillatoria filaments tightly, we called in some of our colleagues in this laboratory to make observations. Six others verified our results by making similar observations. The most conspicuous of these corroborative records was made by Dr. I. F. Lewis. He was given an ameba that had ingested an end of a very long filament, indicated as broken off in figure 3. He took a fine glass rod and bent the plant to contour b (fig. 3), at which point the tension of the alga caused it to spring back as a straight rod. ‘‘Twenty big bends, some like this, others differ- ent, were made as the ameba gradually lost its hold.” No such large filaments have been ingested wholly. The ameba sometimes travels from end to end along such long objects, sometimes making several trips, and then leaves the food behind. Frequently small fragments have been seen in different stages of digestion within amebas (fig. 5, O). It would seem that the ameba seeks the planes of fission of the Oscillatoria filaments to break off fragments for food. Such may not be the case, how- ever, for we have seen an ameba travel along a Mougeotia fila- ment in a similar manner, and there are no fission planes in Mougeotia. No Mougeotia filaments or fragments were ever seen completely ingested. Large desmids were also ingested in part and then rejected. On one occasion, January 28, 1919, two amebas began to ingest opposite ends of a large Penium syn- chronously. The lower half of the desmid was ingested within twenty minutes by one of the amebas. During this period the upper ameba ingested about one-third of the desmid. Both amebas were closely embracing the plant, but they eventually rejected the object by withdrawing from it. Small desmids have been observed by us being ingested by Ameba proteus. A Chla- mydomonas within its gelatinous sheath was also ingested. Nei- 400 WM. A. KEPNER AND W. CARL WHITLOCK ther the small desmids nor the encysted Chlamydomonas rolled before the advance of the ameba, and they too were ingested _ within a closely fitting food vacuole. The observation, based upon the ingestion of an encysted Chlamydomonas, makes an interesting contrast with Jennings’s (’04) observation of an ameba ingesting an encysted Euglena. In the latter case the encysted alga rolled ahead of the advance of the ameba, and here Jennings saw a cup form behind the algal cysts. This con- trast between our observation and that of Jennings suggests that even the type of reaction involved in ingesting non-motile objects may be modified to meet an unusual turn of events. There are some non-motile food objects which give off carbon dioxide. Of these, bacterial gleas form common examples. February 16, 1918, Dr. R. D. Mackay observed an ameba glide over a glea. As it was about to leave the glea, two embracing pseudopods were sent out about the bacterial mass. These pseudopods lay close up to the sides of the rounded mass and eventually con- stricted a small portion from the glea as the enclosing pseudopods began to converge (fig. 4, a and b). In the above reactions we have the ameba responding to non- motile objects that gave off either oxygen or carbon dioxide. In reacting to this class of food, the amebas seized the objects in an intimate embrace. The following constitute a list of motile objects to which Ameba proteus has been seen reacting: a) Euglena viridis, Peridinium, and diatoms; b) rotifers, Paramecium caudatum, Urocentrum, Glaucoma scintillans, Colpidium, and two species of unidentified ciliates, Chilomonas, Codosiga, Euglena acus, and two species of unidentified flagellates. Of this group of motile food objects, a) forms a subdivision of forms that give off oxygen, while b) forms a subdivision of forms that yield carbon dioxide to the surrounding medium. The reaction of ameba to diatoms has been rather indefinite. The ameba seems to react to these motile plants as if they were non-motile. We have, however, obtained but two observations based upon diatoms, and in both of these cases the diatoms, while they were being intimately embraced, escaped. FOOD-REACTIONS OF AMEBA PROTEUS 401 Except for the diatoms, we have seen that there is a wide range of motile food bodies to which Ameba proteus displays a general type of response. ‘The following observations have been chosen as examples of the ameba’s second type of food reaction and also to display the range of adaptive modification this type of reac- tion may present. The ameba seems to have a marked preference for Chilomonas paramecium. It will readily accept one of these little ciliates, though it has been feeding on a non-motile object or other motile objects. On March 19, 1919, we observed a specimen that had been feeding upon Oscillatoria. A Chilomonas swam into a bay between two stout, short pseudopods and lay in the position shown in figure 5. The ameba immediately sent two secondary pseudopods, A and B, out toward each other and behind the Chilomomas. These pseudopods met and fused; the ciliate was thus surrounded on all sides. It was next overarched by a thin sheet of ectoplasm. When all lines of retreat were thus cut off from the Chilomonas, the ameba reduced the size of the large vacuole, within which the prey had been captured, to that of the usual food vacuole. Both ectoplasm and endoplasm entered the formation of the pseudopods A and 8B in this reaction. ‘This is the manner in which the enclosing pseudopods are usually con- structed. But even the structure of the pseudopods may be modified to meet the needs of a peculiar situation. In one instance we observed an ameba approach two Chilo- monases in the shallow margin of a hanging drop. In this case ectoplasmic pseudopods a and a’ were sent out about the Chilo- monases (fig. 6). As a grew down to contour b, an overarching layer of ectoplasm, c, was formed above the prey. The internal margins thus formed eventually fused as b grew down to divide the enclosed space into two food vacuoles. The animal then moved out into deeper water. The unusual feature of this reac- tion is not that the overarching protoplasm is ectoplasmic, for that and the underhanging wall of the forming food vacuole are usually ectoplasmic. The unusual feature is the fact that the ectoplasm formed all sides of the forming food vacuoles. These vacuoles were thrown into the endoplasm when the animal moved 402 WM. A. KEPNER AND W. CARL WHITLOCK out into the deeper regions of the drop after capturing the two flagellates. During the course of this observation it was noticed that an ameba does not of necessity react to an object that is setting up currents in the surrounding water or that is colliding with the ameba repeatedly; for before, during, and after the reaction of the ameba to the above two Chilomonases, a very active, dense swarm of bacteria plied to and fro against the side of the ameba making frequent contacts with it. At none of these contacts did the ameba react to this highly motile mass. It mattered not whether the contact were made at an angle between pseudopodia, as at A, figure 6, or at the tips or sides of the pseudopods. A newly formed pseudopod that is taking part in the forma- tion of a food vacuole may further react to cooperate with a part of the body proper to construct a second food vacuole. That such is the case is shown by the following example. Two Chilomonases were being surrounded by pseudopods a and a’ (fig. 7). When a had grown to contour 6, a third Chilomonas came up by the side of a’. In reacting to this third Chilomonas, the body proper threw out pseudopod c’, while pseudopod b sent out c to meet c’. In this manner all three flagellates were captured. On March 19, 1918, we saw an advancing pair of pseudopods, a and 6b, encounter a relatively large piece of foreign matter as they advanced about a Chilomonas which lay in position indi- cated in figure 8. At this synchronous contact of the two pseudopods the one, b, was arrested while a advanced to contours c and d, d finally fusing with the body proper. The Chilomonas was next overarched and captured. Perhaps a more striking example of a reaction involving for- eign matter is presented in our observation of an ameba ingesting a paramecium that lay in a shallow bay by the side of a large brown mass of detritus (fig. 9). The ameba was advancing in a general way toward the paramecium along pseudopods 1, 2, and 3. As it approached the ciliate, pseudopods 1 and 2 widened and partly fused to form a large bi-lobed extremity, m-—m’. When this extremity had nearly touched the paramecium, it sent FOOD-REACTIONS OF AMEBA PROTEUS 403 out a small secondary pseudopod, a, beneath the prey, and } anterior to it (fig. 10). When the pseudopods a and b came in contact with the detritus, they moved apart and became much stouter (fig. 11). In the meantime a third pseudopod, c, appeared projecting from between a and b over the dorsal side of the para- mecium, while a pocket was formed within the body proper of the ameba at the bases of these three pseudopods. The para- mecium first jumped to position 2, figure 11. The excited para- mecium next backed into the pocket of the body proper, 3, and a, b, and c closed in and surrounded it completely. Usually ameba reacts to a free-swimming Euglena viridis by sending out pseudopods that widely embrace it. Sometimes, however, the embracing pseudopods close in upon the Huglena to hold it in a tight grip behind the position of the gullet, and this though the flagellum be quite active. On March 17, 1919, we saw a Euglena caught in this manner at its anterior end. The projecting part of the flagellate’s body was passive, but the fiagellum was very actively lashing within the enclosed bay. All movement for the time being had ceased in the gripping: pseudopods. This observation had lasted for but a minute more: or less when a large Paramecium, coming up at right angles to the Euglena, collided with it at the point indicated by the arrow in figure 12, and dragged the Euglena free from the ameba’s grip. This was apparently the first step in the process of chang- ing the second type of reaction into the first type. Mr. C. O. Dean, a student in this laboratory, observed an ameba that had thus gripped a Euglena viridis and thereby cut off its chance of escape. After the ameba had thus laid hold of the Euglena, its “eetoplasm flowed out around the Euglena” on all sides and so close to the wall of the Euglena that there was no water present between ‘‘the surfaces of the two organisms.”’ This is not com- parable to the food-taking by means of invagination as Prenard (05) and Grosse-Allermann (’10) have described for Ameba terricola. In 1900 the senior author observed a relatively small ameba ingest a relatively large Paramecium caudatum. In this case the ciliate was surrounded by pseudopods that were sent out. 404 WM. A. KEPNER AND W. CARL WHITLOCK about it, but not touching it, about as Blochmann (’94) and Mast and Root (’16) indicate to be the usual method of ingesting paramecium. ‘The latter authors saw some very interesting exceptions to this method of swallowing paramecium. We, too, have observed departures from this type of reaction. On May 2, 1919, we had a hanging drop in which there had been many Colpidia, but which were now dying off. ° The dead ones, though frequently encountered by the ameba, were not in any case ingested. The living Colpidia were frequently accepted in wide embraces. The paramecia in this hanging drop were peculiar in that they were wider than normal ones and rather sluggish. Then, too, their bodies were so pliable that an ameba’s pseudo- pod, advancing against the dorsal side of one of them, would indent it. Moreover, when the paramecia were crowded between two amebas, they became greatly flattened and even in some instances bent upon themselves at right angles. The cilia and contractile vacuoles of these peculiar paramecia were active. The amebas attacked these relatively inactive paramecia over and over; but in each instance their attack was peculiar in that they attempted to surround these ciliates closely or intimately. Because of this unusual method of attempting to capture the paramecia they caught none, for after two-thirds or less of the length of the paramecium’s body had become involved in the embrace of the ameba, the paramecium would slowly glide out and remain by the side of the ameba until it would again be partially enclosed in a second embrace, when it would move out of the enclosing arms of its would-be captor. The conduct of the amebas toward these unusual paramecia is itself peculiar and exceptional. Here for some reason the ciliary disturbance of the water by the paramecia has not resulted in stimulating the amebas in such manner that they sent out about the prey remote encircling pseudopods. A further departure from the usual method of ameba in cap- turing paramecium was observed March 19, 1919. This ameba was first seen at 10:10 a.m. It was then perfectly quiet, spending all of its available energy upon the partly constricted para- mecium. The ameba showed no cytoplasmic movement (fig. FOOD-REACTIONS OF AMEBA PROTEUS 405 13). At 10:15 a.m. the paramecium was further constricted and the ameba quiet (fig. 14). At 10:25 a.m. the cytoplasmic isth- mus of the paramecium’s body was stretched and the ameba dis- played a little movement along pseudopod c and threw out pseu- dopods a and 6 about the projecting portion of the paramecium, the cilia of which were quite active (fig. 15). Pseudopods a and b were soon withdrawn. At 10:35 a.m. the constricting and stretching of the isthmus of cytoplasm were increased and the isthmus was flexed (fig. 16). By 10:48 a.m. the flexing of the enclosed cytoplasm had become very conspicuous (fig. 17). Two minutes later a pseudopod, d, was sent out along one side of the projecting lobe of the paramecium’s body, the cilia of which were yet quite active. This secondary pseudopod was at once with- drawn, while the paramecium was further stretched and bent. At this phase of the reaction a Cyclidium darted into the field and lay near the free end of a large ‘anterior’ pseudopod. The ameba reacted to this animal at once by sending out pseudopods e and f and capturing the smaller ciliate (fig. 18). The para- mecium was now released by the ameba as it ingested the Cy- clidium. The constricted, elongated portion of the mutilated paramecium shortened greatly and the large ciliate swam off under its ‘own steam,’ having a contour about like the outline given in figure 19. No trace of cilia could be seen on the part of the paramecium’s body that had been ingested by the ameba. An ameba may ingest food at different parts of its body syn- chronously. We have observed one ingesting five Chilomonases at one time and at five different regions of its body. Moreover, the two types of food reactions may be carried on simultaneously. On January 28, 1919, while an ameba was ingesting a quiet fila- ment of Oscillatoria, a Chilomonas came to lie beneath the fila- ment at a position indicated in figure 20. The