ho Ger aol ie. ee a a Gt, Phe ext t thy “Sot bis Tey rereiey. part ph mun ; hea taratatas ft ( CGE oerieas Pio tate : atelbletelteterstarete tates caress pester hi here ort isis Ae ali She nee 3 fae Pea aa ~ Ap Terarue “ Sieiits tek Cette seiiaretctetar acaba etait Meas as = aes Picked ety : ty ssrotrerpticetnitenon steterae) aatesetetetstasecettaten Sieteetariat eet pict Se : > 3 et teh, ort y ecbine saa ect Re riety arena? ; psteteters rela taraey ebebatebrrerstet Gehry peget eit ; PEN GCEL Batata tes rit % See Me ae Pele ieee nie ttt Gite Slotatits tastes tel etatet rth gtas yy es: i cates bi ef: nk Tee as ‘ é | etper ky pecs seek 5 Ay me) ; Wt Keak fits ee a ‘Pan ote =P Es + 2 es & t; rig Perel ate. ete . if nn tierra preteen i Reuceteat i vig! erat 2 arey Phe eh a at resis es a + - THE JOURNAL OF EXPERIMENTAL ZOOLOGY HDLTED BY WILLIAM E..CastLEe , Jacques LorB Harvard University The Rockefeller Institute Epwin G. CONKLIN Epmunp B. WiLson Princeton University Columbia University CHARLES B. DaveNPORT THomas H. Morean Carnegie Institution Columbia University HeRBERT S. JENNINGS Grorce H. Parker Johns Hopkins University Harvard University FRANK R. LILLIE RAYMOND PEARL University of Chicago Johns Hopkins University and Ross G. Harrison, Yale University Managing Editor VOLUME 31 JULY—NOVEMBER, 1920 THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA. CONTENTS Nori, JULY Maruitpe M. Lance. On the regeneration and finer structure of the arms of the cephalopods. Thirty-nine figures (eight plates).................. 1 W. H. TautarerRo. Reactions to light in Planaria maculata, with special reference to the function and structure of the eyes. Eighteen figures.. 59 S. R. Detwiter. Experiments on the transplantation of limbs in Amblys- toma. The formation of nerve plexuses and the function of the limbs. MewvieIity=tWO rll UTESs..sg: crass .lh a fakes oe she Seas uGsete NO. say AARERE woae sale No; 25 AUGUSE M. F. Guyer anp E. A. SmitH. Studies on cytolysins. Il. Transmission of induced eye defects. Seven figures and four plates.................. 171 Georce H. BisHorp. Fertilization in the honey-bee. I. The male sexual organs: their histological structure and physiological functioning. ineewexturounesandeuhneenplavest=ne sas. wneeee ae aac sas 225 GerorGceE H. BisHop. Fertilization in the honey-bee. II. Disposal of the sexual fluids in the organs of the female. Two text figures............. 267 Gary N. Cauxins. Uroleptus mobilis Engelm. III. A study in vitality. OneschartanGktwordlaenamsia aan vem eeneeie aor cient Graco ccs 287 No. 3. OCTOBER C. C. Lirrnte. Factors influencing the growth of a transplantable tumor in TOS, ALO) Cheer ONS BNC Ose WANK s. oocccensounaccocoMMesoasuuoucaonen 307 Epwarp F. Apotex. Egg-laying reactions in the pomace fly, Drosophila... 327 G. H. Parker. Activities of colonial animals. I. Circulation of water in Renilla. One text figure and one plate (four figures)................... 343 No. 4. NOVEMBER J. M. D. Outmsrev. The results of cutting the seventh cranial nerve in Amiurus nebulosus (Lesueur). Four plates (twenty-six figures)........ 369 GustaFr Fr. GOrHitINn. Experimental studies on primary inhibition of the cilvaryamovementun croc cuCUmMiIsyas-— 40ers oeee en eee to ae 403 H. J. Mutuer. Further changes in the white-eye series of Drosophila and their bearing on the manner of occurrence of mutation. Three figures. 443 G. H. Parker. Activities of colonial animals. II. Neuromuscular move- ments and phosphorescence in Renilla. Twelve text figures and one jolEe (Suet METER) Bae tere nee stot oles aol ate Oh oe ie OU SPIO Cioran ate 475 oa yn 8 Ae Be ee i ae 3 \4 Pat 7 ORs | 1 Ves ARG siete fi : a : stain Ou ee | aaa ry } ; erihe \ RT ON ral sf Wh! rane ; i At ee Ati ‘ ; is ’ : : A s % if r i Kae . i > ¥ r . f t « rayne: oe 7 : . 4 ‘ é ne ‘ fl : ayes — are Ait < ay Say wh ' a Silt) Pauwels yb 44 : vy bo perm ’ Ai " if Pm | { i met y ce ae oy » aa THE JOURNAL OF EXPERIMENTAL ZOOLOGY, Vou. 31, No. 1 JUNE, 1920 Resumen por la autora, Mathilde M. Lange. Nueva York. Sobre la regeneracion y fina estructura de los brazos de los cefal6podos. A. Fina estructura. El brazo consta de una sola capa epitelial, una capa de tejido conjuntivo subcutaneo y tejidos muscular y nervioso. La musculatura esta dividida en tres grupos: el fasciculo muscular central, que contiene varios grupos de fibras diferentes, la musculatura de las ventosas y los mtsculos que reunen a ambos grupos. El sistema nervioso consta de un nervio axial colocado centralmente (a menudo comparado con la médula espinal de los vertebrados), de un grupo de ganglios relacionados con cada ventosa y de cuatro cordones nerviosos intramusculares. El] nervio axial esta provisto de tejido neu- roglico. B. Regeneracién. 1) Cambios externos: El nervio axial sobresale del brazo después de la operacién. Las ventosas distales se recurvan tendiendo a cerrar la herida, permaneciendo en esta posicidn anormal hasta que la herida cicatriza comple- tamente. ‘Tan pronto como las ventosas vuelven a adquirir su posicién normal el nédulo de regeneracién, que es cupuliforme, aparece en la mitad externa del brazo. Este nédulo se desar- rolla en una especie de apéndice flageliforme, que lleva pequenos pliegues transversos en el lado interno. Estos pliegues se trans- forman mas tarde en ventosas. Originariamente aparecen en una sola fila, pero mas tarde adoptan la posicién en doble fila. Los primeros cromat6foros aparecen en la parte regenerada al cabo de unas tres semanas después la de operacién. 2) Estudio histol6gico. La sangre no aparece en la herida immediatamente después de la operaci6n, sino generalmente varias horas (tres a cinco) mas tarde. Forma un tejido de cicatrizacién que se retiene durante la vida y produce tejido conectivo. Los demas tejidos nuevos se forman a expensas de tejidos preexistentes de la misma clase. En los sarcoblastos se observaron mitosis pero no en los neuroblastos. Es posible que las células neurdglicas contribuyan a la formacién de nuevas células nerviosas. Translation by José F. Nonidez Cornell University Medical College, N. Y. AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, APRIL 19 ON THE REGENERATION AND FINER STRUCTURE OF THE ARMS OF THE CEPHALOPODS MATHILDE M. LANGE THIRTY-NINE ILLUSTRATIONS CONTENTS Initio cuetiomm sparta Arie beaaee tee cee ce Sewn eee TS tT 1 IEYUOVETP QUIMTIGONCES ceuctao ory ion oe tats oe Sits Ge ees BGS Basa. Eee See eRe iG oe eee 3 ENGR CHET tO Meese rene. ees were Lme ee toast el cet arte vores even seal ahey sasnguete et anes ee ch.) 6) average aieore a'e,o- 5 1. A study of the external changes taking place during regeneration.... 5 22 Ashistological study: of TegeneratiOnir 6. é Jesh sao 4 swe jcorvs ed oie ays a = 14 GAO UT CRM BATT Oa sailed he ccredtns Stars ts Ea Nm ced tbone Seatac B svete Bia sic 14 RPE SITEUES ORGS race ceccate eich s Bin my Se atc ois Nae eels ashen o Madders, acoek ack onala wb Oe 22 Colne meERvOuUSSySUCIMbes cee whichis chown toa aie IS nla om omit ee tes 26 SHLUTTODAIAY 5 ebeuchs did ore) a cri Beceies oO toaenccles coe eee kaon tage ince iG Ee daeerio ar et ran ta Oris Scan 32 ANTUIOSINGSES SG & AURIS, FAK hol Rae ORE DEN CDSE OMEREE Sean ORR Ce SeRPRIEE ES SR ME SEMA 35 INTRODUCTION The ability of the cephalopods to renew lost parts of their arms is a fact which has been revealed to us through the dis- covery of several specimens whose injured tentacles were in the process of regeneration. Verrill (82) found such cephalopods off the northeastern coast of North America and Brock (’86) reported the presence of similar ones in the Indian Ocean. Eisig and Riggenbach (’01) observed the formation of a regeneration bud on the tentacle of Octopus de Filipi after autotomy, and Hanko (713) published a paper describing an octopus tentacle which at the upper end had been split into two parts, each part retaining the function of a normal arm. But all these reports lack a detailed description of the process of regeneration, no . experimental investigation of this subject having been published up to the present. At the suggestion of Prof. C. Chun, I decided to make a closer study of this phenomenon. I feel indebted to the late Professor Chun for this suggestion. Unfortunately, he died just as I began my investigations. His assistant, Doctor Grimpe, kindly assisted me during the early stages of my work. 1 2 MATHILDE M. LANGE I wish to thank him for the interest he took in its progress. I also wish to express my thanks to Professor Hescheler, in whose laboratory I finished this study, and to Dr. Marie Daiber. Their - valuable suggestions were of great help to me. The specimens necessary for the work were gathered at the Zoological Station of Naples and at the Musée Océanographie at Monaco. Octopus vulgaris, Eledone moschata, and Sepia officinalis were the three species chiefly employed. At first I encountered some difficulty in keeping the animals. My experi- ence taught me that non-transparent aquaria were better suited to the purpose than plain glass basins and that the water must flow into the basin slowly, as a strong current is harmful to the well-being of the animals. If the basin is large enough, it is advisable to turn the water off for a couple of hours every day. The food consisted mainly of live crabs. In case the basin is inhabited by more than one animal, it is best not simply to throw in the food, but to feed each animal singly, in order to prevent fighting, as the animals often injure their arms in this way, and such injuries are liable to retard regeneration. Ani- mals whose lens was extirpated were narcotized in a solution which consisted of four parts of 25 per cent alcoholic chloreton, and 96 parts water (sea-water). The animal remained in this solution from three to five minutes. In order to hasten revival after operation, air was pumped into the mantel-cavity and pressed out again. This proved quite a stimulant to respiration. The narcotic poison which is secreted by the gills generally gathers in the funnel. It is advisable to rid the animal of this poison by inserting a probe into the funnel. The specimens were fixed in the following solutions: Flemming’s strong mixture, Hermann’s solution, a mixture of formalin, alcohol and acetic acid, and also in a mixture of mercuric chlor- ide, alcohol and acetic acid. Several pieces were fixed in 10 per cent formalin and several in neutral formalin. If enough ani- mals are available, it is advisable to fix an entire animal for each successive stage, but if the scarcity of animals necessitates the use of one and the same animal to produce several stages, it is imperative that regenerated ends be cut off under water. Special — OCTOPUS ARM, REGENERATION AND STRUCTURE 3 attention must be paid to this, as otherwise air penetrates the tissues. The presence of air in the tissues renders their impreg- nation with paraffin or other media extremely difficult, and thus the microtome work very unsatisfactory. Combinations of celloidin and paraffin and also of collodium and paraffin proved the most practical substances for embedding. The pieces after being well drained in alcohol (100 per cent) were placed in a mixture of equal parts of aleohol and ether, and remained there for several hours. They were then put into a diluted solution of celloidin or collodium for 24 hours. The pieces impregnated with collodium were then left in oil of origanum for 24 hours, and the celloidin pieces were submitted to the same treatment in cedar oil. Later they were immersed in a mixture of oil of origanum plus 40° paraffin, and cedar oil plus 40° paraffin, respectively, and remained in these mixtures for twenty-four hours. Thereupon they were placed in several baths of paraffin of different degrees, and finally in 58° paraffin, in which they were embedded. The 100 per cent alcohol had hardened the tissues to such a degree that microtoming was exceedingly difficult. It was therefore necessary to employ mastic-collodium. No albu- minous glycerin, only distillated water was used for mounting. The slides were further treated in the usual way. Photoxolin was not used; on the contrary, the mastic-collodium was removed by a solution of equal parts of aleohol and ether. The sections were stained on the slide with haemalaun (Mayer) haematoxylin (Heidenhain), eosin, orange G several of the specimens fixed in osmic, were stained saffranin plus emerald green. The best stains were obtained by a combination of haematoxylin (Heiden- hain) plus eosin. Some specimens were stained by way of impregnation, according to the method of Bielschowsky and Maresch. THE FINER STRUCTURE OF THE ARM The anatomy of the arm of the cephalopods has often been made the object of closer study. Cuvier’s publication 1817 gives quite a minute description of it. Since then a number of authors have devoted their attention to the same subject. Colo- 4 MATHILDE M. LANGE santi (76) was the first to make the tentacle of the octopus the subject of a microscopical examination. The best paper pub- lished on this subject up to the present was written by Guérin (08). The arm of the cephalopod consists of four distinct parts: first, the skin or integument; second, the muscles; third, the nervous system, and, fourth, the vascular system. . The skin is composed of two layers, the epidermis and the dermis. The epidermis consists of a single sheet and is covered by striated cuticle. The nuclei of its cells are quite large and contain granules. The unicellular glands are more or less pear- shaped in comparison with the ordinary epithelial cells, their plasm contains fewer granules and they are much smaller. The dermis of the octopus arm consists of connective tissue, which surrounds the chief muscle bundle in equal thickness on all sides. It is permeated by many blood-vessels, by muscular and nerve strands, and also contains chromatophores and lumi- nous organs. The muscles of the arm can be divided into three distinct groups, viz., the central muscle bundle, the muscles of the suckers, and the muscles which serve as a connection between these two groups. The central muscle bundle consists of six longitudinal muscle strands, one transverse set of fibers, and six oblique or diagonal muscle strands. The musculature of the suckers consists principally of radiating fibers interspersed with circular muscles, the latter being more numerous in the musculature of the adhesive part than among the muscles of the sucking cavity. At the juncture of the adhesive part and the wall of the sucking cavity the circular muscles are especially well developed, forming a so-called sphincter. The connecting muscles connect the suckers with the central muscle bundle and also with the dermis (fig. 1). Ballowotz (93) made a closer study of the finer structure of the muscle fiber and found that it forms a narrow cylinder taper- ing at each end. This cylinder consists of spiral fibers and a granulated protoplasmic substance, which probably serves as a connection between the fibers and holds them together. OCTOPUS ARM, REGENERATION AND STRUCTURE 5) The nervous system of the arm is quite complicated, and is composed of three distinct parts, viz., the central or axial nerve, the group of ganglion cells situated above each sucker, and the intramuscular nerves. The complex structure of the axial nerve has given rise to much scientific discussion. Van Beneden (’90), Cheron (’66), Owsjannikow (95), and Kowalewsky claims that the axial nerve is a part of the peripheral nervous system, whereas Colosanti (76), Uexkull (93), and Guerin (’08) maintain that its qualities in structure, as well as in function, are such that it would easily be compared to a central nervous system. The three components of this complex axial nerve are: 1) a layer of ganglion cells; 2) a centrally located mass of nerve fibers, and, 3) two myelin cords running along the back of the arm. The ganglion cells are surrounded and supported by glia tissue. This tissue also forms a sheath around the processes of the ganglion cells and is present in the mass of centrally located nerve fibers (fig. 3). Each arm is provided with one main artery embedded in the connective tissue, which les between the two. myelin cords. Two large veins (venae brachiales superficiales) running along the external side of the arm in the dermal layer, form the two main components of the brachial venous system. Little veins from the inner side of the arm convey the blood from the vicinity of the suckers to the two large afferent vessels. The blood of the Cephalopoda is a thin liquid containing only one kind of blood-corpuscles. The latter have some similarity to the leuco- cytes of the vertebrates. Kollman (08) gave a detailed descrip- tion of them (fig. 4). The blood has no fibrinogen. ’ REGENERATION 1. A study of the external changes taking place during regeneration In the introduction attention has been called to the fact that the regenerative power of the octopus arm has been revealed by the discovery of many animals having regenerated arms. The authors who have reported on such specimens have also been mentioned. The embryonic development of the arm has 6 MATHILDE M. LANGE up to the present not been made a subject of special study. There are several papers on the development of the Cephalopoda (Kolliker (44), Grenacher (’74), Bobretzky (77), Ussow (’74, ’81), Viallton (’88)), but most of them treat only of the earlier stages or of the development of some particular organ, and none of them enter into a detailed account of the normal growth and development of the arm. (Guérin has given a short sketch of the histological differentiation of the arm musculature, but does not mention the morphological changes which take place during the process of normal growth. A thorough and detailed report on the development of the arm has not yet been published. A. Naef’s monograph on the development of the Cephalopoda (now being printed) will surely contain a detailed report on this subject. The histological structure of the arm is the same at the base as at the distal end (with the exception of the tip, where the tissue is in an undifferentiated embryonic stage). I therefore did not pay great attention to the level of the cut or to the amount of the arm I severed from the proximal end. In the course of my experiments, however, it became apparent that the regen- eration of arms cut off near the base required more time (in some cases two to three weeks passed before even the slightest sign. of regeneration proper became visible)... As my stay at the Zoological station at Naples was limited, I generally amputated only about one-third of the arm (rarely half of it), as I wished to have quite a number of specimens which were already in an advanced stage of regeneration. In cutting off the distal portion, care was taken that the section plane was as vertical as possible to thé longitudinal axis of the arm. I was often surprised that no visible traces of blood could be found on the wound immedi- ately after operation. In order to be quite certain on this point, I carefully dried the tentacle with a towel before cutting and then 1 At the tip the tissues of the arm are still in an embryonic stage. Distal parts of the tip probably regenerate more quickly because they do not require so much time to transform their tissue into an embryonic blastema. The proxi- mal parts probably require more time for this process, as their tissues are more differentiated. OCTOPUS ARM, REGENERATION AND STRUCTURE 7 placed a piece of filter-paper on the wound immediately after the operation had been performed. I could not detect any moisture on the paper. ‘This retention of the blood after amputa- tion is in all probability associated with the ability of the arm to cast off distal portions by means of autotomy. According to EKisig and Riggenbach, Octopus de Filipi frequently casts off the greater portion of several tentacles in this manner. Riggen- bach also mentions the ability of the octopodes to free their dis- tally held arms by simply casting off the held portion. TI also observed similar cases of autotomy of the arm of Octopus vulgaris, but I believe that the ability to autotomize is confined to the distal portion. I was not able to find any portion of the arm which was modified or in any way arranged for autotomy. Neither do any of the numerous publications treating of the structure of the arm mention the presence of any mechanism especially adapted to autotomy. (It is a well-known fact that many arthropods are provided with such mechanisms.) Gener- ally four-fifths of the arm is cast off. But this is not always the case, and the distance between the base of the tentacle and the point of rupture is by no means always the same. Riggenbach mentions some cases where this distance averages about 2 cm., sometimes more, sometimes less. Immediately after operation the external rim of the wound contracts spasmodically; this contraction is especially noticeable in the dermis. The external parts of the wound are thus covered, but the central musculature and the axial nerve remain unpro- tected. The axial nerve even protrudes beyond the surrounding tissues. Figure 5 shows a wound about one and a half hours after operation. The protrusion of the axial nerve is quite obvious. With the help of a magnifying glass I was able to see the myelin cords, the central nerve-fiber mass, and even the main artery quite plainly. This fact shows that this part of the wound was still without any covering whatever. Figure 6, showing a later stage (about ten hours after operation), presents quite a different picture. The dermis has contracted more closely over the wound, but has not succeeded in covering it completely. The axial nerve no longer extends beyond the 8 MATHILDE M. LANGE surrounding tissues and its components are no longer visible, not even with the help of a magnifying glass. The hitherto unprotected portion of the wound has been covered by a sub- stance, the nature of which could only be ascertained by means of a histological examination. This examination disclosed the fact that this covering consisted of blood. I am not able to state the exact amount of time which expires between the oper- ation and the bleeding. However, I was able to detect blood only on such pieces which had been fixed five or six hours or more after operation. Pieces which had been preserved previous to that time showed no traces of blood. Here is a case where the section of blood-vessels is not immediately followed by bleeding, but where the bleeding takes place a considerable time after operation. This, I think, is a fact which deserves notice. At first I thought that the blood which might have covered the wound at an earlier stage could have been washed away or that the animal bled from four to five hours before the bleeding stopped. But both these cases seem rather improbable. In the first place, I could not detect any moisture on the filter-paper which I placed on the open wound directly after operation; secondly, the conditions in which the Octopoda live would be very harmful to them if they were subject to prolonged bleedings from wounds in the arm. Brock believes that to a certain degree some rela- tion between the oecology of the animal and the relatively great regenerative power of the arm exists. He wrote as follows: Ohne die sehr niitzliche Eigenschaft, die Arme schnell und voll- standig zu regenerieren, wiirden die Cephalopoden wohl schwerlich die Konkurrenz mit anderen Mitbewohnern auf dem Riff aufnehmen kénnen, trotz der unvergleichlich reichen Jagdgriinde, welche es ihnen bietet. Es ist nicht schwer zu begreifen, dass Wohnorte von der Natur eines Korallenriffes den Armen eines Cephalopoden geradezu verhiingnisvoll werden miissen. So kann es nicht ttberraschen, bei den Octopoden der Korallenriffe so haufig versttimmelten oder in verschiedenen Stadien der Regeneration begriffenen Armen zu _be- gegnen. Von langarmigen Arten ist es tiberhaupt nicht mdglich gewesen, ein Exemplar mit unverletztem Arm zu erhalten. OCTOPUS ARM, REGENERATION AND STRUCTURE 9 Among the animals which were delivered to me for experimental purposes at Naples and Monaco, I also found a great many whose arms had been injured before capture, and quite a number of them showed advanced stages of regeneration. This shows plainly that the loss of an arm is by no means a rare or adangerous occurrence. If, however, an animal, which is subject to frequent injuries on a certain part of its body, would each time bleed from five to six hours, the loss of blood would in the end probably prove fatal. The fact that the animals easily survive frequent injuries of their arms indicates the improbability of such pro- longed bleedings. It is difficult to explain the tardy appearance of blood on the wound. The only explanation I can give is the following: The minute the arm is cut off or cast off by autotomy the blood- vessels contract at the wound, later the muscles of the blood- vessels relax and allow blood to flow. The blood-corpuscles soon form a clot (agglutinate), and this clot serves as a prelimi- nary covering for the wound. Figure 7 which in comparison to figure 6 presents quite a differ- ent picture, exhibits a completely covered or closed wound. The time in which complete healing of a wound is achieved varies greatly. Some wounds were healed within less than twenty-four hours after operation, others showed no healing after thirty hours and were at that time only covered with a blood clot. The differences in the time necessary for the complete healing of the wound are probably caused by various factors. Generally a wound in the distal portion of the arm healed more quickly than one located in the middle or at the base. It is quite likely that the age of the animal also plays a part, for the wound healed more rapidly in a younger animal than in an older one. The season of the year may also have some influence on the progress of wound healing. In Naples, where I experimented in the spring, I found that the wound healing took less time than in Monaco, where I carried on my experiments in the fall of the year. Figure 7 shows a perfectly smoothly healed arm stump. This smooth appearance is probably due to the wound’s having been 10 MATHILDE M. LANGE completely covered by epithelium. That the wound was actually covered by epithelium was proved by a microscopic examination of sections made of the piece in question. Another fact worth noting in this picture is the position of the two suckers at the obtuse end of the arm. These two suckers are somewhat drawn up as if they were also helping close the wound. ‘This abnormal position of the suckers, which I observed as a regular occurrence during the process of wound healing, pushes the section plane out of its original position (vertical to the brachial axis) toward the exterior side of the arm. The suckers con- tinued in this abnormal position for some time—in most cases from two to three days, in some cases ten days, and some even more. The same factors which cause the difference in time necessary for wound healing probably also play a part here: viz., location of the wound (distal or proximal), age of the animal, and season of the year. During my stay at Naples an octopus which measured a total length of 13 meters was placed in one of the basins of the aquarium. Before its