Chilomonas was lying beneath the plane in which the filament of Oscillatoria lay. The ameba advanced about the plant until pseudopod b was formed. This pseudopod then sent out an encircling wall of cytoplasm about the Chilomonas and then overarched it with an ectoplasmic film. The space within which the Chilomonas was thus taken was next divided into a larger and a smaller 406 WM. A. KEPNER AND W. CARL WHITLOCK vacuole, the prey being in the smaller vacuole. The Chilo- monas was not disturbed until it was thus enclosed within the smaller vacuole. The filament of Oscillatoria was further in- gested, but it was finally rejected. ‘Thus, while a reaction to a non-motile object was being carried on, the ameba completed a food reaction of the second type, in capturing a passive motile object. Chilomonases have been seen to swim in beneath unattached regions of amebas’ bodies. In such cases, when the amebas react positively to the flagellates, a curtain of cytoplasm is dropped down around the prey, the lips of which turn in beneath the food body and fuse without disturbing the Chilomonas. Perhaps the most interesting reaction we have seen was that of an ameba reacting to a Chilomonas that had come to lie against the tip of a pseudopod (fig. 21, 7). The ameba sent out two pseudopods in response to the stimulus. The smaller pseudopod arose from the side of the parent pseudopod and a little behind its end, while the larger secondary pseudopod came out quite a distance behind the tip of the parent one. ‘The interesting fea- ture of this reaction is the fact that the parts reacting to the source of stimulation are parts least stimulated; indeed, the greater reaction was displayed by the least stimulated part. The quiet Chilomonas could stimulate the parent pseudopod in two ways: either chemically by means of its metabolic by-products, or physically by means of slight vortices that the play of its flagella may set up. In either case the end and not the sides of the parent pseudopod would be most affected by these stimuli. Moreover, we have studied the types of vortices set up in the water by quiet Chilomonases. This study showed that in all cases the strength of the currents thus set up was greatest at the anterior end of the Chilomonas. Finally, as the two secondary pseudopods were coming out by the sides of the Chilomonas, a second Chilomonas came to lie at position 2, figure 21, and thus double, or at least increase, the sources of stimulation; but this did not modify the conduct of the two secondary pseudopods. These facts indicate that the ameba’s reaction is a qualitative and not a quantitative one. FOOD-REACTIONS OF AMEBA PROTEUS 407 DISCUSSION It has been the tendency of recent work on the ameba to reduce the conduct of the ameba to simple terms. For example, Loeb (05) says—‘‘As a criterion for ‘living matter’ we might use the irritability or spontaneity. But as the ‘spontaneity’ of living matter is in its simplest form (in Amoebae) apparently not different from the physical phenomenon of spreading, for this criterion the limits of divisibility of living matter coincide with the limits of purely physical phenomena”’ (p. 321). McClendon (09) tries to explain food-getting of ameba in the following man- ner: ‘‘Chemical and physical influences of the medium cause a hardening and shrinkage (by loss of water) of the ectosare (Rhumbler’s ‘Geletinisrungsdruck’). Chemical processes within prevent this hardening from extending to the endosare, and dis- solve portions of the ectosare that are displaced inward. The medium affects different portions of the surface to different degrees, causing regional differences in degree of hardening and shrinking, thus producing amoeboid movements. . “413 _ THE JOURNAL OF EXPERIMENTAL ZOOLOGY, Vou. 32, No. 3 PLATE 1 EXPLANATION OF FIGURES 1 First position of specimen with two small fragments of Oscillatoria within its body and in the act of ingesting a second filament of Oscillatoria. X 100. 2 Second position after the ameba had been turned through 180 degrees by pushing against the partly ingested algal filament, a. The ameba was given time to fix itself to the substratum in this position, when the Oscillatoria filament was a second time pushed upon. This time the body of the ameba was not torn from the substratum and turned, but the tip of the ameba bent as the filament was pushed to position b. X 100. 3 A very long, quiet Oscillatoria filament was being ingested when the pro- jecting part of the filament was bent about the ameba to about the position shown in contour b. When bent to this extent, the filament would slip from the glass rod and spring back as a straight rod. The filament was thus bent and released twenty times before the ameba released its hold on the plant. X 100. 414 FOOD-REACTIONS OF AMEBA PROTEUS PLATE 1 WM. A. KEPNER AND W. CARL WHITLOCK PLATE 2 EXPLANATION OF FIGURES 4 Specimen reacting to a non-motile bacterial glea by constricting it with the pseudopods a and b. The larger lobe of the glea was ingested and broken up and the fragments delivered to small food vacuoles within the endoplasm of the ameba. X 200. 5 Specimen with fragments of Oscillatoria (O) in various stages of digestion (as shown by color) within the endoplasm. The ameba is cutting off the retreat of a quiet Chilomonas by means of the advancing pseudopods A and B. XX 100. 6 An ameba against the side of which a motile mass of bacteria (A) was play- ing. The animal did not react to this stimulation. In reacting to some Chilo- monases, that lay at the margin of a hanging drop in water that was shallow, the ameba sent out ectoplasmiec pseudopods, a and a’. When a had grown to contour b an ectoplasmic wall was thrown over the top of the flagellates. These animals were thus caught in an ectoplasmic enclosure instead of one that was formed of both ectoplasm and endoplasm. X 200. 7 As the ameba was sending pseudopods a—b, and a’ about two Chilomonases, a third Chilomonas came to he along the outer margin of a’. In reacting to this third flagellate pseudopods c¢ and c’ were thrown out. X 200. 8 While pseudopods a and b were advancing along each side of a Chilomonas, they collided at the same time with a solid body. The growth of pseudopod b was now inhibited, while a advanced to contours ¢ and d and finally surrounded com- pletely the prey. X 200. 416 FOOD-REACTIONS OF AMEBA PROTEUS PLATE 2 WM. A. KEPNER AND W. CARL WHITLOCK 417 PLATE 3 EXPLANATION OF FIGURES 9,10, 11 y, margin of a mass of detritus by which a paramecium was lying. 9, shows ameba advancing toward paramecium; 10, ameba sending pseudopods a and b forward until they made contact with detritus, after which they were moved apart and enlarged. In the meantime a pocket was formed within the body of the ameba into which the paramecium moved as it moved to positions 2 and 3. X 200. 12 A ciliate being captured by pseudopods a, 6b, c, d, while a Euglena has been grasped as it was retreating from a forming food vacuole. X 200. 418 FOOD-REACTIONS OF AMEBA PROTEUS PLATE 3 WM. A. KEPNER AND W. CARL WHITLOCK 419 ; PLATE 4 ; or Me, EXPLANATION OF FIGURES @ 13, 14,15 Figures show phases in the process of an ameba tearing a parame-_ cium into pieces. X 200. ; 420 FOOD-REACTIONS OF AMEBA PROTEUS PLATE 4 WM. A. KEPNER AND W. CARL WHITLOCK 421 PLATE 5 EXPLANATION OF FIGURES 16, 17, 18 Show process of constricting and stretching the paramecium con-_ tinued. e and f, pseudopods advancing about a ciliate. As this small ciliate was being captured, the paramecium was released by the ameba. X 200. 19 Shows the shape the living paramecium assumed after it had been released by the ameba and swam away. X 200. FOOD-REACTIONS OF AMEBA PROTEUS PLATE 5 WM. A. KEPNER AND W. CARL WHITLOCK 423 PLATE 6 EXPLANATION OF FIGURES 20 As the ameba was advancing about an Oscillatoria filament, a Chilomonas came to lie to the side of and beneath the alga. When the ameba had sent out the large pseudopod, b, it sent down beneath the alga a part of its body which enclosed the Chilomonas within a large vacuole before the prey was disturbed. Both types of food reactions were here carried on by the ameba synchronously. x 200. 21 An ameba capturing two Chilomonases which lay off the end of the parent pseudopod. XX 200. 424 FOOD-REACTIONS OF AMEBA PROTEUS PLATE 6 WM. A. KEPNER AND W. CARL WHITLOCK Resumen por el autor, Alfred O. Gross, Searles Biological Laboratory, Bowdoin College. La alimentacion y el sentido quimico en Nereis virens Sars. Nereis virens no es un gusano carnivoro, como han creido varios autores. Se alimenta principalmente de plantas que crecen en las proximidades de los agujeros que habita, o de frag- mentos de plantas transportados cerca del gusano por la marea. El sentido del gusto o sentido quimico no juega en Nereis una parte muy activa en el hallazgo o la seleccién del alimento. Nereis es fuertemente quimotropico negativamente hacia los Acidos, hidréxidos y sales. Es estimulado con mayor intensidad por el cloruro potisico que por el cloruro sédico, al contrario de lo que sucede en el caso de la lombriz de tierra. Esta diferencia esta relacionada con el tanto por ciento elevado del cloruro sddico en el agua de mar, a la cual esta adaptado Nereis. El tegumento general de Nereis es sensitivo a la accién de la estimulacién quimica, pero existe una localizacién o concentra- cidn del sentido quimico en los palpos y tentaculos, cuya con- dicién esta relacionada con la rica inervacién de estos apéndices y la relacién de sus nervios con el cerebro. Aunque existe una tendencia hacia la localizacion del sentido quimico, este animal no posee receptores especializados para recibir los estimulos quimicos. La localizacién del sentido del gusto en los palpos y tentaculos debe explicarse mediante la existencia de alguna cualidad especifica diferenciada del protoplasma de_ estos apéndices. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, MARCH 14 THE FEEDING HABITS AND CHEMICAL SENSE OF NEREIS VIRENS, SARS ALFRED O. GROSS Searles Biological Laboratory, Bowdoin College, Brunswick, Maine Nereis virens is a very common marine worm distributed along the Atlantic coast from Virginia northward to the Arctic regions. On the Pacific coast of America it is less common, but there are records of its occurrence from California northward to Puget Sound, Washington. In all favorable places of its range it occurs under stones or in burrows in the sand and mud of the intertidal areas. Nereis is a very favorable animal for use in experimental work because of its abundance and the ease with which it may be kept alive in the laboratory for long periods of time. Since it is commonly used in the zoological laboratories as a type for dissec- tion, a study of its habits seems desirable. The experimental work on the chemical sense was conducted at the Marine Biological Laboratory, Woods Hole, Massa- chusetts. J wish to express my gratitude to Prof. G. H. Parker who suggested the problem and who has given me helpful criticism. The fishermen and clam diggers along the New England coast believe that Nereis is dependent on the clam for its existence, hence the common name, ‘clam worm.’ Situations favorable for the clam are also attractive to Nereis, and as many of the worms find their way into the interior of dead snail shells or into the mud and sand between the two valves of the dead clams, the layman concludes that living molluscs are preyed upon and killed by Nereis. Zoologists, if they have any con- ception at all of the feeding habits of Nereis, believe it to be a carnivorous worm, whose powerful jaws are for the purpose of capturing and tearing other marine animals. In all probability, 27 428 ALFRED O. GROSS this idea has arisen from certain published statements such as the following made by Prof. A. E. Verrill on page 318 of his report upon the invertebrate animals of Vineyard Sound. ‘‘It is a very active and voracious worm, and has a large, retractile proboscis, armed with two strong, black, hook-like jaws at the end, and many smaller teeth on the sides. It feeds on other worms and various kinds of marine animals. It captures its prey by suddenly thrusting out its proboscis and seizing hold with the two terminal jaws; then withdrawing the proboscis, the food is torn and masticated at leisure, the proboscis, when withdrawn, acting somewhat like a gizzard.” This statement apparently was taken at its face value, and we find it copied into the various text-books and natural histories, of which the follow- ing taken from the Standard Natural History (vol. 1, p. 229) is one of many examples: ‘‘It is a very active and voracious worm terrible to smaller animals upon which it preys capturing them by its large proboscis which it suddenly thrusts out seizing its victim with the two large jaws which arm the tip of its efficient weapon of attack,” etc. Verrill’s statement has also misled investigators who have taken it for granted that the food of Nereis is animal. Prof. 8. 8. Maxwell, in his paper on the physiology of the brain of annelids, quotes Verrill, and later, on page 283, he describes the normal feeding reactions of Nereis virens as follows: ‘‘ Wenn man ein Stick Futter, z. B., ein kleines Stick von einem Wurm, auf eine Nadel spiesst und vorsichtig einem normalen Wurm reicht, kann man den Fressvorgang leicht sehen. Wenn man das Futter den Spitzen der vorgestreckten Fihler nihert, kommt der Wurm gewohnlich ruhig niher. Dann zieht er den Kopf ebenso ruhig ein wenig zuriick, legt die Fiihler an den Korper und 6ffnet den Rachen, um die Nahrung zu fassen.” It was with the above conception that Nereis virens was a carnivorous worm, that the author began experiments on the sense of taste. A voracious worm whose food is other animals would be expected to have well-developed organs of taste. Various experiments were subsequently devised in an attempt to study the normal feeding reactions of the worms. Entire, as FEEDING HABITS OF NEREIS VIRENS, SARS 429 well as extracts and ground-up messes of marine worms, crus- taceans, molluscs, fish, etc., would not tempt even a semistarved individual to eat. Though Nereis never utilized the animal substances provided as food, it ate freely of the sea lettuce which had been introduced into the dishes to aerate the water. Think- ing that possibly laboratory conditions had so altered the physio- logical conditions of the worms, that its feeding habits were abnormal, observations were made in the field. In no case was a Nereis seen to prey upon living animals, but many were observed to eat vegetable matter. To substantiate these obser- vations examinations were made of the intestinal contents of a number of worms collected from various situations in several localities as shown in the following table: TABLE 1 - NUMBER LOCALITY OF WORMS EXAMINED CONTENTS OF THE ALIMENTARY TRACT GIVEN IN APPROXIMATE PERCENTAGES Naushon Island, Woods 35 Eel-grass, 75 per cent Hole, Mass. Various algae, 15 per cent Sand, mud and miscellaneous material, 10 per cent Juniper Point, Woods Hole, 10 Nemaleon (an alga), 75 per cent Mass. Sphacelaria (an alga), 15 per cent Sand, egg masses and miscellaneous material, 10 per cent Eel Pond and Buzzards 38 Roots and blades of eel-grass, 95 per cent Bay, Mass. Sand, mud, sponges, Bryozoans and mis- cellaneous material, 5 per cent Lynn Beach, Lynn, Mass. 12 Eel-grass, 55 per cent Algae, 25 per cent Sand and mud, 15 per cent Miscellaneous material, 5 per cent New Meadows River (salt), 20 Rock weed, 95 per cent Brunswick, Maine Mud and miscellaneous material, 5 per cent The results of this examination show conclusively that Nereis virens is not an animal feeder, but is primarily a vegetarian. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32. NO. 3 430 . ALFRED O. GROSS Furthermore, this worm is able to adapt itself to a large range of plants for food and utilizes that which is abundant and most convenient to its burrows. The jaws and proboscis are used extensively in excavating burrows, but, as compared with the earthworm, a relatively small amount of sand and mud is ingested by Nereis. The animal materials, such as bryozoans, sponges, and egg masses, found in the intestine were originally attached to the plants eaten by Nereis and were probably an accidental element of the food. The worms seemed to exhibit no preference for eel-grass covered with bryozoans and. egg masses, nor did they shun such material when they chanced to come upon it. When a number of Nereis are crowded into a small dish they may, especially if mechanically or chemically stimulated, vio- lently thrust out the proboscis, extend the jaws, and bite the body of a fellow worm so severely as to sever it in two parts. I have seen a worm bite its own body in two when placed under pressure or treated with a strong acid or alkali. In such cases it may incidently take into its proboscis some of the flesh which is grasped. Very often, when ejecting extracts of animal juices from a pipette toward the head of the worm, it would thrust out its proboscis, Just as it did when treated with an acid or alkali. These thrusts I soon learned were not attempts at securing food, but were acts of self-defense and, it is very probable they often serve the worms as an effective protection against enemies as large or much larger than itself. The feeding response is a much more deliberate act. Is it not possible that an observation, such as noted above, and the fact that other species of Nereis have been reported as animal feeders, may be primarily respon- sible for Professor Verrill’s erroneous statement, a record which has been copied so many times without any attempt at veri- fication? The jaws, though not used in capturing animal prey, are employed in tearing out bits of the plants used as food. In the intestines of some of the larger individuals it was not uncommon to find pieces of eel-grass or other vegetation 1 to 2 cm. in length. A number of experiments were made with the natural food in an effort to localize the sense of taste, but the worms showed no FEEDING HABITS OF NEREIS VIRENS, SARS 431 consistent responses to food of any kind. They fed freely upon sea lettuce and other plants placed in the aquaria, but the find- ing of it was more or less accidental. Food hidden in sand, placed in cheese-cloth bags, or otherwise concealed was, as far as could be determined, never detected by the worms. Ani- mals from which one or all of the pairs of cephalic appendages, such as the tentacular cirri, palps, and tentacles, were removed, fed and thrived as well as normal animals. It is evident that the sense of taste, or chemical sense, of Nereis virens does not play an important role in locating and selecting food. It is conceivable, however, that a chemical sense may be developed which enables the worm to detect certain unfavorable environmental conditions of the water and mud in which it lives. To test the chemotropism of Nereis, simple reagents, such as HCl, KOH, NaOH, KCl, NaCl, and NH,Cl, were used. The worms were tested by the various methods used by Parker, Hurwitz, Shohl, Crozier, and Irwin. on the earthworm. The fence method used by Shohl proved to be the most satisfactory for the experiments on Nereis. For this purpose a rectangular, shallow glass tray was divided into two compartments by a paraffin partition, a quarter of an inch wide. A notch about three-quar- ters of an inch long and reaching within a half inch of the bot- tom, was cut in the middle of the partition. The entire tray was covered over with a thin layer of paraffin, to prevent the liquids from wetting the walls. Sea-water was placed on one side of the fence and sea-water containing the stimulating sub- stance on the other side. The worms were transferred from the individual dishes in which they were kept to the notch in the fence by means of two paraffin-covered wooden spatulas. The worms thus placed were free to crawl into the liquid toward which their anterior end was directed or to withdraw into that on the opposite side of the fence. Nereis was found to be very strongly negatively chemotrophic to all the reagents used. The reaction times of the worms, which increased inversely as the strength of the stimulating substances, were recorded by means of a stop-watch controlled by foot pressure. 432 ALFRED O. GROSS When the worms were placed on the fence with sea-water on one side and ordinary tap-water or distilled water on the other, the worms quickly withdrew into the sea-water, indicating that the latter has a marked disturbing effect on Nereis. Because of this condition, the special substances used as stimuli were always added. to the sea-water. It may not be safe, by this method, to make a comparison of the relative stimulating efficiency of one acid with another or with an alkali, hydroxide, or salt, because of the many substances in solution in sea-water which might affect the reagent. One can, however, make comparisons of the rela- tive sensitiveness of the worms under different conditions. As long as there is a constant stimulating liquid in the mixture of a measured quantity of sea-water and a definite amount of the chemical, it makes no difference for this purpose what the result- ing chemical combinations and mixtures may be. For convenience of comparison the various reagents were made up in molecular solutions and these solutions were added in definite quantities, by means of a burette, to the sea-water in the following proportions. TABLE 2 1 ee. mol. HCl to 300 ec. sea-water lec. mol. KOH to 10 ec. sea-water 1 cc. mol. NaOH to 10 cc. sea-water 1 ec. mol. KCl to 10 ec. sea-water 1 ec. mol. NaH,Cl to 3. ec. sea-water 1 ec. mol. NaCl to + ec. sea-water The worms exhibited a marked reaction when tested with mixtures of sea-water and molecular solutions in the proportions shown in the above table. The reaction times of the worms when tested with these solu- tions were short, but not too short to be accurately measured by means of a stop-watch. Though these concentrations of salts produced only approximately similar reaction times, it is inter- esting to note that it required about fifty times as great a concen- tration of NaCl as KCl to produce an approximately similar reaction time on Nereis; whereas Parker and Metcalf found NaCl to be more stimulating than KCl to the dung earthworm, Allolobophora foetida. This striking difference is probably FEEDING HABITS OF NEREIS VIRENS, SARS 433 correlated with the high percentage of NaCl and the low per- centage of KCl in the sea-water to which Nereis is adapted. The author hopes to perform experiments on this interesting and important aspect of the problem which involves the rela- tions of osmotic pressure and sense of taste to the stimulating substances used as well as the relative stimulating efficiency of the various reagents. This paper involves only those experiments made on Nereis in an effort to determine whether the chemical sense is localized in certain cephalic appendages or in other parts of the worm. For a preliminary test twenty-four worms of a uniform size (10 to 12 cm. long) were numbered and: placed in separate finger- bowls, each containing 20 to 25 ec. of sea-water and a small piece of sea lettuce. The latter aerated the water and provided food for the worm. Each individual of the whole series of Nereis was given one test in its turn with the HCl, then one test with the KOH, and so on until the entire set of readings for each of the two reagents was obtained. The order of the worms in the test was reversed each time a new series of readings was taken. All the experiments were made under conditions controlled for temperature and light, and, as far as possible, free from mechan- ical stimulation. After each individual test, the worm was rinsed in fresh sea-water before being returned to its bowl. The sea-water containing the stimulating substance, as well as the plain sea-water, was renewed after each set of readings, since a small amount of the liquid was carried from one side to the other by the worm. After the reaction time of the worms had been determined, the tentacular cirri, palps, and tentacles of the first twelve of the twenty-four Nereis were removed, while the other twelve of the series were retained in a normal condition to be used as a control. The entire series was left undisturbed for a period of six days, a length of time more than sufficient for the wounds made by the operations to heal. Readings were then made as before to determine what effect, if any, the removal of the cephalic appendages had on the sensitiveness of the worms to chemical stimulation. 434 ALFRED O. GROSS The average reaction time was very much lengthened in the ease of the twelve individuals from which the appendages were removed, but it remained practically unchanged in the unoper- ated animals used as a control. After these determinations were made, the worms were put aside until the appendages were completely regenerated. With the regrowth of the appen- dages the sensitiveness of the worms to chemicals was restored, as evidenced by the reaction time which became about equal to that of the control animals and to that of the same worms before the operations were made. The results of this preliminary experiment indicate that certain of the appendages of the head region are more sensitive to chemical stimulation than the gen- eral integument of the worm. In order to determine whether the chemical sense is shared equally by all the appendages or more strongly developed in some than in others, the experiments were repeated, but. with this difference, only one of the three pairs of cephalic appendages was removed from the worms of any one series. The anal cirri were likewise tested for their sensitiveness to chemical stimulation. The following tables contain the results of the tests made upon Nereis virens under the various conditions indicated. In each case the number of animals used, the number of readings made, and the mean of the reaction times with the probable error is given. It is deemed impracticable to publish the individual readings which, with the preliminary tests, involve more than 2000 determinations. For convenience in comparing the reaction times of the worms under two conditions, the difference of the means of the reaction times and the ‘significance factor’ are also given in the tables. The significance factor involves a comparison of the means of the reaction times including their probable errors. The value of this factor is obtained by dividing the difference of the means of the reaction times of the normal and operated animals by the square root of the sums of the squares of the probable errors of these two reaction times. As an example, take the case of the palps in table 3 in which the worms were tested with KOH. 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GROSS animals with their palps removed is increased to 21.57 seconds with a probable error of 1.45. Substituting the values in the formula as above stated, we have Zip — 9.44 = 12437) * A879)? 4- (145/212 A significance factor greater than about 3 signifies the results are to be considered of scientific value. No importance is to be attached to the difference in reaction times if the signifi- cance factor falls below 3. Furthermore, if this factor becomes greater than 3 in the two sets of readings of the control, then the results of the experiments become questionable, either because of lack of care in performing them or because certain factors, such as light, temperature, etc., were not properly kept under control. An examination of the tables at once reveals the fact that the palps and tentacles are so highly sensitive to chemical stimulation that their removal causes a marked change in the reaction times of the animals. The tentacular cirri, which together have a much greater exposed surface than the ten- tacles and palps combined, are sensitive to a much less degree; indeed, in only the HCl test was there a noticeable change in the reaction time when the eight tentacular cirri were removed. — The significance factor in this case is only 4, so even here the difference in reaction time becomes of doubtful value. The anal cirri, though they are sensitive to chemical stimulation, are not sensitive to the degree that their removal causes a meas- urable change in the responsiveness of the worms. DISCUSSION The fact that the palps and tentacles are much more sensitive to chemical stimulation than the tentacular cirri becomes of more interest when the innervation of these appendages is con- sidered. The palps and tentacles are supplied with well-devel- oped nerves, which arise directly from the supra-oesophageal ganglion or brain, whereas the two pairs of tentacular cirri are innervated by nerves which have a very different origin. The FEEDING HABITS OF NEREIS VIRENS, SARS 439 nerves of two pairs of tentacular cirri arise from the sub-oesoph- ageal ganglion, and those of the others take their origin from the circumoesophageal connectives. In the higher animals, the nerves of special sense, such as sight, taste, ete., are directly connected with the brain. It is reasonable to infer that in a highly organ- ized worm like Nereis, we have the beginnings of a concentra- tion of sense receptors into more or less limited regions which have become secondarily but directly related to a centralized brain. This localization of the chemical sense has not pro- gressed to any great degree, for the whole general integument of Nereis, though less sensitive than the palps and tentacles, is open to chemical stimulation. The same is true with the light sense. The general integument of Nereis is sensitive to light, yet there is a tendency toward a localization of the light sense in the presence of two pairs of relatively well-developed eyes. These eyes are innervated by large nerves which connect directly with the brain. The conditions of these sense organs in Nereis are intermediate between those forms in which there is only the general integumentary sense and the higher