capture the animal had lost the greater portion (about three-fourths) of one of its arms, thus placing the. wound in the proximal portion of the arm. The wound was completely healed and the two distal suckers were drawn up at the end in the abnormal position mentioned above. It required a period of three weeks before these suckers were again in their normal position. As soon as the two last distal suckers regain their normal position, the first sign of a beginning regeneration becomes visible in the shape of a little knob, lying near the external side of the arm (fig. 8). Figure 9 shows a more advanced stage, the knob has already developed into a small process. On the interior side of this process a little groove becomes noticeable. This groove is plainly seen in figure 10. The same picture also shows the formation of little transverse folds within the groove. These folds later develop into suckers. In the further course of regen- eration the folds take a form similar to little warts (figs. 11 and 12). The cavity of the sucker and the adhesive part are formed later by means of invagination. The beginning of this process can already be noticed in the most proximal of the suckers shown OCTOPUS ARM, REGENERATION AND STRUCTURE 11 in figure 11. All suckers are at first formed in the shape of little transverse folds arranged in single file. Later they are rounded off to little papillae. This process of rounding off seems to start in the center and progress sideward, so that the papillae are in quite a different position from the original folds. The latter were quite centrally located, whereas the papillae or warts have a lateral position. As the folds are rounded off alternately, once to the right and once to the left, the double row of suckers characteristic of the arm of Octopus vulgaris is thus gradually formed. But the above is only true of the suckers, which origi- nate in the regenerated process. Attention has already been drawn to the fact that the regenerated process is attached to the external half of the arm. The process is considerably thinner than the stump of the arm, and in comparison to it looks like a thin lash-like appendage. Therefore, a considerable portion of the obtuse end of the stump remains free. On this free end the first regenerated suckers are formed in the shape of little transverse folds, but their further development differs somewhat from the development of the suckers located in the regenerated process. While the final double-rowed arrangement of the latter is already visible at an early stage of their development, the suckers of the free end remain arranged in single file till they are a great deal further advanced, and some of them even remain so permanently. The above is very well illustrated in figures 13 and 14. The four proximal suckers are arranged in single file and already show the invagination, which eventually leads to the formation of the sucking cavity and the adhesive part. Above these four suckers are ten to eleven newly formed suckers belonging to the regenerated process. ‘These still show the form of papillae and are not nearly as well developed as the lower four, but their position already indicates their final arrangement in two rows. Above these papillae we can detect two to three small transverse folds arranged in single file. The latter are suckers at a very early stage of development. How does the regeneration of the arm compare with its normal development? The arm of the octopus embryo can be divided into two parts—a rather thick proximal part, provided with THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 31, NO. lL 12 MATHILDE M. LANGE three well-developed suckers (arranged in single file), and a distal part which compared to the proximal part is exceedingly thin. Naef has called this lash-like appendage a flagellum. The three suckers are arranged in single file and remain so perma- nently. In a more advanced stage the arm is provided with a larger number of suckers, of which the first three still are in single file, while the others are arranged in zig-zag fashion. Here we have an arrangement similar to that in figures 13 and 14. So we may safely say that the arrangement of the first newly formed suckers in the course of regeneration does not differ greatly from the arrangement of the first suckers in the course of normal development. In regenerated parts, however, only one sucker remains permanently without a partner. In some cases I have found from two to three suckers arranged in this manner. The single sucker at the base of the regenerated part was to be found in even such advanced stages of regeneration where it was difficult to distinguish between the old stump and the newly formed part. Brock believes that this single sucker only appears after the lost part has been completely regenerated. On page 592 he speaks of it as follows: Ist die Einschniirung (an der Amputationsstelle) bereits bis auf die Furche verschwunden, und geht der regenerierte Arm schon ganz unmerklich in den Stumpf tiber, so verr&t sich der Vorgang der Regen- eration noch lange durch eine mehr oder minder breite Liicke in der Reihe der Saugnapfe gerade an der Amputationsstelle, welche erst ganz zuletzt von einem an dieser Stelle hervorsprossenden Saugnapf ausgeftillt wird. Brock arrived at these conclusions through the study of several regenerated arms which he found on animals caught in the Indian Ocean. He never tried to prove his assertions by experiment. T should like to draw attention to the fact that never in the course of my investigations (and I had quite a quantity of material at my disposal) was I able to detect an interval in. the row of suckers such as Brock both illustrates and describes. The sucker which Brock believes to be the last is probably identical with the single sucker at the base of the regenerated part (figs. 15 and 16). This sucker, however, is by no means the last, but OCTOPUS ARM, REGENERATION AND STRUCTURE 13 on the contrary the very first sucker to be formed in the course of regeneration. Three or four weeks after operation, the first chromatophores appear in the regenerated part. They are lighter in color than the chromatophores of the arm stump. Figures 15 and 16 show regenerated pieces in an advanced stage of development. The only differences between the stump and the regenerated part consist in the different thickness and the different coloring of the chromatophores. The arm of Eledone regenerates in the same way as the arm of Octopus vulgaris, with the exception that the suckers remain arranged in single file; this beg their normal and permanent position. I did not study the regeneration of the arm of the Decapoda very closely. JI was able to observe the complete healing of the wound and the formation of a dome-shaped regeneration knob,? but not able to follow up the development of the suckers, as the animals generally died before they reached this stage. Brock claims that the arms of the decapodes lack all ability to regen- erate. As the arms of the decapodes are considerably shorter than the octopus arms, they are not subject to such frequent injuries. Nevertheless, the Decapoda are able to replace a lost arm; only the manner in which they do so differs from the way in which the octopus repairs the same damage. Doctor Naef showed me a sepia (at Naples) which had lost almost a whole arm. The animal had already begun to replace the lost part, but not by means of regeneration from the old stump, but by developing that rudimentary buccal arm’ which was correlated to the lost arm. ‘This arm developed from a rudiment had the same structure as a normal arm and differed from the latter only * The regeneration knob of the sepia arm is centrally located, whereas the knob of the Octopus vulgaris is nearer the exterior side. No lash-like append- age or thin process is developed. The difference in thickness between the stump and the regenerated part is not so great. Perhaps the different arrangement and location of the myelin cords is the cause of this. ’ The buccal funnel of the decapods is a set of rudimentary arms and corre- sponds to the inner circle of arms of the Nautilidae. The buccal funnel of the decapods possesses seven to ten rudimentary arms, which, according to Naef, are provided with two rows of suckers. 14 MATHILDE M. LANGE in length (it was shorter) and in its position nearer to the buccal funnel. In the further course of development this arm would probably have replaced the lost one. This case would indicate that sepia does not always renew the lost part of its arms by direct regeneration, but in some cases resorts to compensatory regulation to replace the lost organ. 2. A histological study of regeneration The course of regeneration which takes place after injury can easily be divided into three distinct stages, viz., the healing of the.wound, the degeneration of the tissues, and the renewal of the same. However, I wish to draw attention to the fact that these histological processes need not necessarily follow each other in the same order as here mentioned. a. Wound healing. It is not possible to detect many changes in the wound shortly after operation. The only obvious alter- ation consists in a contraction of the edges of the wound, by which they form a kind of raised rim around the same. The greater part of the wound with the exception of the axial nerve is in some degree protected by this rim. The histological examination showed that the raised rim was formed by the connective tissue of the dermis, in its endeavor to contract over the wound. How- ever, only the externally located muscles are covered by the con- nective tissue, while the center of the arm, comprising the axial nerve with its surrounding connective tissue layer and the inner muscles, remain unprotected. Figure 17, which shows a longi- tudinal section through the center of an arm one and one-half hours after operation, illustrates these conditions. Only the external portions of the wound are covered, and the axial nerve even protrudes beyond the surrounding tissues. The only alteration visible in the tissues immediately adjacent to the wound is a slight disintegration in the inner transverse muscle in the central mass of nerve fibers (neuropil) and in the ganglion layer. This subject will be considered below (pp. 22 and 27). The main brachial artery is closed. It is hard to tell whether this is a natural condition or one produced by fixing. The artery OCTOPUS ARM, REGENERATION AND STRUCTURE 15 contains some blood in its most distal portion, most of which consists chiefly of blood-corpuscles without plasma. Cuénot claims that such blood-corpuscles are degenerated. All the blood-vessels leading toward the wound contain very little blood, nor is there any trace of blood on the wound itself. I believe that up to the time at which this piece had been fixed, no bleeding had yet taken place. The reasons which led me to have this opinion have been discussed in the previous chapter (p. 8). Young stages of regeneration (such as are shown in fig. 17) are always open, no covering or closing of the wound having yet taken place. Techow (10) found that the wounds of the gastropods were provisionally closed by a clot which was formed by the contents of the blood-vessels. I found that the wounds of the Cephalo- poda were closed in the same manner. About five or six hours after operation all blood-vessels leading to the wound are filled with blood, which after leaving the vessels spreads over the wound, thus forming a protective covering for the same. Atten- tion has already been drawn to the fact that the blood of the Cephalopoda contains no fibrinogen (p. 5), therefore the clot which preliminarily closes the wound cannot be formed through blood coagulation, as is the case in wounds of vertebrates and arthropods. The wound can only be closed by the agglu- tination of the blood-corpuscles, and a histological study of the sections proved this to be the case. In agglutinating the blood- corpuscles form a kind of network (figs. 19, 20, and 21). In many other invertebrate animals the wounds are also closed by means of blood- or lymph-corpuscles. Techow (710), Hanko (’13),4 Cucagna (’15), and Nusbaum (’15) found it so for the Mollusca. Hescheler (’98), Nusbaum, and Friedlander (’95) claim that wounds of worms are closed in the same manner. Ost (’06), Friedrich (’06), and Reed (’05), who have studied the regeneration in Arthropoda, found that the wounds of these 4Hanko studied the regeneration of Nassa mutabilis and speaks of blood coagulation as a means of closing the wound. This is probably an erroneous statement, for, according to Kollman, the blood of the gastropodes contains no fibrinogen, therefore coagulation is not possible. 16 MATHILDE M. LANGE animals are also closed by the agglutination of the leucocytes, but the agglutination is accompanied by blood coagulation, as the blood of the Arthropoda contains fibrinogen. The leucocytes of the Cephalopoda do not undergo any great change during agglutination. I could not observe any formation of pseudopods such as Loeb (’09) and Geddes (’01) describe. The only alter- ation which I could discover in the blood-corpuscles was hyalino- sis resulting from the disappearance of a great number of granules. The change wrought by hyalinosis can be plainly seen in com- paring figures 4 and 19. The blood-plasm seems to disappear soon after the bleeding stops, for I could not find any trace of it on most of the sections. Were it not for a few cases such as one exhibited in figures 19 and 22, where blood plasm is still present, one could easily suspect that the plasm flows away immediately after leaving the blood-vessels. If we examine the plasm in the blood-clou closely, we can detect a difference between it and the plasm in the blood-vessels. In the first place, the plasm of the clot is not as uniform in structure as the plasm in the blood-vessels, and, secondly, it does not stain the same intense brown-red, when the combined stain of Heidenhain and eosin is employed. But in spite of these differences, it still retains the character of blood-plasm. In some sections made of pieces which had been fixed after a period of five hours after operation, I even found the plasm of the blood-clot almost identically the same in appearance as the plasm in the blood- vessels. Gradually the blood-plasm entirely disappears from the cicatricial tissue. As no signs of degeneration were visible, the plasm probably becomes absorbed, but unfortunately, I was not able to find out how this is done. After the disappear- ance of the blood-plasm the cicatricial tissue looks like a very close network of thin threads, in which numerous nuclei are embedded (figs. 20 and 21). The fact that these nuclei increase in numbers would indicate that they multiply. Migration of nuclei from the subjacent tissue is not likely, for the cicatricial tissue is separated from all tissues, with the exception of the musculature, by a very distinct boundary line. The muscles, however, being in a state of degeneration, which-begins before OCTOPUS ARM, REGENERATION AND STRUCTURE iL the formation of the blood-clot, cannot possibly contribute cells for the tissues covering the wound. ‘The increase in the number of nuclei must be the result of nuclear division. In spite of careful search, I was not able to find any mitosis. The nuclei probably multiply by means of direct or amitotic division. The cicatricial tissue which is formed by the agglutinated blood-corpuscles is never cast off, but retained. The same may be said of the cicatricial tissue of the Pulmonata, Nudi- branchia, and Prosobranchia. Several authors have been able to establish the same facts for worms. ‘The wounds of the Vertebrata and the Arthropoda (with some exceptions), however, heal under a scab of coagulated blood. The scab soon degen- erates and is cast off as soon as the healing has been accomplished. In time the leucocytes, which constitute the cicatricial tissue, become more and more hyalin. The granules, which were at first equally “distributed in the protoplasm, gather around the nucleus and along the cell wall and finally disappear. The cells change their shape—formerly round, they now become elongated. At the same time the nucleus also becomes elongated. At times this alteration begins in the most superficial layers of the cicatricial tissue, but the study of a greater number of sec- tions showed that generally the most exterior cells retained their round shape longer than those below the surface, the latter becoming elongated very soon (figs. 20 and 21). These changes take place in the cicatricial tissue before it is covered by epi- thelium. MHescheler, Friedlander, and Rievel found similar elongated spindle-shaped cells in the cicatricial tissue of worms. Friedliinder and Rievel claim that these cells are evolved from leucocytes, whereas Hescheler doubts it. What finally becomes of this cicatricial tissue? There are two points worth notice in connection with it. In the first place, I was never able to find any sign of degeneration in this tissue, and, secondly, the regenerating epithelium does not grow under or through it, but covers it. Both these facts show plainly that it is retained and not cast off. It therefore may be calied a blastema, and in order. to distinguish it from the second blastema, which appears later and consists of neuro- and sarcoblasts (fig. 31), I would designate 18 MATHILDE M. LANGE it as primary blastema. As this blastema does not degenerate, it is used to supply the material for some regenerating tissue. It is not likely that the primary blastema contributes any ma- terial for the construction of the nerves or muscles, for the nuclei of the primary and second blastema differ greatly in structure. The nuclei of the former are generally elongated and provided with several nucleoli, the nuclei of the latter are larger in size, round in shape, only have one nucleolus, and show a greater affinity for staining agents. All these differences indicate that it is quite impossible for the nerves or muscles to draw any material for their regeneration from the primary blastema. However, it is very likely that the primary blastema contributes a great part of its material to the construction of the new con- nective tissue, especially the dermal layer. There are two facts which further strengthen this theory. First, the similarity of the nuclear structure and, secondly, the fact thaf the primary blastema is dislocated by the second blastema’s being pushed sideward, so that it gradually occupies the place of the dermal connective tissue. Most of the papers dealing with the subject of regeneration do not definitely state what ultimately becomes of. the cicatricial tissue. This applies to the three papers here- tofore written on the regeneration in Mollusca. Nusbaum and Cucagna (’15) mention the presence of connective-tissue cells in the cicatricial tissue before the same is covered with epithelium, but they do not mention the origin of these cells. Even though the literature treating of regeneration in worms is very volumi- nous, I was not able to find very much information on the further utilization of the cicatricial tissue. Many authors never even mention its presence. Others explain its formation, but do not say what becomes of it later. Friedlander intimates that muscle fibers might arise from the cicatricial tissue, but does not state this as an actual fact. Rievel, who believes that the cicatricial tissue (he calls it granulation tissue) has its origin in the meso- derm, claims that later it becomes mesenchymatous and finally forms the unstriated muscles. According to Hescheler, the cicatricial tissue becomes fibrous (in the course of regeneration) and arranged in layers which run parallel to the front external OCTOPUS ARM, REGENERATION AND STRUCTURE 19 contours of the body, occupying a place which would naturally be filled by the continuation of the longitudinal muscles. As the cicatricial tissue of the animals whose blood contains fibrinogen soon becomes scabby and is cast off, it is quite natural that most of the publications on the regeneration in vertebrates and arthropods do not give any great attention to the cicatricial tissue. This applies almost without exception to all the reports on the regeneration in arthropods. Among the many authors who have studied the regeneration of vertebrates there are a few who point out that not all of the cicatricial tissue is merely a preliminary covering for the wound, but that some of it at least is utilized for some future purpose. Aufrecht (’90), Billroth, and Rindfleisch claim that the leucocytes enclosed in this tissue are the origin of connective-tissue fibers. Among the more recent reports on this subject I should like to draw: attention to a paper published by Baitsell (16). This paper treats of the processes connected with the healing of skin wounds in frogs. Baitsell points out that the blood-clot formed by the coagulation of the blood acts as a kind of connective tissue for the time being by holding the edges of the wound together. He found that the coagulated blood formed a typical fibrin net. Later on this net was changed into a fibrous tissue, consisting of separate fibers and fiber bundles. This alteration was affected in a few days’ time. This change could not be traced to the activity of any other cells, as it took place before any connective-tissue cells had migrated into the blood-clot. The new tissue had the appearance of regenerating connective tissue. It soon ex- hibited the same reactions to staining agents as the old con- nective tissue, but differed from the latter in its attitude toward pancreatin, as it was digested by it. Baitsell points out that embryonic connective tissue is also digestible by pancreatic juice. The summary of all these reports shows that the theory according to which connective tissue is evolved from cicatricial tissue has already been advanced by several investigators. The blastema is separated from the subjacent tissues by a well-defined boundary line, only where it touches the musculature does this line disappear, and the tissues seem to pass into each other. 20 MATHILDE M. LANGE When a short distal piece is amputated, very little blood leaves the blood-vessels. In such regenerations there is little primary blastema (fig. 23). In his study of the regeneration in Gastropoda, Techow describes processes very similar to those which I have just de- picted in Cephalopoda. He found that the wound remains open without any covering whatever during the first few hours after operation. Then the blood-vessels leading to the wound become filled with cells, which later form a layer over the wound. Te- chow gave a detailed description of these cells, but did not classify them histologically. In my opinion, these are haemolymph cells, for it would be rather strange for the blood-vessels to be suddenly filled with a great number of cells which normally did not belong there. An appearance of strange cells in the blood-vessels would denote a pathological condition. But these cells later on form a blastemal tissue, so they cannot be path- ological. These cells cannot be anything else than blood-cor- puscles, and from Techow’s paper we can clearly see that Gas- tropoda, like the Cephalopoda, do not bleed immediately after operation, but only after a period of several hours has elapsed. During the formation of the cicatricial tissue, which acts as a preliminary covering for the wound, the epithelium which is destined to form the final covering remains inactive. It is diffi- cult to state just how much time must elapse after operation before the regeneration of the epithelium is initiated. It varies from ten hours to two days. The causes of this fluctuation are probably the same as already mentioned on page 9. Before the epithelium begins to stretch over the wound, the basal membrane of the uninjured epithelial cells immediately adjacent to the wound draws back a little. According to Hescheler, the same thing happens before the regeneration of the epithelium in Lumbricidae. The cells from which the basal membrane has withdrawn are no longer in such close connection with the more proximal epithelial cells, and probably on that account able to aiter their form. They become flat, and their nuclei, which formerly were vertical, are now in horizontal position. Figure 24 shows a picture of such horizontal nuclei. These OCTOPUS ARM, REGENERATION AND STRUCTURE P| flat epithelial cells then proceed to crawl over the wound until the latter is entirely covered by an exceedingly thin epithelium, which seems almost no thicker than a membrane. The nuclei, however, are easily visible and the cuticle shows the characteristic striae. I could not detect any nuclear division at this stage. Here we have a case of rearrangement of old material, which is generally called morphalaxis. Cucagna and Nusbaum found that the final healing of wounds in Nudibranchia was accom- plished in the same manner. Lang, Hescheler, and Stevens (’06) state that the same is the case in worms, but Techow and Hanko, who both worked on Gastropoda, claim that mitosis takes place in the epithelium while the latter spreads over the wound. ‘The cells of the newly formed epithelium vary greatly in shape (fig. 24). Sometimes they are long, sometimes short and _ broad. Some of them are funnel- or pear-shaped, and others again have appendages extending into the subjacent tissue. In general the epithelial cells are larger than the cells of the adjacent tissue. The variety of form among the young epithelial cells is perhaps due to the absence of the basal membrane. The absence of such a membrane permits the direct connection of the epithelium with the subjacent embryonic tissue. This makes it possible for cells of this tissue to migrate into the epithelium in order to increase more rapidly the rather small number of its cells. But in spite of very careful search, [ was not able to find a single case of such migration. Wound healing having been accom- plished, the epithelial cells, which constitute the covering, undergo a change. They no longer remain flat, but become cubical, and their nuclei regain their former vertical position, at the same time growing more voluminous. At this stage the young epithelial cells begin multiplying by means of nuclear division. This activity is not confined to a certain part, but spreads all over the young epithelium. The division is so rapid that the new epithelium is soon filled with a large number of nuclei, and on the third or fourth day after operation looks like a syneytium (fig. 26a). There are relatively a greater number of nuclei in the new epithelium than in the old. As I could not find any cases of mitosis, | am inclined to think that the increase in the Be MATHILDE M. LANGE number of nuclei is due to amitotic or direct division. This opinion was confirmed by the discovery of several nuclei in different stages of amitotic division (fig. 26b). Lang (’09, 710) found that the epithelium of the Turbellarians also regenerates by means of Amitosis. 