forms in which the chemical sense is vested solely in special sense organs innervated by cranial nerves. Maxwell has attempted to show that the feeding responses, that 1s responses due to chemicals or substances given off by food, cease when the supra-oesophageal ganglion is removed. Maxwell’s statement is as follows: ‘‘Operirte Wiirmer beachten dagegen angebotenes Futter gar nicht, es sei denn, dass man es unsanft auf sie wirft und sie dadurch erschreckt. Sie kriechen uber Stiicke Futters, die in ihrem Wege liegen, als ob es Steine oder anderes lebloses Material wire. Obschon ich diese Wiirmer viele Wochen hindurch gehabt habe, ist es mir nicht in einem einzigen Falle gelungen, sie zu fiittern. Mit dem Verlust des supradsophageschen Ganglions scheint das Thier die Fahigkeit verloren zu haben die spezifischen Reaktionen auf die chemischen Reize, die vom Futter ausgehen, zu zeigen.” Maxwell’s experiments show that the removal of the supra- oesophageal ganglion changes the responsiveness of the worms to chemical stimulation—a result which is in direct line with what 440 ALFRED O. GROSS I have found. When the above ganglion is removed, the animal is less sensitive to chemicals, because that part of the chemical sense which resides in the palps and tentacles is lost. The nerves of the tentacular cirri were left intact, as evidenced by the fact that the cirri were still responsive to mechanical stimula- tion. Unfortunately, Maxwell’s experiments are of less value from the standpoint of their bearing on the sense of taste because his observations, as before noted, are not of the feeding reactions of Nereis. The responses he secured by holding a piece of worm flesh in front of Nereis was merely the characteristic defensive thrust, due to chemical stimulation or irritation. These responses are, at best, very irregular and erratic and cannot be used in careful comparative work. In the tests on the operated worms Maxwell placed the stimulating substances, 1.e., pieces of worm flesh in the sea-water containing the Nereis. Under such conditions it is difficult to detect and impossible to meas- ure quantitatively the effect of chemical stimulation on the worm. ‘To such a liquid the worms soon became adapted and not stimulated at all. That the operated worms exhibited no feeding reactions under these conditions is perfectly obvious, because even a normal Nereis does not feed upon flesh, with which Maxwell tested the worms. I have found that worms still respond to chemical stimulation after the brain is removed if tested by the method previously described. This chemical sense of the general integument evidently works through a ganglionic reflex, that is, through the ganglia of the ventral nerve cord. In addition to the rich innervation of the palps and tentacles, as shown by Retzius, there is an abundance of diffuse integu- mentary sense organs to be found on these appendages. Lang- don has shown these organs to be especially numerous on the tentacles and on the tips of the palps. But since these organs are also abundant on the tentacular, parapodial, and anal cirri, their significance in connection with the sense of taste is at least a doubtful one. From the standpoint of distribution, the evidence is to the contrary, and I am inclined to believe these integumentory sense organs, which are also abundant FEEDING HABITS OF NEREIS VIRENS, SARS 441 in the earthworm, are purely tactile. Any evidence that the so-called ‘spiral organs’ are chemical receptors is also lacking. The localization of the sense of taste in the palps and tentacles must be explained by some differentiated specific quality of the protoplasm of these appendages. In Nereis there is a beginning of the localization of the sense of taste or chemical sense, but there have not as yet developed specialized receptors (taste buds) for the reception of chemical stimuli. CONCLUSIONS 1. Nereis virens is not a carnivorous worm as stated by Verrill and others. 2. Nereis feeds chiefly upon plant life. 3. The sense of taste or chemical sense of Nereis plays a small part, if any, in locating or selecting food. 4. Nereis is strongly negatively chemotrophic to acids, hydrox- ides, and salts. 5. Nereis is stimulated much more by potassium chloride than by sodium chloride—a reverse of the conditions found in the earthworm. This difference is correlated with the high percentage of sodium chloride in the sea-water to which Nereis is adapted. 6. The general integument. of Nereis is sensitive to chemical stimulation, but there is a localization or concentration of the chemical sense in the palps and tentacles—a condition correlated with the rich innervation of these appendages and the relation of their nerves to the brain. 7. Though there is a tendency for a localization of the chem- ical sense, there are no specialized receptors, taste buds, for receiving chemical stimuli in Nereis virens. 442 ALFRED O. GROSS BIBLIOGRAPHY Crozier, W. J. 1916 Cell penetration by acids. Jour. Biol. Chem., vol. 24, pp. 255-279. Hurwitz, 8. H. 1910 The reactions of earthworms to acids. Proc. Am. Acad. Arts and Sciences, vol. 46, pp. 67-81. Irwin, M. 1918 The nature of sensory stimulation by salts. The Amer. Jour. of Physiol., vol. 17, pp. 265-277. Lanapon, F. E. 1900 The sense-organs of Nereis virens Sars. Jour. Comp. Neur., vol. 10, pp. 1-78. Maxwe tt, 8.8. 1897 Beitrige zur Gehirnphysiologie der Anneliden. Archiv fiir die gesammte Physiologie, Bd. 67, S. 263-297. Parker, G. H., anp Metcatr, C. R. 1906 The reactions of earthworms to salts. Amer. Jour. Physiol., vol. 17, pp. 55-74. Retzivus, G. 1895 Zur Kenntniss des gehirnganglions und des sensiblen Ner- vensystems der Polychiten. Biol. Untersuchungen, Bd. 7, S. 6-11. Suout, A. T. 1914 Reactions of earthworms to hydroxyl ions. Am. Jour. Physiol., vol. 34, pp. 384-404. Sranparp Naturat History 1885 Lower invertebrates, vol. 1, pp. 1-889. Boston, S. E. Cassino & Co. VerRILt, A. E. 1873 Report on the invertebrate animals of Vineyard Sound and the adjacent waters. U.S. Comm. of Fish and Fisheries, part 1, pp. 295-778 (Nereis, p. 318). et pyc iia A seit: ao su arasttersyy “het - van he oe +t : i my! - da Neel ue Ms * : aa r 4 mn Hy omy ri he } : eel is i eta): oy a i ih, Aes ay aul * 7, ita or an Oy oe Fis TEE smbyhe bide, vai» TERE ee 5 age aged al ve ae We tes Hee Pe ie ae | ; eb" ALE Ni sab binif airs ial UAT IAT) ethan ia _ i al | Tye vel it nice tin olan NTH UBTH the eee a BL, ih plang ba 9 Mayssetig ioehivi eget a MIMO TAS abt bron s ile "1, tee . a rif" Welaelie i ee 9 nee uy i dtobdy THC NTL ee Bete Sa 2 per bis wien rit 1a eth al Wr oye | "i $ | ; = ur oT, oh anid: a wih BUTS rie we a a ao RTA SODA) Voy Aa 2 oS Ah i pi j i. ‘ ‘ J a J ‘ 5 a f ‘ 1%) ree fj ey 4, * at \ eae APR | i it i f wey pe) by v t c ¥ : a) i 7 , ( . rT) | . i i Thi j Aa i) bin ¥ : Vale ; , ; i Dy Z iv : : : ’ ire ‘ - valet a P ’ 4 S au 1 @ * ‘i ee f j , 1 bate he aa Ley ekg, j ie _— ‘ ms ; te as pdr ral . * = * ' bey ' mr a ia az ( y ; ae | i _? : on wei? na -uarin ety ad y poids ou } ra ¢ io ; diate i } : ; ' : on Aare ay i vy ; up § , . a i q a f nt { {) oy 4 ‘ i. ie " ‘ 4 ; 4 iy K teat a er ve . 4 i a nepdluth + os hye suey Dy ii inet. ith a, ais } | ne ¥ pr lp'aiys nly: LAI! 0 LAMA AEN t=. Ps ee pul rare Py pate bis ‘iss yn oteleisdeetiat - Resumen por el autor, W. J. Crozier, Rutgers College. Sobre la historia natural de Onchidium. El] presente trabajo contiene una descripcién de las activi- dades de Onchidium floridanum, miembro de un grupo de pul- monados semi-marinos notables por sus rasgos estructurales de naturaleza enigmatica, y, como demuestra este trabajo, también por sus costumbres no menos notables. A una discusién de las reacciones sensoriales sigue un andlisis comparativo del heliotropismo y especialmente de la conducta de este animal al buscar una habitacién. El autor insiste especialmente sobre el cardcter no adaptativo del heliotropismo en Onchidium y sobre la interpretacién mecanistica de los fendmenos que exhibe al buscar un albergue. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY i ON THE NATURAL HISTORY OF ONCHIDIUM! LESLIE B. AREY anp W. J. CROZIER Northwestern University Medical School and Hull Zoélogical Laboratory, The University of Chicago CONTENTS [AGRE CHICIT GLO 5 APNE GT RRR. che, Semper 4 Nc Ane Oe Se OR 0 Co a 443 Occurrence; ‘homing’ activities; coloration; repugnatorial glands.......... 445 SEMSOLVALCSDONSCSI ae hs eee epee ioe -scie ay chkosrebererne cae arches cto. saetetstereis efelees 459 feae\ViechsmicaitexcheahlOMs. Oe e112 too ae. - «Skee ANRENAT 22 2 = Sel aa « dees 459 PMO GCL RCIA 1 ON spt sient Sete oak MELA Se 2h oS 2) ase ceene heks Sen oy hey Sevrey A 464 SMM eMM alex CUDA LION skis she Asia ects, << w a's repens aie NS «os vuole Rich see tole Gack deste 474. Ham OLeMiC Alege xCloAULOM 1 ae tematic sorte cists cco cue errors ie nker oe te Sere NE saps al os 47 Ontthe- analysis of the hontmg behavior.).........2 000. fh. Se 479 MICO RTO fe Mot ot. oh artis « I MER: CARRE s oo SARS Dae b oie Set See e. MeR« eee 489 ie Origimuor One brain: AM eek certs k aen,0 Sethe, vf? Qaereree hia eos se Se ee 489 2. Heliotropism and the analysis of conduct. FR ee ryt Aah Sa Sts, AEE OD 3. On the nature of heliotropic inhibition nl SE UeEEl biccel 2 omket bese AON 4, The question of persisting rhythms of behavior.................... 493 5. The comparative physiology of homing movements................ 493 SLID hla b Gtr Gere Poe BEG DOE DOH GOS Rei anircoecing Soe Horror Eyer 497 Literature ade dictate ae REE, Cit BU ch OE aI or RIA eS toh Cee aA rte 499 INTRODUCTION Onchidium (Onchidella) floridanum Dall belongs to a group of naked pulmonates (Pelseneer, ’01, p. 21) which, after the long discussion concerning the obscure morphology of their respira- tory apparatus (cf. Joyeux-Laffuie, ’82; Bergh, ’95; v. Wissel, 98), have been chiefly remarkable for their littoral marine habitat, and, more conspicuously, for the eyes—of a structural type unique among gastropods (Semper, ’77; Stantschinsky, 07; Hirasaka,? ’12)—developed by some of them upon their ‘ Contributions from the Bermuda Biological Station for Research, no. 126. 2 We are indebted to Mr. T. Minoura, of the zodlogical department, Univer- sity of Chicago, for a knowledge of Hirasaka’s paper, and for his kind translation of essential portions of its contents, as well as for his translation of Fujita’s (’97) account of the respiration of the Japanese Onchidium. 443 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 32, NO. 3 444 LESLIE B. AREY AND W. J. CROZIER dorsal surface. In connection with these strange eyes and their supposed functional significance (Semper, ’81; Bretnall, ’19) a few observations and some suppositions have from time to time been recorded with reference to the behavior of Onchidium; but knowledge of its life and habits, which we find to exhibit some perplexing features of curious interest, has been strangely meager; nor has proof been offered that the ‘eyes’ are indeed photo-sensitive (cf. Crozier and Arey, 719¢e). At Bermuda, O. floridanum is quite generally distributed along the shores of the islands, commonly inhabiting protected loca- tions where the intertidal shore zone is covered by a layer of sun-bleached algae, although it is also found in intertidal habitats of other kinds (Crozier and Arey, ’19a). O. floridanum is an Antillean species closely resembling others of this genus found in the Pacific (Dall and Simpson, ’01). Like most of its rela- tives, this species is strictly intertidal; some forms have been recorded as dwelling beneath low water. Onchidium is a good laboratory animal. It can be maintained in small dishes with a little sea-water for a month or longer, even if starved. In one case several individuals were kept in small (50-ce.) bottles, tightly stoppered, and half full of sea- water, in which diatoms had been planted. They were alive, active, and of normal appearance at the end of six weeks. The mollusks were observed to creep above the water in the bottles at irregular intervals, later returning under water and feeding upon the plant grow “lhe there. For experimental observations it is nevertheless desirable that freshly collected animals be employed, and this has been the rule in the work forming the basis of the following discussion. Our material came, in 1914-17, from Little Agar’s Island, and in 1918 from the shore of Dyer Island, in each instance very close to the locations of the Biological Laboratory in these years. In addition, much of what we have learned concerning the behavior of Onchidium is the result of sun-baked vigils on the shore rocks in other parts of Bermuda. The work was initiated in 1914 by L. B. A., and has since then been extended by W. J. C. The observations here collected pretend to no NATURAL HISTORY OF ONCHIDIUM 445 exhaustiveness; they do, however, throw valuable light upon some vexed questions, particularly in connection with the theory of adaptation. It is therefore important to remark that upon some of the peculiar features in this account of the behavior of Onchidium our respective observations, secured in an entirely independent manner, have been found in essential agreement at every point of overlapping. Elsewhere we have already com- mented on certain points in the behavior of Onchidium (Arey and Crozier, 718; Crozier and Arey, 19a, 1919c). It is our purpose here to bring together in a unifying way the results of our previous communications, presenting fully the evidence concerned and indicating its place in a more systematic account of the natural history of this animal. OCCURRENCE;. HOMING ACTIVITIES; COLORATION; REPUGNATORIAL GLANDS If, during the period of low water, on a sunny day, one inspect the shore rocks at Bermuda laid bare by the tide, at a place protected from the dashing of the surf, one frequently has little difficulty in discovering numbers of small blue-black slug-like Onchidia, about 15 mm. long or smaller. They feed quietly upon the thin coating of felted algae or creep about in a manner which at first sight seems aimless. Favorable localities are found on the lee shores of smaller islands within the semi-enclosed sounds (Great Sound, Castle Harbor, but not in Harrington Sound, where there is practically no tidal rise and fall and where no Onchidia occur). The animals also live, however, in bays on the north and south shores of Bermuda, but not where they would be directly exposed to the ocean surf. The body of the adult Onchidium when resting undisturbed is dome-like, oval in outline, at most 17 mm. long xX 12 mm. broad X 6mm. high. At the anterior end, two slender tentacles, with knobbed ends, project from under the mantle-fold. Two large ‘oral lappets,’ the ‘cephalic lobes’ of Pelseneer (’01, p. 20), overlie the mouth region; in creeping they are constantly in slight contact with the substratum. The margin of the encircling mantle-fold is serrated; certain of the marginal projections rep- 446 LESLIE B. AREY AND W. J. CROZIER resent the