'Techow, however, claims that the increase in epithelial cells of the Gastropoda is due to both mitotic and amitotic division. At the time when this lively nuclear division of the epithelium begins, the latter is already provided with a well-developed basal membrane. It is difficult to say where this membrane has its origin. It is possible that the subjacent tissue takes an active part in its formation. The presence of a basal membrane probably prevents any epithelial cells from migrating into the subjacent tissue. At any rate, I could not find any such cases of migration. In this point the epithelium of the Cephalopoda differs greatly from the epithelium of the Gastropoda. Accord- ing to Techow, the latter contributes cells to the subjacent tissue, this being made possible by the tardy appearance of the basal membrane (it took eight days before it was formed). b. The muscles. Soon after operation disintegration sets in in those muscles immediately adjacent to the wound. This is due to a degeneration of muscular tissue, which comes to pass in the following manner. The sarcoplasm breaks down, the spiral fibers seem to expand or grow thicker, thereby filling the gaps left by the decaying sarcoplasm, and crowding out the granulated plasm of the core (fig. 27). In spite of this degener- ation, the muscle fibers still stain deeply when eosin is employed. Later on in the course of further degeneration the colorability decreases, the axial tube disappears, and the muscle fiber loses its cylindrical shape. In the end all that is left of the muscle fiber is a clotty mass, which stains very slightly. The nuclei of the muscle fibers do not all degenerate in the same way. In some the disintegration becomes noticeable in the chromatin, the latter massing together in little lumps, but the exterior form of the nucleus is not changed during this process, neither were there any visible signs of shrinkage. In other nuclei the shrinkage and the concentration of the chromatin seem to OCTOPUS ARM, REGENERATION AND STRUCTURE 23 take place simultaneously; at any rate, they alter their form, appearing round instead of elongated. These two processes continue until the nucleus has the appearance of a solid mass of chromatin. Then it breaks up into two, sometimes three parts, a process which has been called fragmentation (fig. 28). These nuclear fragments probably have great qualities of resist- ance, for they endure the whole process of degeneration, are present in its very first stage, and are still visible when the tissue has become quite necrotic. I was not able to find any phagocytic formations consisting of sarcoplasm and parts of the nucleus, such as Metchnikoff (’92) discovered in the degen- erating muscles of vertebrates. Neither was I able to detect a fatty degeneration which Bordage (’14) found to be the case in the muscles of Orthoptera. Soon after the wound has been covered by blood, corpuscles from the blood-clot migrate into the degenerated muscles and begin to dissolve and absorb the disintegrated parts. The cloddy remnants of the muscles, which up to the appearance of the blood-corpuscles, consisted of a solid mass, begin to dis- integrate, and at the same time the nuclear fragments (described above) decrease in number (fig. 29). Finally so many blood- corpuscles collect in the degenerated muscle tissue that the latter appears like the primary blastema and is hardly to be distinguished from it. The first sign of regeneration in the muscle tissues is the ap- pearance of large cells, which have very little protoplasm and seem to consist only of a large nucleus (fig. 30). These cells first appear in that part of the blastema which occupies the place formerly filled by the degenerated muscles. These cells are most probably sarcoblasts, and like the sarcoblasts of the verte- brates originate in the old muscle tissue. It is difficult to give any exact information as to how they are formed or as to whether the whole muscle fiber or only a part (and which part) contributes the necessary material. But there can be no doubt about their actually being sarcoblasts, for their development into muscle fibers can easily be followed up in later stages. The sarcoblasts have a rounded or oval nucleus containing very fine granules 24 MATHILDE M. LANGE and one nucleolus. They migrate to the distal portion of the wound, and in combination with the neuroblasts form the second blastema (fig. 31). They multiply very rapidly by means of mitosis (fig. 32). About twelve to fourteen days after operation the sarcoblasts of the proximal portion of the regenerated piece begin to transform into the definitive muscle fibers. But this differentiation does not begin simultaneously in every part of the musculature. The longitudinal muscles are the first to begin this process, and in these the very first differentiation takes place in those parts which are nearest to the perimuscular con- nective-tissue membrane. The differentiation progresses distal- ward in the longitudinal muscles. The transformation of the transverse muscles into their final form probably takes more time, for they are still in the sarcoblast stage at a time when the surrounding longitudinal muscles already appear as quite well-developed muscle cells. Most likely this is due to the differ- ent intensity of growth in the different muscle fibers. The muscle fiber can only grow by means of sarcoblasts. The best proof of this assertion is the fact that at the distal end of every regenerated piece as well as of every normal arm there are no muscle cells, but only sarcoblasts. As the difference in thickness between the regenerated piece and the stump is particularly great at the juncture of these two parts, there must be an active and intense growth in breadth at this point, in order to equalize this difference in thickness. The transverse musculature will probably take an active part in this growth. Whereas in the longitudinal muscles the point of active and intense growth is always carried farther and farther away from the juncture the more the regenerated piece grows in length, the transverse muscles of this part remain in a stage of active and intense growth for a much longer time. Hence the transformation of the sarcoblasts into muscle fibers begins later in the transverse muscles than in the longitudinal muscles. That the sarcoblasts of the Cephalopoda are a product of the old muscle fibers has already been mentioned. This is a case of new formation from preexisting tissue and verifies the statement that a new tissue can only be formed by the same kind of old tissue. According 5 OCTOPUS ARM, REGENERATION AND STRUCTURE 2 to Techow, however, this rule is not applicable to the muscles of the Gastropoda. He claims that in the course of regeneration the new muscles are also evolved from large cells (sarcoblasts), but believes that these cells are originally epithelial cells which have migrated into the subjacent tissue. There are very few reports on the histological processes connected with the muscle regeneration in Mollusca. I could only find three in all—Te- chow’s, Hanko’s, and the paper published by Cucagna and Nus- baum on the regeneration in Nudibranchia. Techow’s opinion on the subject has already been given. Hanko claims that the new muscles are a product of the old, but he does not mention the presence of any sarcoblasts. He states that certain cells (Wanderzellen), whose origin he does not explain, gather around the distal and somewhat inflated end of the muscle stump. These cells form a kind of bridge between the muscle stump and epi- thelium, and along this bridge the new muscle fibers, which are formed by proliferations of the old, develop. According to Cucagna and Nusbaum, the new muscles are evolved from sareocytes (sarcoblasts) formed of the sarcoplasm and nucleus of the old muscles. Schultz, Bordage (14) and Friedrich state that the same is true of the muscles of the Arthropoda. Barfurth (91), Nauwerk (’90), and Fraisse (’86) found that the muscles of the vertebrates also regenerate by means of sarcoblasts. The musculature of the suckers is formed by lateral pro- liferations of the central muscle bundle (fig. 33). These buds are soon separated from their seat of origin by connective tissue. The invagination of external tissue which causes the formation of the sucking cavity also indents the muscle-bud, consisting of sarcoblasts. In the course of further development the sarco- blasts are grouped around the cavity in two distinct parallel layers (fig. 34). Later these sarcoblasts develop into the radi- ating and circular muscles of the sucker. As in the transverse muscles, the formation of the final muscle fibers begins later in the sucker muscles than in the longitudinal muscles of the central muscle bundle. 26 MATHILDE M. LANGE c. The nervous system. Up to the present no histological study of nerve regeneration in Mollusca has been published. Techow refrained from doing so on account of technical diffi- culties. Hanko states that the nerves which innervate the eye of Nassa mutabilis regenerate, but does not state how. He refers to a paper by M. Kiipfer which was in preparation when his own was published. I tried to obtain this paper, but as it has not yet been published, I was unable to see it. Nerve regeneration in worms has been studied by Lehnert, Bardeen (04), Hescheler, Lang, Flexner (’98), Schultz, and Stevens. The two first authors claim that the new nerve fibers simply grow out of the old. Lang, Flexner, Schultz, and Stevens found that the new brain is formed by parenchym cells. The regeneration of the nervous system of vertebrates has often been made the subject of histological study. Experiments were made chiefly on the spinal cord of tritons and lizards, in some cases on the spinal cord and also on the brain of birds (pigeons) and mammals (rabbits and dogs). Dentan (73), Eichhorst (’75), Naunyn (74), Keresztzsegy (92), and Hanns (’92) could not find any regeneration in the central nervous system of vertebrates. Strébe (’94) claims that the injured spinal cord of the rabbit makes an effort to repair the damage, but that an actual regen- eration does not take place. Contrary to this, Walter (753), Cattani (85), Brown-Sequard (’50), Miiller (’64—65), Masius (’70), and van Lair (’70), Caproso (’88), all claim that the central nervous system is able to regenerate. Tedeschi (97) found regenerated ganglion cells and nerve fibers in the brain of mam- mals. There is quite some diversity of opinion on the manner in which the nerve cells multiply. Walter, Cattani, Modino (85), Friedmann (’88), Ziegler (95), Coen (’88), Sanarelli (’96), Marinescu (’94), and Tedeschi, all claim that the nerve cells multiply by means of mitosis. Caproso and Barfurth, on the other hand, believe that the new nerve cells are evolved from neuroblasts, which originate in the epithelium of the central canal. Mihlmann (’08, ’10), who has made the structure and growth of the nerve cell an object of special study, is of the opinion that already at an early stage of its development certain OCTOPUS ARM, REGENERATION AND STRUCTURE 2t inhibiting elements appear in the nerve cell which prevent its further division. In the following I shall try to give a short histological study of nerve regeneration in the arm of Octopus vulgaris. An examination of the wound with a magnifying glass im- mediately after operation reveals the fact that the axial nerve protrudes beyond the surrounding tissues (fig. 4). H. Miiller found similar conditions in the spinal cord of a lizard whose tail had been amputated. An intense and active degeneration is initiated in the protruding part of the axial nerve. This ‘ disintegration progresses so quickly, that it is very difficult to make a thorough examination of its various stages. It begins in the layer of ganglion cells. The first signs of degeneration become visible in the nucleus. The chromatin, which in the normal nucleus is generally thickest along the periphery, moves toward the center and gathers around the nucleolus, the nuclear membrane still retaining its original form while this process is going on (fig. 35). Later on this membrane also degenerates, and the nucleus, which has in the meantime shrunk to a homo- geneous little lump, lies in a kind of vacuole. Most of these nuclear remnants soon disappear, but some of them seem to be endowed with a great power of resistance, as they are still present even after a few days. I was not able to observe carefully the degeneration of the protoplasm. On the whole, I found the endoplasm resisting longer than the ectoplasm. The nuclei of the glia tissues shrink and are changed into homogeneous chro- matin globules, resembling the reduced ganglion-cell nuclei, only somewhat smaller in size. The degeneration in the neuropil is at first not as marked as in the ganglia layer, and is only noticeable by a slight disintegration of the tissues. When stained with eosin, the neuropil no longer exhibits the same intense coloring as before, and the glia nuclei distributed in it have noticeably shrunk. The fibers of the myelin cords are swollen and unduly enlarged at the distal end of the stump. Sometimes they are from four to six times as voluminous as the normal fiber. The myelin, which in the normal fiber is so equally distributed as to give it a homogeneous appearance, THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 31, No. 1 28 MATHILDE M. LANGE clots together to granules which either form a network or larger ‘lumps of granules (fig. 36). Strébe found that the white sub- stance of the spinal cord of vertebrates also swells up as a result of degeneration. This fact would to a certain degree strengthen Colosanti’s assertion that a kind of analogy existed between the myelin cord and the white matter. The degeneration reaches deeper into the myelin cords than into the ganglia layer and the neuropil. About ten