location of large mantle-glands, which give rise to a repugnatorial secretion; there are fourteen of these glands in O. floridanum, seven on either side. Although at about the time of low water Onchidium is often abundant upon shore rocks (ef. Semper, ’81; Eliot, 99), and in many places even conspicuous, during high tide it is invisible. It never wanders above high-water mark nor beneath the water level. In these respects it differs sharply from a much smaller species (Onchidiella) of intertidal habitat also found at Bermuda (Arey and Crozier, 719 a, p. 163), the latter creeping about when covered by the sea, but being sheltered within dead Serpula tubes, barnacle shells, and the like during low tide. Eliot (’99) has briefly referred to the occurrence of O. tonganum on the tidally exposed reefs at Apia, Samoa. O. floridanum also lives upon more or less isolated rocks and islets, within Great Sound, but does not occur on the Bermuda ‘coral’ reefs, which are not exposed at low water. We were soon struck by the fact that sometimes, even when the tide was not high, no Onchidia were procurable in places known from preceding and from subsequent observations to be thickly populated by them. There is in fact a very exact rela- tion between the appearance of the Onchidia upon the rocks and the state of the tide. This relation involves some curious and precise ‘homing’ habits, of a kind hitherto unsuspected among mollusea (Arey and Crozier, ’18). Onchidium lives in communities numbering a dozen or more individuals, with pocket-like ‘nests’ in the eroded shore rock. The openings to these nests are as a rule very inconspicuous. The mollusks creep out of their nest only when the tide has a little more than half ebbed, that is, about 2 to 2.5 hours before low tide. For any particular nest the time of emergence depends upon the nearness of the nest to the high-water mark. Those Onchidia living in nests located nearest to the high-water level appear in the open sooner than do those situated farther down. It is the actual position of the cavity of the nest itself, in rela- tion to the tidal level, rather than the location of its external opening, which regulates the moment of emergence. NATURAL HISTORY OF ONCHIDIUM 447 A common habitat of Onchidium is on isolated rocks which are more or less completely submerged at high water and are covered by a yellowish-brown felt-work of algae. On such islets, and on more extensive protected shores of similar appearance, numbers of tiny crevices are almost invariably lined with a layer of Modiolus,? and these frequently contain a passageway to an Onchidium nest. The eroded cavity in the limestone forming the irregularly shaped nest is sometimes of the bigness of a man’s head, though usually much smaller. The external openings of the passageways are quite inconspicuous, for they are not only small, but they are further masked and partially choked by the growth of Modiolus. It is astonishing through how small an opening an Onchidium can slowly make its way, as it insinuates itself into the tiny spaces between the mussels. An individual that is 5 mm. high when normally creeping can squeeze through the space between the edges of two glass slides held 1 mm. apart. In nature the process seems even more startling. When a group of Onchidia emerges from the nest the individuals appear one at a time in continuous series. Colonies were also found established at sheltered spots where loose stones were held together by red clay, the nest being here a deep crevice between two stones. This type of habitat is less common than that afforded by eroded limestone. Following the emergence from its nest, a colony of Onchidia wanders in various directions over the rock. Some individuals may creep a meter from the nest. They remain exposed for a certain length of time, and then, s¢multaneously, return directly to the nest from which they came. The individuals emerging from one nest sometimes become more or less scattered, separated, and even somewhat mingled with others derived from other nests. Among the components of any one community, however, the coincidence of the return to the home nest is in most cases, if not indeed in all, remarkably 3 This Modiolus is sometimes found underneath small stones and on the under sides of rock slabs, where numbers of stones occur piled together. In such dark- ened situations the color of the mussel is not black, but, on the contrary, retains the reddish-brown cast of the juvenile shell. 448 LESLIE B. AREY AND W. J. CROZIER close; one is tempted to compare this rather startling exhibition to the effect of a thunder-storm in causing human families to retire to their respective homes. On the other hand, the colonies which, in any given place, are the first to appear are likewise the first to retire, so in this way a separation of the different communities is effected, favorable to their exact observation. Independent study has convinced us that there is probably no ‘mixing up’ of the individuals from different communities; in fact, we have never seen an instance in which an individual coming from one nest and carefully watched during the whole of its unmolested perambulations, failed to return to its original home. The process of return to the nest has a highly determinate aspect. The return course is direct and as straight as is per- mitted by the physical imperfections of the substratum. In creeping back to the nest an Onchidium may move toward the sun, although if removed from the rock and immediately placed on a glass plate it will be found negatively heliotropic. Helio- tropism has nothing to do with the direction of normal creeping (Crozier and Arey, 719 ¢). In the immediate vicinity of the entrance to the home cavity, the Onchidia frequently make use of a natural groove in the rock. In the case of small colonies, all the individuals may utilize this trail. The members of larger colonies, however, may simul- taneously approach the nest from several widely separated points. Arriving at the entrance to the nest, they crowd about it in an absurd orderly fashion, and without restlessness ‘await their turn’ to enter. Owing to the fact that the opening of the nest is usually quite small, several minutes may be required for an individual to insinuate its body into the opening. Hence the disappearance of the whole colony within the nest usually occupies some time. Once, however, they have collected about, or upon, the mass of Modiolus which commonly surrounds or even partially occludes the entrance to the nest, the Onchidia, because of the similarity of their coloration to that of Modiolus, may readily be overlooked. NATURAL HISTORY OF ONCHIDIUM 449 The following notes of one emergence of the Onchidia inhabit- ing a section of Little Agar’s Island will give a sufficient general idea of the phenomenon, which we have repeatedly observed: July 6, 1914. A cloudy, but fairly bright day. Low tide at 11:30 A.M. 9:57 a.m. A few (4 or 5) Onchidia ‘out,’ others*in process of emer- ence. 10:02 About a dozen out. Many at once begin to climb straight upward. Others wander in devious paths; if on a flat rock- shelf, may start toward the water, but do not actually go downward. 10:12 In one cove on S. E. side of island 24 individuals seen. Over 100 seen on flat rocks at opposite side of the cove, to the west- ward; at 10:02, only 1 or 2 were to be seen here. 10:17 A few stragglers are still appearing. 10:32 Cl NO; >) Br The cation series is practically the inverse of that usually found in such eases, and of that which we have found for the sensory activation of several other mollusks (Arey and Crozier, 19; Crozier and Arey, ’19b), employing comparable criteria. The anion series is more like that commonly reported. The relatively great stimulating power of NaCl may be related to the fact that Onchidium creeps upon rock surfaces wet with sea- water, but exposed to the evaporating power of the sun. In this connection it should be noted that the mouth region is decidedly the most sensitive part, and also that in an actively creeping animal it is not possible, as a rule, to obtain sensory responses from activating substances at concentrations so dilute NATURAL HISTORY OF ONCHIDIUM 479 as in the case of resting individuals. These features may play a part in directing the movements of Onchidium in nature. There is some evidence for the view that receptors for general ‘irritating’ activation are distinct from chemoreceptors proper. Many substances which provide powerful sensory excitants for Onchidium do not induce discharge of the repugnatorial glands. Methyl or ethyl alcohol, however, at 5 M concentration in rain- water, do lead to such discharge; whereas pure amyl alcohol, directly applied, does not, although the general responses of the animal are in the case of the amyl aleohol much the more vigor- ous. Chloretone also, and urea, in relatively concentrated solu- tions, do not lead to discharge of the poison glands, although they do induce powerful reactions on the part of the animals as a whole. An effort to effect physiological separation of tactile- and chemo-reception was without decisive result. It may be noted that, according to Joyeux-Laffuie (’82, p. 238), O. celticum exhibits in its feeding a certain amount of pref- erential selection of Ulva from among a diversity of algae. He regarded this fact as evidence of a certain degree of ‘gustatory’ discrimination, associated with the mouth parts, but pointed out in addition that the oral lappets were employed in feeling over bits of algae before their being submitted to the radula. This latter observation we can confirm for O. floridanum, but we have seen no evidence of:selective activity respecting food, perhaps because the thin algal carpeting of the rock in Onchid- ium habitats presents so uniform a field. Some of the older writers have referred to the ‘eating’ of silt by Onchidium; we have already pointed out (Crozier and Arey, ’19a) that shore-line silt is ingested, but in a purely fortuitous manner, through its adherence to the seaweeds. ON THE ANALYSIS OF THE HOMING BEHAVIOR An attempt to interpret or to reconstruct the natural activi- ties of Onchidium on the basis of analytical study of its modes of response when removed from its habitual environment is con- fronted at once by some profound inconsistencies and by the 480 LESLIE B. AREY AND W. J. CROZIER puzzle of ‘homing’ behavior. Although we have to offer some suggestions toward such an interpretation, they are presented with due reserve as to their probable finality. No more striking instance is known to us of apparent incompatibility between the results of controlled experimentation, repeatedly verified, and the most obvious activities of the same animal’s natural life. First, as to heliotropism. In the laboratory Onchidium behaves as a typical negatively heliotropic animal. In nature its behavior is in every essential at variance with this finding (Arey and Crozier, ’18; Crozier and Arey, 719 ¢). Not only does this mollusk creep out of its dark nest into the glaring sun, when the tide is falling, to feed, but it does so only during daylight hours, and never at night, no matter how bright the moon. During the period of its emergence an Onchidium’s movements seem not in any degree influenced by the sun. An individual quietly creeping in brilliant sunlight may be shaded by a board, and after re-expansion following its response to the shading, it creeps on as before. If now sunlight be thrown suddenly on this animal from a new direction, with a mirror, it may momen- tarily ‘hesitate’ somewhat, perhaps turn very slightly to one side, but soon it continues as before. It is not merely the presence of a normal kind of shore sub- stratum which determines this suppression of heliotropism, because animals brought from laboratory stock (secured several days before) and placed upon the same rock are oriented pre- cisely by the sun. The presence of the specific rock surface normal to the Onchidia emanating from a given nest, namely, of that surface within a certain small radius of their nest entrance, is the deciding influence. An Onchidium moving toward its nest entrance, but heading directly into the sunset, may be picked up and then replaced upon the rock without interfering with its course. But if a glass plate be slipped under the ani- mal before replacing it, it is now at the mercy of its heliotropism. If an Onchidium from one section of the shore be quickly trans- ferred to a strange zone, again the animal is oriented by the light. Obviously, there is no question here of the mere rapid exhaustion of the heliotropic mechanism or other kind of ‘light adaptation.’ NATURAL HISTORY OF ONCHIDIUM 481 It is not that the Onchidium, ‘seeking its best interests,’ comes out into the light to feed and in so doing ignores the dictates of its negative heliotropism. Definite evidence, on the contrary, is available to the effect that under normal circum- stances the central nervous mechanism of heliotropic movements is inhibited, perhaps ordinarily by impulses originating in the oral lappets which compete successfully for the control of the body musculature. ‘This inhibition can be abolished (‘reversed’) by means of strychnine (Crozier and Arey, ’719c). Reversal of inhibition within the ganglia of mollusks is known in Chiton, in Chromodoris, and in Cephalopods (ef. Crozier, ’20). A possible explanation for the existence of negative heliotro- pism under any circumstances may be found in an imperfection of the photoreceptive system. The response of Onchidium to sudden shading is clearly of conceivable survival value; it leads to the retraction of the tentacles, the cessation of locomotion, and the depression of the mantle-fold to the substratum; the mantle is thus enabled to assist as a hold-fast, for the suction power of the foot is but poorly developed, and in any event the algal-covered surface affords a difficult field for contact attach- ment by the small foot of an animal so light in weight that it does not flatten out the algae. Our suggestion as to the nature of the heliotropic irritability, depending on the continuous: chemical activity of incident light, involves the assumption that the stimulation so produced is in this case bound up with sub- stances forming a necessary part of the receptive mechanism for the response to shading. The obviously efficient manner in which heliotropic impulses are normally blocked, in such fashion that they play no detectable part in controlling the creature’s movements, may account for the fact that this deficiency in the photosensitizing mechanism has failed to suffer selective elimina- tion. A balanced system of photocatalizable reactions was postulated to account for the phenomena of photic irritability in Holothuria (Crozier, 714). The general features of such a system have been (in Mya) admirably treated in a quantitative manner by Hecht (19). For Holothuria it was suggested that both phases of activity within the photosensitive system— 482 LESLIE B. AREY AND W. J. CROZIER photolysis in light of given intensity, and resynthesis in light of lower intensity—might be involved in stimulation by light and by sudden decrease of light intensity, respectively; here, both forms of irritability play rdles of bionomic importance (Crozier, 15), whereas, according to this notion, but one phase of the matter, namely, sensitivity to shading, is permitted to exert an influence upon the normal behavior of Onchidium. The mechan- ism whereby the possibility of heliotropic response is normally prevented has already been discussed (Crozier and Arey, ’19¢e). It appears to depend upon specific impulses originating in the oral lappets, at their points of contact with the substratum, for when these lappets are removed or are anaesthetized by MgSOs, the Onchidium becomes photonegative, even if replaced upon the specific rock surface from which it was taken. The rhythm of the tides, ordinarily well defined, controls the emergence of Onchidium upon its feeding ground. Under cer- tain conditions the orderly succession of periods of low water is seriously interfered with. High winds and accompanying ocean currents, during times of storm, not infrequently cause such a ‘piling up’ of water within the semi-enclosed sounds at Bermuda that the water may fail to fall appreciably for several tides; such a period is followed, also, by a certain irregularity in the tidal sequence. Only when the water level has become lowered to the proper degree, previously indicated, do the snails emerge. Conversely, extensive periods of low water, notably occurring at spring tides, may leave the rocks uncovered along the Onchid- ium zone for an uncommonly lengthy interval. The duration of the emergence of a given colony is practically fixed, however, and gives not the least indication of being normally terminated by the rising of the tide; rather, the duration of the feeding time is determined in a quite different way, which we shall shortly consider. The facts thus far presented do not exhaust the curious intri- eacies of the behavior of our snails. It has been mentioned that Onchidium comes out from its nest only during the daytime, and never at night. Were it not for certain serious obstacles, all this might be understood in terms of an hypothesis advanced NATURAL HISTORY OF ONCHIDIUM 483 by Joyeux-Laffuie (82), namely, that the tentacular eyes enabled the mollusk to distinguish between the darkened interior of its shelter and the illuminated outside feeding ground, thus guiding its emergence.’ The difficulties facing such interpretation are: 1) the fact that the tentacles of O. floridanum, although there are tentacular eyes, seem non-photosensitive; 2) the fact that the creature is never positively heliotropic and, 3) the fact that emergence does not always occur when the tide is out during daylight hours. Good instances of the sort last mentioned were available in midsummer, when for several days at each lunar interval both tides were seen to expose the beach zone while the illumination was still quite good. At these times the snails were always found to emerge during but one tidal ebb, never during both. Once in each twenty-four hours is the maximum frequency of emergence. Even in the absence of conditions imposed by winds and currents impeding the escape of tidal water from the sounds, however, this rhythm does not represent the minimum frequency of emergence. For at neap tides the Onchidium nests highest above water may fail for some twenty-four hours to be submerged at flood tide, and in that case the Onchidia there located do not emerge until after their nest has been submerged. Moreover, in winter both morning and even- ing tides may fail for a day or so to occur during daylight hours; in this event, so far as we have studied the point (Dec., Feb., 1918), the Onchidia do not emerge at all until one tide occurs while the sun is up. The diurnal rhythm thus clearly established ‘in defiance of? the snail’s heliotropism completely disappears in the laboratory. Fair-sized slabs of stone, a foot or so in breadth, were placed in aquaria containing freshly gathered groups of Onchidium. ‘The animals always collected after a short time on the shaded, under side of such a slab, whether under water or in air. In the dark (artificial darkness or at night), they crept actively over the surface of their stones, feeding; the admission of light quickly caused typical photonegative retreat to the dark, under surface 8 The fact that emergence occurs only during the daytime was not known to Joyeux-Laffuie. 484 LESLIE B. AREY AND W. J. CROZIER of the rock. It was not possible, although several times the attempt was made, to establish an artificial tidal rhythm by periodically lowering the water level in such aquaria; this phase of the work will, we hope, be continued. Experiences of this sort plainly indicated the existence of some very specific corre- lation between the natural behavior of an Onchidium and the features of its ordinary home. In our opening description of the chief phases in the daily life of O. floridanum we have already given a brief account of the most remarkable aspect of this specific correlation, namely, the snail’s ‘homing’ behavior. It was obviously necessary to inquire into the nature of this peculiar activity. Although our results are not in any sense exhaustive, for we were unable to complete the series of experiments planned, the evidence we do command nevertheless permits a fairly precise characterization of the major aspects of the homing process. Involved in this matter are: 1) the fact of almost simultaneous commencement of the return to the ‘nest’ on the part of the scattered members of one colony, 2) the fact that the duration of the feeding interval seems automatically fixed without reference to the rising of the tide, and 3) the evidence concerning central inhibition, already referred to in connection with the normal abeyance of heliotropic movements. To these points we shall return, after dealing with the directed creeping toward the nest. The fact that Onchidia are able to return to their nest after being picked up and replaced at some other point within a cer- tain radius of the nest aperture is best appreciated from the perusal of such records as the following: July 1, 1914. Little Agar’s Island. Five individuals were seen returning to a nest. Two of these were removed to the rock surface above the high-water mark (where, in our experience, these animals never wander naturally); the distance of the new point of departure was perhaps 40 cm. from the aperture of the nest. One of the displaced Onchidia turned directly toward the nest and crept straight back to it; the second one ‘lost its way,’ and wandered off in a strange direction. The three other members of the original group of five marched in a sort of triangular formation toward the nest aperture, two going to one side of the opening, the remaining one to the other side, then all three crept into the nest. NATURAL HISTORY OF ONCHIDIUM 485 The nest was now broken into with a chisel. The three individuals inside were removed, and placed on a flat rock surface above the nest opening, and at three different points each some 46 to 50 cm. from the nest. All three Onchidia succeeded in effecting a return to the mouth of the nest. Near the nest two of them followed a slightly grooved trail; this trail or channel had also been used by the one return- ing individual described in the preceding paragraph. The channel led directly to the mouth of the nest. In getting into this trail, each indi- vidual had to change its course greatly. When they had reached the region about the nest aperture where the rock had been broken in examining the interior of the cavity, the Onchidia became much ‘con- fused’ and merely wandered about on the outside rock, where they were left as the tide rose. July 2, 191 4: A group was noted returning to a nest, and one individual was picked up and replaced on the rock on the opposite side of the mouth of its nest at a distance of 1 meter therefrom. The animal returned directly to its nest. In subsequent years many trials of this sort were carried out, and always with essentially the same result. It is possible, but not probable, that some interesting results would have been obtained by comparing carefully the homing capacity of Onchidia of different ages. In the autumn three. groups of fairly distinet size are noticeable in the Onchidium population, so these snails probably live two years at least, if not more. Nevertheless, our experiments did not disclose any differences in homing ability among the individuals of different sizes. Factors which more noticeably affect the ability to ‘home’ after experimental dis- placement are the natural extent of the normal feeding area and the degree to which this area is populated with nests. These two factors are usually correlated quite closely. Boulder-like rocks more or less isolated from the shore are frequently so eroded as to present a veritable honeycombed aspect; a rock 3 feet by 2 in cross-section, projecting some two feet above m.l.w., was found to harbor about thirty Onchidium nests, if not more; less eroded rocks, often affording considerable expanses of flat surface, were seen to shelter an Onchidium population much less dense. In a habitat of the latter sort the distance limiting successful homing was about 1 meter, while experiments on rocks of the type first mentioned (on the south side of Dyer and of 486 LESLIE B. AREY AND W. J. CROZIER Tucker’s Island) showed that homing from distances greater than 30 to 40 em. was not obtainable. The fact that the course of an Onchidium when creeping out to feed may be quite ‘haphazard,’ zigzagging here and there, while the homeward course is usually as direct as the substratum allows, as well as the findings in experiments just cited, shows that it is not necessary for Onchidium to follow its own slime track. Limpets do adhere to their own tracks (ef. Davis, 795; Bethe, 798; Orton, ’14, ete.); Bethe (loc. cit.) thought that limpets were guided in their return journeys by a sort of chemo- taxis, which led them to follow their own slime trails. An Onchidium picked up when on its homeward journey and placed upon a clear glass plate in diffuse light does not tend to adhere to its own slime track, nor to the slime tracks of other indi- viduals. The same result obtains with paper or the surface of a brick. Nor do these snails ‘favor’ a wet surface over a dry one (glass or filter-paper). An individual from a strange section of the shore put down on rock near an Onchidium nest will creep without hesitation across fresh trails of others. All the facts which we have been able to gather about the homing of Onchidium may be brought into relation according to the hypothesis which we now set forth. Complete demonstra- tion of the validity of this notion involves further experimenta- tion, the nature of which we indicate. The Onchidia in any one colony emerge from their nest after the tide has fallen so far as to have left it above water level for about a half hour to an hour. They scatter over the rock sur- face and feed. In the unfed condition certain sensory impulses otherwise directing and controlling the creature’s movements in such fashion as to cause it to return to the nest are inhibited. — The possibility of such central inhibition is given from the ‘reversal of inhibition’ with respect to phototropism seen under strychnine action while the snail is on the surface normal to it. After having fed for a certain time, substances derived from materials ingested while feeding pass into the juices of the snail’s body and produce a ‘reversal of inhibition’ so far as the ‘homing’ impulses are concerned. Reversals of behavior follow- NATURAL HISTORY OF ONCHIDIUM 487 ing feeding are known in such animals as the Porthesia cater- pillars of Loeb (18, p. 116), and the Planarians studied by Olm- sted (17b). This hypothesis readily accounts for the fact that the period of feeding lasts very nearly the same length of time in all the members of a group. The sensory impulses thus conceived to be released from central inhibition through the results of feeding are regarded as originating in the oral lappets. These well-developed ‘cephalic tentacles’ are constantly in touch with the algal carpet of the stone. If they are cut off, the Onchidium is ‘lost,’ unable to return to its home. The removal of the dorsal tentacles, some- times regarded as the seat of ‘smell’ in snails, has no such effect. These impulses must be regarded as possessing the character- istics of ‘contact odors’ (meaning thereby that perhaps both con- tact and ‘olfactory’ stimuli of a certain kind must be received simultaneously). The reason for this assumption is twofold: in the first place, an Onchidium beneath which there is shpped a glass plate is left thereby at the mercy of its heliotropism; sec- ondly, an Onchidium will ‘home’ from points which it has not previously visited; therefore, the aereal dissemination of some guiding substance must be presumed. The olfactory com- ponent of such a complex must be regarded as more important than the tactile, for the rock surface above high-water mark is not covered by algae as is the surface natural for Onchidium, nevertheless the snails will ‘home’ from points on the former surface, although in the ordinary course of events they never go above high-water mark. They will not home when put under sea-water, even if quite near their nest. The substance providing a tropistic guide for a fed Onchidium must be granted some highly specific quality. In view of Bethe’s findings for ant colonies (’98), such a supposition need not be thought preposterous. Moreover, it is supported by some striking results in our experiments on homing. In a number of trials an Onchidium from one community was so placed that it was forced to creep across the sunken gully leading to the opening of a strange nest. Sometimes such a snail was found to follow the new ‘trail,’ after a certain amount of preliminary turning 488 LESLIE B. AREY AND W. J. CROZIER back and forth or ‘hesitation,’ and even to creep within the new entrance. But never, in these tests, did such a snail remain in the strange nest. Not infrequently several journeys were made about the foreign opening (Arey and Crozier, 18). A further significant result was that in several of the tests made upon Onchidia taken from nests subsequently found to possess two apertures, the successfully homing snails gained entrance by way of a cleft different from that which they had followed in their undisturbed homeward trip. The specificity of the assumed ‘olfactory’ substance is not ‘remembered’ by an Onchidium after twenty-four hours’ con- finement to the laboratory. This point was repeatedly tested at Dyer Island. Such confinement obliterates the possibility of homing to the old nest, even from distances of a few centimeters. Our conception of the rdle of the oral lappets might be taken to explain the functional significance of certain curious glands located on these organs, in certain species. Plate (94), with Oncis, and later Pelseneer (01, p. 20), with Oncidiella patelloides, found on the sides of the oral lappets a pair of symmetrical aper- tures, the orifices of glands compared by Pelseneer to the anterior tentacular glands of Vaginula, but of unknown function. In QO. floridanum, however, these glands are not present, otherwise one might suggest that these peculiar organs furnish a mucous covering for the oral lappets, perhaps containing a material serving as a specific solvent for hypothecated odoriferous emana- tions from the nest. It would be interesting to know how widespread the ‘homing’ may be among these related species. This hypothesis not only accounts for all the facts known to us, as already stated, but obviously avoids reference to such obscurely defined notions as ‘muscular memory’ and the like. The hypothesis could best be tested by means of experiments upon the homing tendencies of Onchidia which had not been permitted to feed, and by the attempted discovery of the sub- stance naturally responsible for our ‘reversal of inhibition.’ It should be noted that we distinctly avoid saying whether or not such substance may be derived from’ the algae ingested, because a certain amount of caleareous mud is also swallowed while feeding (Crozier and Arey, ’19 a). NATURAL HISTORY OF ONCHIDIUM 489 DISCUSSION 1. It is desirable to deal, as briefly as may be, with certain of the more general implications of the conclusions provided by our inquiry into the habits of Onchidium. The Onchidiide are a group well calculated to cause the zoélo- gist trouble. For a long time the taxonomic affinities of these organisms were hazy and in dispute, for it was by some (Bergh, 95; Fujita, 97) supposed that, in addition to dermal respiration accomplished through mantle papillae (conspicuously developed in certain species) when under water, air breathing was also carried out, but by the organ regarded as a kidney—an idea once used as the foundation of von Ihring’s class ‘Nephropneusten,’ but now known to have resulted from an imperfect acquaintance with the difficult morphology of the true lung (Plate, 94; von Wissel, 98; Pelseneer, 01). Thus we are probably dealing with a member of a typical land group, Pulmonata, which has second- arily taken up the habit of living on the seashore. It would be valuable to know whether Onchidella is a more archaic type than Onchidium proper (Plate, ’94), or the reverse. According to Bretnall (19), Onchidium dimelii lives either altogether below low water or between tidal limits. 2. The activities of these animals are not less curious than their presumptive evolutional history. In the case of numerous invertebrates of the shore zone it has seemed possible to provide a clear description of behavior in terms directly stated by the outcome of analytical experiments. In fact, this general method of study has been made the basis of much recently published work in animal ecology. The ethology of Chiton (Arey and Crozier, ’19) and of Chromodoris (Crozier and Arey, ’19b) can be followed with gratifying completeness from relatively simple experimental results. With Onchidium the situation is more subtly complicated, and for the purpose of ecologic inter- pretation the isolated results of such a method are here almost meaningless, as shown conspicuously by the creature’s helio- tropic responses. Michael (’16) and others have recently been to some trouble to emphasize the fairly obvious point that no 490 LESLIE B. AREY AND W. J. CROZIER amount of mere laboratory investigation makes it absolutely certain that we may predict the movements of an animal in nature. In reality, however, it is largely a question of the rela- tive completeness with which experimentation is conducted; nor does it require much penetration to discover that the necessary degree of completeness may differ in various cases. A point of some interest, although perhaps unduly speculative, concerns the historical source of Onchidium’s heliotropic machin- ery on the receptor side. That the possibility of heliotropic orientation does not entrain adaptive consequences, seems ade- quately demonstrated by a previous discussion (Crozier and Arey, 719 c). But most land pulmonates are negatively helio- tropic. Might it then be conceived that the sensory organs involved in this form of irritability are mere ‘holdovers’ from the more ancient stock? Aside from the fact that the skin of at least some snails and slugs is photosensitive (Yung, 710), very little information useful in this connection has been discovered. It must be remarked that the mechanisms for sensitivity to light and to shading are seemingly closely connected, if not identical, in Onchidium; nothing of this sort is known for other pulmonates. More important, however, is the conclusion of Stantschinsky (07) regarding the origin of the dorsal eyes (mantle-eyes) in the family of Onchidia: he has shown it prob- able, on general morphological grounds, that the more highly developed forms of mantle photoreceptors are indeed primary, rather than a secondary development, and that species, there- fore, such as O. floridanum, which lack the dorsal eyes have arrived at this condition through secondary degeneration. Yung (713) holds that certain gastropods are ‘blind,’ their tentacular eyes being non-functional, and that this is due to the fact that the optic nerve fibers fail to penetrate the basement membrane of the retina. The lack of apparent functional activity in connection with the tentacle eyes of O. floridanum might be of interest in relation to this conception, were it not for the fact that the conditions here may not involve a complete absence of innervation of the retinae. So far as they have been made out from carefully studied sections, the relations of the NATURAL HISTORY OF ONCHIDIUM © 491 ‘optie’ nerve in Onchidium seem to be as follows: The tentacular nerve, entering at the base of a tentacle, runs mainly to the periphery of the tentacle, ramifies there, and ends in intimate association with numerous large, clustered nerve cells near the tip of the tentacle; at the level of the eye-cup, a small ramus is split off from the main course of the nerve and passes to the eye, but an actual connection with it, such as is easily seen in many molluscan eyes, is exceeding difficult to demonstrate; our evidence seems to show, however, that a few fibers perhaps do actually enter the optic cup. This structural state may be indicative of degeneration. If the mantle receptors of O. floridanum must be regarded as mere remnants of the original photosensitive equipment of this stock, the possibility of their connection with a primitive helio- tropic mechanism in ancestral pulmonates acquires an unprofit- able vagueness. We have thought it necessary to raise this point because it has sometimes been held that non-adaptive responses ‘‘have been inherited from ancestors in which they were adaptive” (meaning that the mechanism for response has been so inherited). For Onchidium such interpretation is highly improbable. 3. Neither can the heliotropism of Onchidium be dismissed as a mere ‘laboratory product.’ Some writers have endeavored to account for heliotropic orientations as found in various animals on the basis that determinate movements of this character must be the result of ‘abnormal’ conditions (cf. Franz, 713). It will be obvious that notions of this sort cannot affect the analysis of the mechanism of photic orientation, but can refer only to the role of heliotropism as an ethologic factor. It is only in a very limited sense that the heliotropism of Onchidium may be regarded | as ‘unnatural.’ It is not that laboratory conditions artificially imposed determine the orientations so produced, but on the contrary that in surroundings other than the immediate environ- ment of the ‘home’ nest some specific factor producing central nervous inhibition of what may, for convenience, be termed the (sensory) heliotropic impulses, fails to appear. It is sufficient to remember that an Onchidium need only be transferred to a new 492 LESLIE B. AREY AND W. J. CROZIER section of the shore in order to witness the complete unmasking of its heliotropic impulses. Since strychnine has in some instances been shown to produce negative phototropism, even in animals naturally indifferent to light (Moore, 12), it should be clearly understood that the strychnine effect in Onchidium cannot be regarded as of this sort.® Certain animals are known to become photonegative upon immersion in sea-water. Isopods of the genus Ligia, which in certain places occupy territory also frequented by Onchidium, have been said by Abbott (18) to reverse their phototropism, perhaps under control of humidity, in the sense that at low tide they come out from hiding places above flood-tide level and wander over the exposed intertidal zone. That this behavior really involves phototropism of any kind, and is not rather a case similar in certain features to that of O. floridanum, remains to be proved. It would be of interest to test this matter, for Abbott states that ‘‘in the laboratory they (Ligia) give a nega- tive reaction to sunlight.”” Moreover, an understanding of the situation in Onchidium may be important for the elucidation of other curious cases in which an animal’s heliotropism seems fundamentally at variance with its mode of life (e.g., Para- vortex, described by Ball, 716, p. 464). According to Mitsukuri (’01), the specific habitat of Littorma is determined by changes in its phototropism, from negative in air and under water to positive when splashed by waves. Here, again, the evidence that phototropism is really primarily involved is somewhat defective. The heliotropism of Onchidium is in no respect altered by complete immersion in sea-water. 9 Whether the action of strychnine in producing negative hehotropism with an animal naturally photopositive or even indifferent to light (Moore, 712, 713) can be always referred to chemical modifications within the primary receptors, rather than to some more strictly.central nervous (synaptic) effect, remains unanswered. Even with the human eye, where visual acuity (retinal resolving power) is notably augmented by strychnine, one cannot at present be sure that the removal of certain central inhibitions is not at bottom responsible. As an example of the inhibition of one sensory impulse by another, we might cite the heightened tactile responsiveness of certain de-eyed fishes (Crozier, 718). NATURAL HISTORY OF ONCHIDIUM 493 For reasons already amply set forth, we must reject the notion that the movements of Onchidium involve, or depend upon, any ‘reversal’ of phototropism. From the standpoint of adap- tation, the heliotropic mechanism must be regarded as a most interesting example of a perfectly definite functional character- istic which proceeds automatically from the given physicochem- ical composition of the organism (cf. Loeb, ’16), without refer- ence to adaptive requirements (cf. Arey and Crozier, ’18; Crozier and Arey, ’19c). Since the young of Onchidium (developing within the nest) emerge from the egg capsule with the form of the adult, and not as veligers (Joyeux-Laffuie, ’82), and since we have found very tiny individuals (2 mm. long) emerging from nests with adults, it cannot be said that perhaps at an early stage these animals are normally photonegative, by this means first becoming established in their definitive nest. 4. A number of instances are on record of the preservation in the rhythmic activities of animals of some diurnal or tidal rhythmicity inherent in the environment (Wilson, ’00; Gamble and Keeble, ’00; Schleip, 710; Keeble, ’10; Esterly, ’17; Cary, in Dahlgren, ’16, reprint p. 11, etc.). Unfortunately, a number of such reports, especially those concerning the persistence of environmental rhythms in actinians, have proved to be the result of erroneous observation (Parker, 719). We were inter- ested to discover if, in a form like Onchidium exhibiting such complex responses, there would be found any persistence of either tidal or nycthemeral rhythms of activity and repose, in the absence of the rhythmic excitations normally associated. It can be said with confidence that no rhythms of this character are maintained by Onchidium when removed to the laboratory. 5. For some time it has been known that the limpets and their allies inhabiting the tidal zone may at times wander for some little distance from, and subsequently return to, the ‘scar’ indicating their definite ‘home.’ The literature of this subject has been reviewed in an interesting way by Piéron (’09c). A certain complication enters here, for some limpets creep forth from their scar when covered by the sea, others only when left bare by the tide. Piéron (loc. cit.) has given a plausible account 494 LESLIE E. AREY AND W. J. CROZIER of these differences, though certain of his statements could be better weighted with evidence. The general fact of the wandering of limpets from their scars has been known since the time of Aristotle, and the fact of hom- ing has more recently attracted the notice of a number of natural- ists (Bethe, ’98; Davis and Fleure, ’03; Piéron, ’09a, ’09b, ’09c,’19; Bohn, ’09; Orton, ’14; Billiard, ’14; Wells, 717). It is all the more curious that so little close experimental work has been devoted to the elucidation of the matter. Homing activities are shown by a number of more or less distinctly related forms— Patella, Siphonaria, Helicon, Fissurella, Calyptraea; among these, various degrees of ‘homing’ ability are manifest, and in Acmaea testudinalis, according to Willcox (’05b), there is no evidence of this activity at all. ‘Homing’ is found to be success- fully executed by these animals even when they are artificially removed from their scar or from some point along their feeding path and replaced within a reasonable distance of the scar. For Patella, Piéron (’09c) records successful returns following a displacement of 12 cm., while, in certain localities, the extent of the natural feeding journeys may be as great as 55 to 90 cm., according to various observers quoted by Piéron. For other genera less distances limit the molluse’s successful return to its home subsequent to experimental shifting—with Siphonaria, 2 to 3 inches or perhaps a little more; for Fissurella, 2 inches (Willcox, ’05 a). This kind of ‘homing’ has certain resemblances to that of Onchidium, yet there are noteworthy differences. Piéron notes that some individuals (Patella) wander little or not at all in securing food; these are easily ‘lost,’ and do not succeed in returning to their scars if even slightly displaced. Piéron regards the homing as dependent upon a permanent memory of the topography of the habitual situation, and upon a very exact memory of the relief of the spot on which the Patella orients itself according to the