hours after operation a further disintegration is noticeable in the myelin cords and the neuropil, whereas the , ganglion cells show hardly any further change. There isa marked increase in the number of nuclei in the neuropil as well as in the myelin cords. In the myelin cords this increase is due to the migration of blood-corpuscles. The fibers of the myelin cords are torn asunder, the myelin cords thus occupying more space at the distal end than is normally the case (fig. 23). Prob- ably the blood-corpuscles also migrate into the central mass of nerve fibers, but I could not ascertain their presence there with absolute surety. The increase in the number of nuclei in the neuropil is due to the amitotic or direct division of the glia nuclei, which have entered the central nerve-fiber mass along with the processes of the nerve cells (fig. 37). About one or two days after operation a large number of cells, all provided with vesicular nuclei, appear in the neuropil and then migrate to the most distal part of the stump. Together with the sarcoblasts they form the so-called second blastema (fig. 31). These cells are doubtlessly neuroblasts, for later on the ganglia are evolved from them. It is very difficult to find the origin of these neuroblasts. I am of the opinion that the glia nuclei contribute largely to their formation, for the glia nuclei are the only nuclei (besides a few nuclei of the connective tissue), which are normally embedded in the central mass of nerve fibers. But I must also draw attention to the fact that the nuclei of the neuroblasts are a little larger than the glia nuclei. ‘The neuroblasts are very similar in structure to those ganglion cells lying nearest the neuropil, which have been de- scribed as nude nerve nuclei. Perhaps these cells having been OCTOPUS ARM, REGENERATION AND STRUCTURE 29 stimulated to division by the operation, have multiplied and migrated into the central nerve-fiber mass. Some neuroblasts also originate in the layer of ganglion cells. It is hard to tell whether they are evolved from the glia or produced by the nerve cells. If the latter is the case, we may safely say that only the small ganglia, the so-called nude nerve nuclei, take part in the production of neuroblasts. These small ganglia are probably in a very early stage of differentiation. Therefore they are more like the neuroblasts than the medium-sized or larger ganglia. But in spite of their similarity, I would not say - that the small ganglia and the neuroblasts are identical. I was never able to discover any mitosis in the small ganglia, whereas the neuroblasts exhibited many cases of karyokinesis. The large and medium-sized ganglia do not participate in the pro- duction of neuroblasts. Neither a division nor a reduction of the protoplasm (a process which would bring them in closer relation with the neuroblasts) takes place. It is quite probable that both the glia and the small nerve cells produce the neuroblasts. The neuroblasts are very similar to the sarcoblasts, which makes it difficult to tell them apart (fig. 31). Fine fibers of the myelin cords grow into the second blastema, thus separating the neuroblasts from the sarcoblasts of the ex- ternally located muscle bundle. The myelin cords do not change their appearance very much during regeneration. They do not undergo such a radical change as the rest of the tissues. Their alteration, due to degeneration, has already been mentioned (p. 27), and the only change caused by regeneration was an increase in the number of nuclei of the sustentacular tissue. In consequence of this remarkable behavior, they are more easily and rapidly recognized in the regenerated piece than any of the other parts, with the exception of the epithelium, the main artery, and the two venae superficiales. The myelin cords grow into the regenerated piece, not in the form of blastema cells of any kind, but as a well-differentiated tissue. Many authors have put forth the theory that regeneration is in a large degree dependent on the presence of nerves. The fact that the myelin cords, which serve as a connection between the brain 30 MATHILDE M. LANGE and the axial nerve, grow so quickly and appear as differentiated myelin fibers in the most distal portion of the regenerated piece, while all the surrounding tissues are still in blastemal stage, is a strong argument in favor of this theory. On the inner side of the myelin cords the neuroblasts form the same regular rows as the ganglion cells in the normal arm. Eleven days after operation this regular arrangement and also the tissues separat- ing these rows were visible. The first sign of the new central mass of nerve fibers also appears at the same time. The myelin cords probably produce the first fibers of the new neuropil, for the young nerve cells do not at this time exhibit any processes. Only later are the neuroblasts turned into ganglion cells by forming protoplasm and fibers. As the differentiation of the ganglia progresses, the neuropil naturally gains in size. Three weeks after operation the axial nerve is well developed in all of its three components (fig. 38). Among the ganglia the small cells, the so-called nude nerve nuclei, predominate. But there are also quite .a number of medium-sized nerve cells present, whereas large ganglia are still missing. The latter probably appear very late, for I was unable to find any, even in regenerated pieces, which were already in quite an advanced stage. The young neuropil contains relatively many nuclei, which, however, de- crease in numbers as development goes on. The greater part of the regenerated piece is occupied by the axial nerve, the rest of the tissues being confined to a relatively small space. The same conditions prevail in the embryonic arm. In the normal arm the axial nerve constitutes a fourth part of the whole.® Unfortunately, I was not able to observe the formation of the sucker ganglia and the four nerve cords embedded in the muscles. The sucker ganglia probably appear very late in the regenerated piece, for I was not able to find a single one. I believe their formation is only initiated after the nerves which connect the axial nerves with the sucker ganglia and also inner- vate the suckers have grown out from the axial nerve. It is 5 JT should like to draw attention to the publications of Brynes and Fritzsch. Both of them studied regeneration in water newt (Triton) extremities and found that cartilage is evolved from blastema directly opposite of the growing nerve. OCTOPUS ARM, REGENERATION AND STRUCTURE jl possible that the ganglia arise in the embryonic connective tissue under the influence of the growing nerve. In a piece which had been fixed three or four days after operation and which was just on the point of beginning regeneration, I was able to observe fine fibers of the peripheral nerves, which originated in the uninjured part of the axial nerve, but innervated one of the amputated suckers growing into the primary blastema. At the distal end of one of these fine fibers I could detect some cells which showed a marked similarity to the nerve cells (fig. 39).° The subcutaneous connective tissue is most probably evolved from the primary blastema. At first, however, the latter is separated from the subjacent connective tissue by a sharp boundary line, which later on disappears. Unfortunately, I am not able to give any detailed account of the regeneration of the vascular system. I heard that Minervini had studied this process, but I was not able to find his publication. Authors who have studied the regeneration of the vascular system in other animals claim that the new blood-vessels are formed by a proliferation of the endothelium of the old blood-vessels. I was not able to detect any proliferation of endothelium in the regenerating arm of the Cephalopoda. The main artery and the veins grow rapidly and are visible in the most distal portion of the regenerated piece. In such a stage as is exhibited in figure 8 the blood-vessels form a kind of plexus which is well filled with blood. I did not make the regeneration of the epithelial glands and the chromatophores the subject of any closer study. Chun has published a very detailed account of the development of the chromatophores. SUMMARY The most important morphological changes which occur during the regeneration of the arm of the Cephalopoda (Octopus vul- garis) are the following: 1. Wound healing. After operation the edges of the wound curl inward. The axial nerve protrudes beyond the other tissues. Bleeding does not take place immediately after operation, but ae MATHILDE M. LANGE after a period of several hours (five to six). The wound is then completely overspread with blood which serves as a preliminary covering for the same. After bleeding, the protrusion of the axial nerve disappears. The last two suckers at the obtuse end of the arm are abnormally drawn up, as if they also participated in the preliminary closing of the wound. ‘The final wound healing by epithelium occurs in some animals within the first twenty- four hours after operation; in most cases, however, it takes from thirty-six to forty-eight hours. 2. Change of form. The distal suckers which were drawn up regain their normal position. The first visible sign of regener- ation appears in the shape of a little knob near the external side of the arm. The knob develops into a little lash-like appendage, which appears like a thin rod in comparison to the arm stump. 3. Formation of the suckers. The newly formed suckers must be divided into two groups, those that are formed as suckers of the regenerated piece proper and those formed at the obtuse end of the arm stump. All suckers first appear in the form. of little transverse folds. Later on they are rounded off to little papillae. The newly formed suckers at the obtuse end of the arm are arranged in single file. They remain in a single row during the greater part of their development. On the other hand, the suckers of the regenerated piece exhibit the final double-rowed arrangement at a very early stage of their develop- ment. The sucker cavity and the adhesive part are both formed by invagination. At the base of the regenerated piece, one or two suckers (sometimes three) always remain in single file. 4. The chromatophores. The first new chromatophores appear about three to four weeks after operation. They are smaller and of a lighter shade than the normal chromatophores. The histological study led to the following results: 1. Wound healing. The wound is at first unprotected (five to six hours). Then a preliminary covering, consisting chiefly of a clot of agglutinated blood-corpuscles, is formed. From this blood-clot a primary blastema is gradually evolved, the blood-plasm becoming less and less, and the agglutinated blood- corpuscles forming a fine network. The epithelium remains OCTOPUS ARM, REGENERATION AND STRUCTURE 33 inactive for the first few hours after operation. Later on the epithelium cells at the edge of the wound grow flat, the nuclei which up to that time were vertical change their position, be- coming parallel to the edge of the wound. ‘The flat cells creep over the wound from all sides, till the latter is completely covered by a very thin tessellated epithelium, provided with a cuticle. The tessellated epithelium gradually becomes cubical and later on cylindrical. 2. The musculature. Disintegration begins in the musculature soon after operation. The sarcoplasm degenerates. The spiral fibers expand, thus filling the gaps left by the sarcoplasm, and at the same time narrowing the central duct and crowding out the granulated substance of the core. The result of this degen- eration is a cloddy mass, which shows less affinity to staining agents than the normal muscle fiber. ‘The nuclei become homo- geneous globules of chromatin, and break up into two or three pieces (fragmentation). The degenerated muscles are partly dissolved or absorbed by blood-corpuscles which migrate into the disintegrated tissue. The muscles regenerate by means of -sarcoblasts. The sarcoblast possesses quite a large nucleus, which is provided with one nucleolus. The sarcoblasts later migrate to the distal part of the stump, and in combination with the neuroblasts form the second blastema. They multiply by means of indirect or mitotic division. The sarcoblasts of the external longitudinal muscles are the first to exhibit muscle fibers. In the transverse muscles fibers appear at a much later date. The muscles of the suckers are evolved from lateral proliferations of the sareoblasts of the central muscle-fiber bundle. 3. The nervous system. The first sign of disintegration of the axial nerve is found in the layer of ganglion cells. The ectoplasm of these cells degenerates and the nucleus shrinks, the chromatin gathering around the nucleolus. The nuclei of the neuroglia undergo the same change. The fibers of the myelin cords expand and often grow five times the size of the normal tissue. The only signs of degeneration visible in the neuropil are the shrinking of the neuroglia nuclei embedded there and a decrease of its 34 MATHILDE M. LANGE affinity to staining agents. Later the neuropil becomes vacu- olized and the neuroglia nuclei increase in numbers. Blood- corpuscles penetrate the degenerated portions of the myelin cords, but disappear again. The differentiated nerve cells do not multiply by division, but by means of neuroblasts, which originate in the central mass of nerve fibers as well as in the layer of ganglia. Probably both neuroglia tissue and ganglia contribute to the formation of the neuroblasts. The myelin cords do not pass through a blastema stage. They grow as a well differentiated tissue, and are easily discerned in the regen- erated piece. Together with the epithelium and the blood- vessels, they are the first tissues which can be distinguished, all the remaining tissues still being in a blastematous stage. 4. The connective tissue. The first elements which later on go to construct the new connective tissue are probably evolved from the primary blastema, a product of the agglutinated blood- corpuscles. The general results of this study may easily be expressed in the following two sentences: 1. All new tissues with the exception of the dermal connective tissue are produced by the preexisting tissues of the same kind. 2. Sepia occasionally replaces a lost arm by means of com- pensatory regulation (development of the correlated buccal arm to replace a lost one). APPENDIX In the introduction mention was made of the extirpation of the lens. As, however, not many animals survived this oper- ation for any length of time, there was not enough material at my disposal to enable me to give any kind of detailed account of the regeneration of this organ. After extirpation of the lens the injured eye lost its ability to perceive light. A few animals, however, survived the operation and lived for over ten weeks after it had been performed. On these animals I noticed that the injured eye had regained its sensitiveness to light after a period of about eight weeks. This fact was ascertained in the following manner. It is well known that the circular pad of skin which surrounds the visible parts of the eye acts as a lid. If the eye is suddenly exposed to light, this pseudolid shuts. If I exposed an eye to the light whose lens had been extirpated a few days previously, then the pseudolid did not close. Ten weeks after operation, however, the same eye showed the characteristic contraction of the pseudolid when suddenly exposed to light. Some animals even survive the loss of a whole eye. While at Naples I one day received a sepiola (from Doctor Naef), which had completely lost its one eye. The wound was healed, but I could not detect any signs of regeneration. Perhaps the animal had been injured just a few days before its capture. 36 MATHILDE M. LANGE LITERATURE CITED Aurrecut, E. 1890 Ueber die Genese des Bindegewebes nebst einigen Bemer- kungen itiber die Neubildung quergestreifter Muskelfasern und die Heilung per primam intensionem. Virchows Arch., Bd. 44. Bartrsety, G. A. 1916 The origin and structure of a fibrous tissue formed in wound healing. Anat. Rec., vol. 10. vy. Battowitz, E. 1893 Ueber den feineren Bau der Muskelsubstanz. Erstens die Muskelfaser der Cephalopoden. Arch. f. mikr. Anat., Bd. 39, 8. 592-309. BarpEEN, Cu. R. 1904 American Journal of Physiology, vol. 5. Barrurtu, D. 1891 Zur Regeneration der Gewebe. Arch. f. mikr. Anat., Bd. 37, S. 406-492. Regeneration u. Involution. Merkel u. Bonnet, Ergebnisse der Anatomie. Bauer, V. 1909 Einfiihrung in die Physiologie der Cephalopoden. Mitt. d. zoolog. Stat. zu Neapel, Bd. 19, 8. 149-268. VAN BENEDEN, P. J. 1898 Mémoire sur l’Argonaute (nouveaux mém. de Aad: Roy. de se. de Bruxelles, 1898. BittrotH, TH. Zitiert nach Marchand. Boprerzky, N. 1877 Untersuchungen iiber die Entw. der Cephalopoden Nachr. v. d. k. Gesell. d. Kunde d. Natur. Antrop. u. Ethnogr. Moskau. Bd. 24 Borpace, Ep. 1914 Phénoménes histolytiques observés pendant la régénéra. tion des appendices chez certains Orthoptéres. C. R. Acad. de Se. Paris, T. 161, p. 125. Brock, J. 1886 Indische Cephalopoden. Zoolog. Jahrb., Bd. 2, 8. 591-593. Brown-Srquarp 1850 Régénération des Tissus de la moelle épiniére. Gaz. méd., 1850, p. 250. Brynes, E. F. 1899 On the regeneration of limbs in frogs after the extirpation. of limb rudiments. Anat. Anz., Bd. 15. Capraso, E. Sulla rigenerazione de midollo spinale della coda dei Tritoni. Ziegler’s Beitrage, Bd. 5. CarannI, Fru. S. 1885 Sulla Fisiologia del gran Simpatico. Gazz. degli Osp. Cuiiron, J. 1866 Recherches pour servir A l’histoire du systéme nerveux des Cephalopodes dibranchieux. Ann. Se., T. 5. (Séme série), p. 1-122. Cuun, C. Ueber die Natur und die Entwicklung der Chromatophoren bei den Cephalopoden. Verh. d. deutsch.-zoolog. Ges., 12. Vers. S. 162-182. Corn, P. E. 1888 Ueber die Heilung von Stichwunden des Gehirns. Ziegler’s Beitriige, Bd. 2 CotosantI, J. 1876 Anatomische und physiologische Untersuchungen iiber die Arme der Cephalopoden. Reichert u. Dubois-Reymond’s Arch. f. Anat. ConHuEImM 1869 Ueber das Verhalten der fixen Bindegewebskorperchen bei Entziindungen. Virchows Arch., Bd. 45, 8. 333. Cucaena, G., unp Nusspaum, J. 1915 Regeneration bei Nudibranchien. Arch. f. Ent. Mech., Bd. 41, 8. 558-579. OCTOPUS ARM, REGENERATION AND STRUCTURE Mh Cutnot, L. 1891 Etude sur le sang et les glandes lymphatiques. Arch. de zool. expérim., T, 9, 2éme série, p. 13-90 et p. 593-670. Cuvier, J. 1817 Mémoire pour servir 4 |’Histoire et 4 l’anatomie des Mol- lusques, p. 1-54 (Paris-Déterville). DenTAn, P. 1873 Quelques recherches sur la Régénération fonctionnelle et anatomique de la moelle épiniére. Berne. Dominici, M. 1911 Experimenteller Beitrag zum Studium der Regeneration der peripheren Nerven. Berliner klin. Wochenschrift, Jahrb. 48. Exserspacu, A. 1915 Zur Anatomie von Cirroteuthis Umbellata Fischer und Stauroteuthis sp. Zeitschr. f. wiss. Zool., Bd. 93, Heft 3. Eicunorst, H. 1875 Ueber die Entwicklung des menschl. Riickermarkes u. seiner Formelemente, Virchows Archiy., Bd. 64, S. 425. Ersia, H. Biologische Studien. Kosmos 12. Faussek, V. Ueber die sogenannten ‘Weissen Ko6rper,’ sowie iiber die em- bryonale Entwicklung desselben, der Cerebral ganglien u. des Knor- pels bei Cephalopoden. Acad. Imp. Sc. St. Petersburg. 7éme Série, ARG Gis rate) FERNANDEZ, Mia. 1907 Zur Histologie des Tentacles von Nautilus pompilius. Zeitschr. f. wiss. Zool., Bd. 88. FLEXNER, S. 1898 The regeneration of the nervous system of Planaria torva (maculata), and the anatomy of the double-headed forms. Jour. Morph., vol. 14. Fratsse, P. 1886 Die Regeneration von Geweben und Organen bei den Wir- beltieren. Biolog. Zentralbl. Frépprice, L. Recherches sur la physiologie du poulpe commun. Arch. de zool, expér., T. 7. FRIEDLANDER, B. 1895 Ueber die Regeneration herausgeschnittener Teile des Zentralnervensystems von Regenwiirmern. Zeitschr. f. wiss. Zool., Bd. 60. FRIEDMANN 1888 Ueber progressive Verainderungen der Ganglienzelle bei Entziindungen u. Nervenktankheiten. Arch. f. Psych., Bd. 13, 8. 244. Friepricu, P. 1906 Regeneration der Beine und Autotomie bei Spinnen. Arch. f. Ent.-Mech., Bd. 20. Fritscu, C. 1910 Regenerationsvorginge des Gliedmassenskelettes der Am- phibien. Zoolog. Jahrb. Abt. f. allg. Zool., Bd. 30, Heft 3. GARIAEFF, Wu. 1901 Histologie des Nervensystems der Cephalopoden. Zeit- sehr. f. wiss. Zool., Bd. 92, S. 149-186. GeppeEs, P. On coalescence of amoeboid cells into plasmodia and on the so-called coagulation of intervertebrate fluids. Proc. of Royal Soc. London, vol. 30. GrenaAcHER, H. 1874 Zur Entwicklungsgeschichte der Cephalopoden. Zeit- schr. f. wiss. Zool., Bd. 24, S. 419-498. Grimpe, G. 1913 Das Blutgefissystem der Dibranchiaten Cephalopoden. Teil I, Octopoda. Zeitschr. f. wiss. Zool., Bd. 104, Heft 4, 8. 531-631. Gusrrn, J. 1908 Contribution 4 l’Etude des systémes cutané, musculaire et nerveux de l’appareil tentaculaire des Cephalopodes. Arch. de Zool. experim. T. 8, 4éme série, p. 1-178. 38 MATHILDE M. LANGE Hanxo, B. 1913 Ueber das Regenerationsvermégen und die Regeneration verschiedener Organe von Nassa mutabilis (L.), Arch. f. Ent-Mech.., Bd. 38, Heft 3. 1913 Ueber einen gespaltenen Arm von Octopus vulgaris. Ibid., Babaiaess 2idi—222 Hanns, Dr. 1892 Ueber Degenerations- u. Regenerationsvorgiinge im Riick- enmark des Hundes nach Durchschneidung. Ziegler’s Beitrige, Bd. 12, S. 33: HescHELerR, K. 1897 Ueber Regenerationsvorginge bei Lumbriciden. Je- nache Zeitschrift, Bd. 30. 1898 Ueber Regenerationsvorginge bei Lumbriciden. II. Teil. Jena, Gustav Fischer. Hiuuia, R. 1912 Das Nervensystem von Sepia officinalis L. Zeitschr. f. wiss. Zool., Bd. 101, S. 736-806. Jaxupski, A. W. 1915 Studien tiber das Gliagewebe der Molusken. II. Teil, Cephalopoden. Zeitschr. f. wiss. Zool., Bd. 112, S. 48-68. KERESZTZSEGY, J. 1892 Ueber Degenerations- u. Regenerationsvorgiinge im Riickenmark des Hundes nach Durchschneidung. Ziegler’s Beitrige, IBYel, JZ Kuinz, J. 1914 Experimentelle Schwanzregeneration bei Bilchen, Myoxidae. Arch. f. Ent.-Mech., Bd. 40, S. 344-368. KO6OuiurKER, A. 1844 Entwickl. Geschichte der Cephalopoden. Meyer u. Zeller, Zurich. KKotuMann, M. 1908 Recherches sur les Leucocytes. Ann. Sc. nat., 9éme série, T. 8. KKorScHELT, E. 1907 Regeneration und Transplantation. Jena. Festschrift fiir Leuckart. Entwicklungsgeschichte der Cephalopoden. Lane, P. 1909 Ueber Regeneration bei Planarien.. Arch. f. mikr. Anat., Bd. 79, S. 361-424. 1910 Experimentelle und histologische Studien an Tubellarien. Ibid., Bd. 82. Lors, Leo 1908 Vergleichende Untersuchingen iiber die Thrombose. Vir- chows Arch., Bd. 185, S. 160. 1909 Blutgerinnung bei Wirbellosen. Biochem. Zeitschr., Bd. 24, S. 478-495. Marinescu. 1894 Sur la régénération des centres nerveux. Compte rendu de la soe. de Biolog., 17 Mai. Marcuanp, J. 1901 Prozesse der Wundverheilung. Deutsche Chirurgie, Bd. 16. Masius uv. vAN Latr 1870 Bullet. de l’Acad. roy. de Belgique, AD euPAlle MeEtscHNIKkoFF, E. 1892 La phagocytose musculaire. Ann. de Il’Institut Pasteur, T. 6, p. 1-12. Mopino, C. 1885 Giornale della R. Acad. di Torino, Gennaio-Febr. Moreno, J. 1907 La candés ganglienas en los tentaculos de Cephalopodi. Rev. real. Acad. de Cienzas exactas bisy. nat. de Madrid, T. 5. Morean-Moskowskit 1907 Regeneration. Leipzig. Miutmann, M. 1811 Studien iiber den Bau und das Wachstun der Nerven- zelle. Arch. f. mikr. Anat., Bd. 77. OCTOPUS ARM, REGENERATION AND STRUCTURE 39 Miuimann, M. 1912 Mikrochemische Untersuchungen an der wachsenden Nervenzelle. Ibid., Bd. 79. Mier, J. 1864-1865 Regeneration der Wirbelsiiule und des Riickenmarks bei Tritonen und Eidechsen. Abhandl. d. Senkenberg. nat. Ges., Bd. 5. Narr, A. Cephalopoda. Handwérterb. d. Naturwiss., Bd. 2. Teutologische Notizen. Zoolog. Anz., Bd. 40. Naunyn, Bernu. 1874 Ueber Regeneration u. Verinderungen im Riickenmark nach streckenweiser totaler Zerstérung derselben. Arch. f. exper. Pathol. u. Pharmat., Bd. 2. Nuspaum, J. Vergleichende Regenerationsstudien. Zeitschr. f. wiss. Zool., Bd. 79. Ost, J. 1906 Zur Kenntnis der Regeneration der Extremititen bei den Arthro- poden. Arch. f. Ent.-Mech., Bd. 22, 8. 289-325. OwssANNIKOwW, P. Ueber das central Nervensystem und das Gehérorgan der Cephalopoden. Mem. Ac. St. Petersb. PrerreRKorn, A. 1915 Das Nervensystem der Octopoden. Zeitschr. f. wiss. Zool., Bd. 114, Heft 3. Reep, M. A. 1905 The regeneration of the first leg of the crayfish. Arch. f. Ent. Mech., Bd. 18. ReEICHENSPERGER, A. 1912 Beitrige zur Histologie und zum Verlauf der Re- generation bei Crinoiden. Zeitschr. f. wiss. Zool., Bd. 101, S. 1-69. Rievet, H. 1896 Die Regeneration des Vorderdarmes und Enddarmes bei einigen Aneliden. Zeitschr. f. wiss. Zoolog., Bd. 62, S. 289-341. Riccensacu, E. 1901 Beobachtungen iiber Selbstverstiimmelung. Zool. Anzeiger, Bd. 24, S. 587-593. 1902 Die Selbstverstiimmelung der Tiere. Anat. Hefte, II. Abt. RinpFueiscu, Ep. Zitiert nach Marchand. SANARELLI 1896 I processi riparativi nel cervello e nel cervelletto. R. acad. dei Lincei. Scuutrz, E. Ueber die Regeneration der Spinnenfiisse. Trudy Petersburg. Obes. Estesvoip., Bd. 29. Stevens, N. M. 1909 A histological study of regeneration in Planaria sim- plissima, P. maculata, and Pl. morgani. Arch. f. Ent.-Mech., Bd. 24. 1907 Notes on regeneration in Planaria lugubris. Ibid., Bd. 13. Strorse, H. 1894 Experimentelle Untersuchungen iiber die degenerativen und reparativen Vorginge bei der Heilung von Verletzungen des Riickenmarks, nebst Bemerkungen zur Histogenese der sekundiren Degeneration in Riickermark. Zieglers Beitrige, Bd. 15. Trcnow, G. 1910 Zur Regeneration des Weichkérpers bei den Gatropoden. Arch. f. Ent.-Mech., Bd. 31. Treprescur, A. 1897 Anatomisch-experimenteller Beitrag zum Studium der Regeneration des Gewebes des Zentralnervensystems. Beitr. z. Anat. u. path., Bd. 21, 8. 438-72. Tuutin, J. Beitrag zur Frage der Muskeldegeneration. Arch. f. mikr. Anat., Bd. 79, S. 208-230. UExktiLL, F. von 1893 Physiologische Untersuchungen an Eledone moschata. II. Die Reflexe der Arme. Zeitschr. f. Biolog., Bd. 30. 40 MATHILDE M. LANGE Ussow, M. 1894 Zoologisch-embryologische Untersuchungen. Die Kopffiis- ler. Arch. f. Nat. Gesch., Bd. 40, S. 329. 1881 Die Entwicklung der Cephalopoden. Arch. de Biol., T. 2, | F, 4, VeRILL, A. 1892 Cephalopodes of N. E. America. Transact. of Connecticut Acad., vol. 5, p. 260. VILALLETON, L. 1888 Recherches sur les premiéres phases du développement de la seiche 1888. Ann. de Se. nat., 7éme série zool., T. 6. Wa.ttTeR 1853 De regeneratione ganglionum. Diss. inaug. Bonnae. Were, R. 1910 Ueber den Golgi-Kopschen Apparat in der Ganglienzelle der Cephalopoden. Anzeig. d. akad. d. Wiss. zu Krakau. ZIEGLER, KE. 1895 Lehrbuch der path. Anatomie, Bd. 2, 8S. 354. PLATE 1 DESCRIPTION OF FIGURES 1 Transverse section of an arm of Octopus vulgaris according to Guérin. a, epithelium; 6, subcutaneous connective tissue; c, chromatophores; d, vein; e, dermal musculature; /, perimuscular connective-tissue membrane; g, external oblique muscle; 7, inner oblique muscle; j, inner lateral longitudinal muscle; k, external longitudinal muscle; /, internal longitudinal muscle bundle; m, trans- verse musculature; m, perinervous connective tissue; 0, layer of ganglia; p, cen- tral mass of nerve fibers (neuropil); g, myelin cords; 7, brachial arteries; s, nerve; t, sucker; u, sucking cavity; v, adhesive part; w, sphincter. 