irregularities of its shell.1° He succeeded in demonstrating that a Patella could so orient itself, 10Cf. Piéron, 719. NATURAL HISTORY OF ONCHIDIUM A95 even when the margin of the shell had been chipped away, and concluded therefore that the ‘topographic memory’ involved must be a sensory affair. The deduction is reasonable that the cephalic tentacles are the essential guiding organs, particularly in creeping, and that the pallial tentacles serve this function while the Patella is adjusting its irregularly outlined shell to the depression of the scar. But it should not be forgotten that the experimental test of this conclusion, particularly in so far as it pertains to the somewhat obscure ‘topographic memory,’ has yet to be instituted. With reference to the bearing of these findings upon the analysis of ‘homing’ in Onchidium, we need only point out that the homeward creeping of the latter has a much more flexible cast than is true of the behavior of the limpets, especially in those experiments where an Onchidium removed from one entrance of its nest and replaced upon the rock was found to gain the nest again, but by a second, different aperture. The distances from which a successful return is effected are also notably greater in the case of Onchidium. Nevertheless, the probability of any deep-seated ‘memory’ of the location of the nest is negatived by the fact that confinement to the laboratory for twenty-four hours obliterates the capacity for return to the nest. Tests of this point with Fissurella and Patella are of great interest; according to Piéron (’09b), the ability of Patella to return to a particular ‘home’ can survive some days’ removal from the scene. Even bees are said to lose their memory of specific locations after being anaesthetised (quoted from Min- nich, ’19). The behavior of limpets is of greater significance in connec- tion with the possible evolution of the ‘homing’ capacity. Some- thing of this sort seems to have been in Wells’s (17) mind. It can be pointed out that several grades of increasing precision and complexity of ‘homing’ activity are shown by molluscs (Arey and Crozier, 718). Beginning with Chiton tuberculatus (Crozier, ’21), in which there can be found something like ‘hom- ing,’ but of a rather vague type and pretty certainly the result of immediate stimulations, a series comprising also Patella, 496 LESLIE B. AREY AND W. J. CROZIER Onchidium, and Octopus exhibits more and more highly devel- oped ‘homing’ propensities. The return of a Patella, Fissurella, Siphonaria, or Calyptraea to its specific site cannot be accom- plished beyond a relatively slight distance; these creatures also tend to follow fairly definite paths in their excursions and to adhere to these paths when returning; and some of them creep but slightly, if at all, away from their scars. Onchidium’s behavior is obviously an advance in respect to complexity. Analogous behavior has been described for snails and slugs (as in the famous story of the sick snail and its companion, cited by Darwin, ’71, p. 316, and by others; cf. also Cooke, ’95, and Scharff, 07). The investigation of this matter in snails and slugs holds the possibility of considerable interest. Finally, the behavior of Octopus (cf., e.g., Cowdry, 711); which returns to its nest after extensive forays and from considerable distances, under circumstances such that direct vision of the nest entrance is completely excluded, represents the most complex form of this activity among molluscs. There has been a tendency to regard any series of this kind as exhibiting stages in the evolution of a particular response, or even of an instinct. To speak of a ‘homing instinct’ is little short of a perversion of sense. Such a view-point is very likely quite incorrect. Much more probable is it that this series of forms displays merely stages in the evolution of the central nervous machinery making possible more and more complicated behavior. ‘The phrase ‘evolution of an instinct’ tends to obscure the real basis of the matter. . Moreover, in the special instance under discussion, it is not at all obvious that the actual ‘homing’ performances of the several types named are in any sense geneti- cally connected; any relation with the mechanism of homing in higher forms, birds, for example, is in the highest degree improb- able. Even the homing of ants involves certain characteristics, such as those described by Cornetz (14), which are not in any sense represented in the behavior of Onchidium. We early recognized the simulation of associative memory in the activities of Onchidium (Arey and Crozier, ’18) with refer- ence to its nest. If the notion of such memory or ‘beginnings of NATURAL HISTORY OF ONCHIDIUM 497 intelligence’ be valid for cephalopods (v. Uexkill, ’01; Poli- -manti, 710), it is legitimate to inquire if anything of this nature can be imputed to Onchidium. According to Miss Thompson (17), the snail Physagyrina, although in maze experiments it gives no evidence of learning, does exhibit the establishment of simple associations when tested by Pavloff’s method of ‘con- ditioned responses.’ There is no real evidence favoring the idea of memory as evinced in the ‘homing’ of O. floridanum. One adequate test of the conceivable action of associations or even of primitive intelligence has occurred to us. When an Onchidium is picked up and put down on a strange portion of the shore, it cannot, of course, find its old nest; but other nests and various unoccupied crannies are available for shelter. The fact is, however, that instead of seeking the shelter afforded by ‘strange’ crevices, the snail is on the contrary at the mercy of two chief modes of response: its negative phototropism and its withdrawing reaction when shaded; that specific quality of its own particular nest which probably determines homing makes it possible for the creature to enter its own nest notwithstanding its photic sensitivities. Strayed Onchidia do not find shelter in new cavities of the rock, but on the contrary creep about on the shore until covered and washed off by the returning tide. Evi- dence of intelligence or of adaptive use of associative memory is completely absent, although, as we have elsewhere remarked (Arey and Crozier, ’18), the close simulation of behavior of that order is certainly deceptive. SUMMARY Onchidium (Onchidella) floridanum is a small naked pulmo- nate inhabiting the intertidal shore zone at Bermuda. ‘The indi- viduals of this species are grouped together into colonies num- bering about a dozen individuals, more or less, in each. A colony during high water occupies a ‘nest,’ in the form of an eroded cavity in the shore rock or a cleft between clay-cemented stones. During the day-time only, and at most but once in the twenty-four hours, the Onchidia emerge from their nest after the falling tide has left it above water for about an hour. The animals feed for a fixed period of about one hour, then those 498 LESLIE B. AREY AND W. J. CROZIER individuals emanating from a given nest begin simultaneously to execute a direct return to the nest from which they originated. These animals will not enter a ‘foreign’ nest. When tested apart from their specific normal environment the Onchidia are always negatively phototrophic. In the natural state their movements are entirely independent of heliotropism. This independence can be obliterated by injected strychnine, which produces ‘reversal of inhibition.’ Similarly, the simul- taneous return to the nest on the part of the various members of a colony can be understood on the assumption of a ‘reversal of inhibition’ brought about by substances derived from mate- rials ingested while feeding. The impulses which, on this hypothesis, suffer central inhibi- tion in the outwardly creeping snail may be identified with those which normally control the determinate character of the home- ward course. These impulses probably originate in the oral lappets, and are taken to have the character of a ‘contact odor’ (see text) specific for each particular nest. This is the only hypothesis which can account for the observed peculiarities of the movements of Onchidium and for the out- come of the experiments concerning homing reported in this paper. There is no evidence of associative or persisting memory in connection with homing, nor do other activities of Onchidium point to the existence in this form of anything approaching intelligent behavior. Responses to immediate stimulations are adequate for the analysis of the situation. | The negative heliotropism of Onchidium, apparently devoid of adaptive significance, is accounted for in terms of a photo- sensitive receptor system enabling these snails to respond to shading by an effective use of the mantle as a hold-fast, supple- menting the weak suctional efficiency of the foot. The existence of receptors making negative heliotropism possible cannot be understood as a condition persisting from ancestral pulmonates normally responding in this way. Mantle eyes are absent in this species, and although the ten- tacular eyes are perhaps of normal structure, no photic sensi- tivity has been discovered in connection with them. 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The germ cells of.... 235 Arny, Lestip B., AND Croziur, W.J. On the natural history of Onchidium............ 443 Asilus sericeus Say. Spermatogenesis in the IVA coer aroe iis nists ie le sis siotohorelaysjerais ss) 165 ODINE, Josrern Hau. Factors influenc- ing the water content and the rate of metabolism of certain Orthoptera...... 137 Bures, E. L., Burce, W.E., anp. Anexpla- nation for the variations in the intensity of oxidation in the life-cycle............. 203 Bure, W. E., anp BurcE, E. L. An expla- nation for the variations in the intensity of oxidation in the life-cycle............. 203 ELLS of anurans. I. The male sexual cycle of Rana catesbeiana larvae. The germ Changes. Studies on the retina. The struc- ture of the retina of Alligator mississip- piensis and its photomechanical.......: 207 Chemical sense of Nereis virens Sars. Feeding habits sad. ock ecitec eeeeis sere syeuyaie et 427 Content and the rate of metabolism of cer- tain Orthoptera. Factors influencing the RU LETAN herrea Me ecene i hiatoe siete ne 137 Crossing over. Further studies on the effect _ ofstem perature Ole tncaes case clos stn 187 Crossing over. I. The effect of selection on crossover values. Studies on............. 333 Crozier, W. J., AREY, Lestie B., AND. On the natural history of Onchidium........ 443 Cycle of Rana catesbeiana larvae. The germ cells of anurans. I. The male sexual.... 235 ETLEFSEN, J. A, aND Roperts, E. Studies on crossing over. I. The effect of selection on crossover values........ 333 DerwiteR, 8. R., LaAurRENs, HENRY, AND. Studies on the retina. The structure of the retina of Alligator mississippiensis and its photomechanical changes.............. 207 EEDING habits and chemical sense of Nereis virens Sars. The............... 427 Food reactions of Ameba proteus............. ¢97 ERM cells of anurans. I. The male sex- ual cycle of Rana catesbeiana larvae. AY ce 5 tae coe ene 6 WE esa asta ee 235 Gross, ALFRED O. The feeding habits and chemical sense of Nereis virens Sars..... 427 | eee and chemical sense of Nereis vi- rens Sars. The feeding............... 427 Harrison, Ross G. On relations of sym- metry in transplanted limbs.............. 503 History of Onchidium. On the natural...... 443 Howxanpd, Ruta B. Experiments on the effect of removal of the pronephros of % Amblystoma punctatum................. 355 | (ee es of oxidation in the life-cycle. An explanation for the variations in the.. 203 EPNER, Wm. A., AND WuuitTtock, W. Caru. Food reactions of Ameba pro- ™ | Pig Henry, AND DEeTwILer, S. R. Studies on the retina. The structure of the retina of Alligator mississippiensis _, and its photomechanical changes......... 207 Life-cycle. An explanation of the variations . in the intensity of oxidation in the....... 203 Limbs. On relations of symmetry in trans- plantedhepere cee... '.8 sa a eee 1 ETABOLISM of certain Orthoptera. Factors influencing the water content angdithe ratelOtine.: ccc sae sce sears eels 137 Mzrz, CHARLES W., AND Nonipez, José F. Spermatogenesis in the fly, Asilus sericeus ROT Bs sooenes ou SeOee matoe De saae Soa LOD, Nee eS history of Onchidium. On the 443 Nereis virens Sars. The feeding habits and SERS EE ino nwoc: 2 ODP ARMOR en SO ETS aE SCOOT 165 (@yanenes On the natural history of 443 Orthoptera. Factors influencing the water content and the rate of metabolism of cer- UCN OITA Ae 5 3 ek CORE RR CERIO ce DOS COE 137 Over. Further studies on the effect of tem- perature On crossing..........-.-.---+-+-- 187 Over. I. The effect of selection on crossover values. Studies on crossing.............. 333 Oxidation in the life-cycle. An explanation for the variations in the intensity of...... 203 De ive a changes. Studies on the retina. The structure of the ret- ina of Alligator mississippiensis and its. 207 PLovucu, Harotp H. Further studies on the effect of temperature on crossing over.... 187 Pronephros of Amblystoma punctatum. Ex- periments on the effect of the removal of Proteus. Food reactions of Ameba........... 397 Punctatum. Experiments on the effect of removal of the pronephros of Amblystoma 355 Re catesbeiana larvae. The germ cells ofanurans. I. The male sexual cycle of 235 Reactions of Ameba proteus. Food.......... 397 Removal of the pronephros of Amblystoma punctatum. Experiments on the effect of 355 504 INDEX Retina. The structure of the retina of Alli- gator mississippiensis and its photome- chanical changes. Studies on the........ 207 Rosekrts, E., DETLEFSEN,J.A, AND. Studies on crossingover. I. Theeffect of selection GNICIOSSOVEL) VALUESs--ceee eee annex on 333 ARS. The feeding habits and chemical sense of Nereis virems................0. 427 Selection on crossover values. Studies on crossing over. I. The effect of........... 333 Sense of Nereis virens Sars. The feeding habitsiancrchemical-sseeeereren cs ase. ce 427 Sericeus Say. Spermatogenesis in the fly, ANSI aes, toe ee pole rie Sion 165 Sexual cycle of Rana catesbeiana larvae. The germ cells of anurans. I. The male...... 235 Spermatogenesis in the fly, Asilus sericeus Say 165 Swincie, WILBUR WILuis. The germ cells of anurans. I. The male sexual cycle of Rana catesbeiana larvae................. 235 Symmetry in transplanted limbs. On rela- HONS Of As5s). Mosel ee cee ee ee ERE ES ATURE on crossing over. Fur- ther studies on the effect of............ 187 Transplanted limbs. On relations of sym- Metry- IN). hehe ee eee Vee in the intensity of oxidation in the life-cycle. Anexplanation for the 203 Virens Sars. The feeding habits and chemical sense:of Nereis:04)..4,32h ono eee 427 ATER content and the rate of metabo- lism of certain Orthoptera. Factors ® “influenempqthee». 1 taee aun eee eee 137 Wuittock, W. Cart, Kepner, Wn. A., AND. Food reactions of Ameba proteus........ 397 uN I ) 5 WHSE 020 I | | pa) fe} = = 4 ray = |