2 Muscles according to Ballowitz. A, longitudinal section; B, transverse section; a, spiral fibers; b, core; c, nucleus. 3 Large nerve cell in the axial nerve of the arm of Octopus vulgaris. a, glia fiber; b, glia nucleus; c, ectoplasm; d, endoplasm; e, Nissl granules; k, nucleus; k’, nucleolus. 4 Blood-corpuscles from the brachial arteries. 42 PLATE 1 OCTOPUS ARM, REGENERATION AND STRUCTURE MATHILDE M. LANGE Sy, SSS SSS SSS SSS NS RS SAAN SNS 43 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 31, NO. 1 PLATE 2 DESCRIPTION OF FIGURES 5 Wound of an Octopus vulgaris arm one and a half hours after operation. a, axial nerve; b, myelin cords. 6 Wound of Octopus vulgaris arm ten hours after operation. 7 Healed wound of an Octopus vulgaris arm one day after operation. Distal suckers are drawn up. 8 Little knob first visible sign of regeneration three days after operation. 4 OCTOPUS ARM, REGENERATION AND STRUCTURE PLATE 2 MATHILDE M. LANGE PLATE 3 DESCRIPTION OF FIGURES 9 Dome-shaped knob three days after operation. 10 Regenerated piece eleven days after operation. Little transverse folds are suckers at a very early stage of development. 11 and 12 Regenerated piece three weeks after operation. 46 OCTOPUS ARM, REGENERATION AND STRUCTURE PLATE 3 MATHILDE M. LANGE het , Mase ‘ at a t. ve ve CA ie: . ‘ Sects l2 47 PLATE 4 DESCRIPTION OF FIGURES 13 and 14. Regenerated piece three weeks after operation. 15 Regenerated piece of an Octopus vulgaris arm four weeks after operation. 16 Regenerated piece of an Octopus vulgaris arm six weeks after operation. 48 OCTOPUS ARM, REGENERATION AND STRUCTURE PLATE 4 MATHILDE M. LANGE ¢ F: eg ee ee #8 Se bi! craters . a a a A ML tg ge Pad A is oe Ee Fe a 4 hoe Piss e vie 1 : AY eres ye | ones pa IS 49 PLATE 5 DESCRIPTION OF FIGURES 17 Longitudinal section through the wound shown in figure 5 (one-half hour after operation). a, epithel; 6, subcutaneous connective tissue; c, chromato- phores; d, musculature; e, artery; f, myelin cord; h, neuropil; 7, layer of nerve cells; 7, sucker. 18 Open brachial artery six hours after operation. 19 Agglutinating blood-corpuscles. 20 Brachial artery closed by a clot of agglutinating blood-corpuscles. 21 Primary blastema evolved from blood-corpuscles. 22 Longitudinal section through an Octopus vulgaris arm ten hours after operation. Preliminary covering of the wound formed by agglutinating blood- corpuscles. 50 OCTOPUS ARM, REGENERATION AND STRUCTURE ; PLATE 5 MATHILDE M. LANGE ? a) | Ca iE ae 51 PLATE 6 DESCRIPTION OF FIGURES 23 Healed wound near the tip of the arm. a, very thin layer of primary blastema. 24 Octopus vulgaris arm forty-six hours after operation. Newly formed epithelium. 25 Octopus vulgaris arm 1 to 2 days after operation. Wound completely healed, newly formed epithelium with subjacent primary blastems. 26a Newly formed syncytial epithelium one day after operation. 26b Amitotie division of epithelial nuclei. 52 PLATE 6 OCTOPUS ARM, REGENERATION AND STRUCTURE MATHILDE M. LANGE 53 PLATE 7 DESCRIPTION OF FIGURES 27 Degenerating muscle fibers one and a half hours after operation. 28 Degenerating muscle nuclei. 29 Blood-corpuscles among degenerating muscle fibers and nuclear frag- ments ten hours after operation. 30 Sarecoblasts two days after operation. 31 Primary and second blastema. 32 Mitosis of sarcoblasts. 33 Formation of the sucker muscles eleven days after operation. 34 Invagination of a sucker three weeks after operation. Section made through the lowest newly formed sucker shown in figures 13 and 14. PLATE 7 OCTOPUS ARM, REGENERATION AND STRUCTURE MATHILDE M. LANGE 55 B02 B10) Bf at) PLATE 8 DESCRIPTION OF FIGURES Degenerating nerve nuclei one and a half hours after operation. Inflated myelin fibers six hours after operation. Increase of nuclei in the neuropil. Tip of a regenerated piece three weeks after operation. through piece shown in figures 13 and 14. Nerve growing into the primary blastema. a, young nerve cell. 39 56 Section made PLATE 8 OCTOPUS ARM, REGENERATION AND STRUCTURE MATHILDE M. LANGE 57 Resumen por el autor, W. H. Taliaferro. Universidad Johns Hopkins. Reacciones de Planaria maculata a la accién de la luz, con especial mencién de la funcién y estructura de los ojos. 1. El ojo de Planaria maculata consta de dos partes: las re- tinulas y las células accesorias que forman la copa pigmentaria. 2. Los ejemplares normales son negativos a la luz y se orientan exactamente en un rayo luminoso horizontal. 3. Los ejemplares con ambos ojos extirpados son negativos a la luz, pero no se onientan. 4. Los ejemplares con un ojo extirpado no exhiben movimientos circulares. Se orientan como los ejemplares normales cuando se les ilumina el lado normal, pero no se orien- tan cuando la iluminaci6on se dirige al lado “‘ciego,’”’ a menos que se muevan hasta recibir la luz en el ojo funcional. 5. Las reac- ciones de jos ejemplares con un ojo normal y ia mitad posterior o anterior del otro ojo extirpada se describen en el trabajo. 6. La extirpacién de ios ojos, a diferencia de lo que sucede al cortar el extremo anterior, no produce efecto sobre la marcha de la locomocidn en la luz directiva o no directiva. 7. El ojo consta de dos regiones sensoriales localizadas; la estimulacién de una e ellas causa el movimiento del animal en un sentido opuesto al ocupado por el lado que contiene el ojo, mientras que ia estimu- lacion de ia otra produce el efecto opuesto. 8. Aunque el pigmento puede localizar a la estimulacion fotica en cierto grado, probablemente no es el principal agente localizador de la estimu- iacion f6tica, como ha indicado Hesse. 9. La localizacién del estimulo luminoso esta relacionada con ja estructura y posicién de los rabdomas. 10. Una vez que el animal se orienta en un rayo luminoso horizontal no recibe estimulacién orientadora hasta que abandona el eje de orientaci6n. Translation by José F. Nonidez Cornell University Medical College, N. Y. AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, MAY 5 REACTIONS TO LIGHT IN PLANARIA MACULATA,! WITH SPECIAL REFERENCE TO THE FUNCTION AND STRUCTURE OF THE EYES W. H. TALIAFERRO Zoological Laboratory of the Johns Hopkins University EIGHTEEN FIGURES CONTENTS Pe pEGHMCEIONE LE NGSt Ls ou eeee ee eee ceeded cote sonic deed bce tobe ge 60 Msi be rials Mader e GAO Secrecy yaya ciorb oeauay ortse hae et aRLa OOS wstwNe blepz's foe's: obra tod eae 63 PE RMe NUNEr Gib RE TOUO tr gel tgs Ns ctrl las Save cir Kasecsiccre' AAt uote My Fig. 5 Diagram representing orientation to light in normal specimens and ‘wandering reflex.’ The arrows x and y indicate the direction of the rays of light. A, B, and C, indicate path of specimen. 1, 2, 3, 4, and 5, successive posi- tions of specimen; w, point of beginning of ‘wandering reflex.’ When specimen reached position 2 the light y was intercepted and light x turned on. significant part in the normal process of orientation, will be de- scribed here because they later become of great interest in other reactions to light. The first of these I have designated the ‘wandering reflex.’ After an animal is oriented, it takes a fairly straight course for a certain distance (1 to 4 em.), then it begins to wander toward the right or left. If the animal in this wander- ing turns far enough to allow the rays of light to enter the pig- REACTIONS TO LIGHT IN PLANARIA MACULATA ras) ment-cup of the eye, it suddenly reorients and again proceeds directly from the source of stimulation as represented in figure 4, R. In referring to this figure one might ask why this ‘wander- ing reflex’ with the subsequent reorientation occurred only after the direction of the rays was changed at 6 and not after the pre- ceding changes. The answer to this lies in the fact that after the preceding changes in the direction of the rays the animal was not allowed to proceed far enough, for, as previously stated, the animal always proceeds a certain distance before the wandering commences. In practically all of the numerous records made of the orientation to light in Planaria maculata this wandering re- action and reorientation occurred one or more times. The wandering of specimens from the path of orientation and the subsequent reorientation as soon as the animal turns enough to let the light rays enter the pigment-cup suggests strongly that once an animal is oriented it receives no orienting stimula- tion unless it leaves the path of orientation. This will be con- sidered in detail in another section. The second type of réaction alluded to above has been desig- nated the ‘twisting reflex.’ Whenever a planarian is proceeding from a source of light, it pauses at irregular intervals and twists the anterior end so that the ventral surface tends to be directed upward. Under strong illumination this response is exhibited at intervals of approximately 3 to 4 cm. It is interesting to note that decapitated specimens never give the twisting reflex. When the anterior ends of such specimens are allowed to regenerate, this reflex is not exhibited until after the anterior end is almost fully developed. From a study of the sections of such animals, considerable evidence was obtained in- dicating that it is necessary for the regeneration of the ‘brain’ to be practically complete before the twisting reflex occurs. Neither the twisting reflex nor the wandering reflex apparently plays any significant part in the normal process of orientation, but, as we shall see later, both play a paramount réle in the orientation of forms with one eye removed. 2. Character of turning in animals during orientation in different intensities of illumination. In the description of the orientation THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 31, No. 1 76 W. H. TALIAFERRO of normal animals to a horizontal beam of light, it was noted that animals usually turn directly away from the light without any preliminary trial movements. This is certainly true in the ma- jority of cases. A study of orientation in different intensities of illumination, however, reveals, among other things, that a given specimen may even turn first toward the light and then away from it. If the lateral illumination is of a very low intensity, the animal orients by describing a rather broad arc of a circle with barely any perceptible bending of the body. If the intensity is some- what higher, the animal does not describe this arc, but turns its head directly away from the source of stimulation, making a rather sharp angle in the contour of its body just posterior to the cephalic lobes. As the light intensity is increased, the animal still bends its head away, but the angle, i.e., the point of bending, becomes situated more and more posteriorly until it reaches the region of the pharynx. When the animal bends its body in the region of the pharynx, as it does only under the influence of very intense light, a peculiar reaction takes place. The specimen raises the anterior half of the body and violently turns it, first toward the light, then in the opposite direction until it faces directly away from the light, after which the head is lowered and the animal proceeds as usual. The reaction of turning the head first toward the light and then away under the influence of strong stimulation will be taken up later. In studying the relation between the character of turning during orientation and the intensity of the ight, no attempt was made to measure accurately the illumination because of the great individual variation and the great variation in the same speci- men at different times. Then, too, in working continuously with a given animal, it becomes more and more indifferent to stimulation by light. The following detailed description will illustrate the character of the results obtained in all of the the numerous observations made. On September 7, 1916, a specimen tested two hours after collecting described a rather broad arc of a circle in orienting in an illumination of 52 meter-candles (fig. 6, 4). When the REACTIONS TO LIGHT IN PLANARIA MACULATA GG specimen was then illuminated laterally with stronger light, viz., 75 meter-candles, the animal bent its body in the region just posterior to the cephalic lobes, thus turning the anterior end sharply away from the light. The point of bending occurred somewhat farther back (fig. 6, 6) when the animal was subjected to a lateral illumination of 208 m.c. When the lateral illumi- —___> > Fig. 6 Diagram representing orientation of planarians in different illumina- tions. Arrows indicate the direction of light. A, path of specimen in 52 meter- candles; B, path of specimen in 208 m.c.; 1, 2, successive positions of specimen; C, path in 3328 m.c., 1, 2’, 3’, successive positions of specimen. nation was increased to 3328 m.c., the bending occurred in the region of the pharynx, but first toward and then away from the light, and the reactions were very violent (fig. 6, C). These observations on the character of turning in specimens under the influence of increasing intensities of light are interesting when considered in relation to the nature of the nerve impulse from the eye to the musculature which causes the bending. When 78 W. H. TALIAFERRO a planarian bends its body in a lateral direction, this can be conceived to take place either by a lengthening of the side toward the light or by a contraction of the side opposite the light, or possibly both. If the animal bends by lengthening one side, this is probably due to a contraction of the dorsoventral muscles of that side. This contraction would tend to flatten the body in a given region and hence elongate its contour. On the other hand, a contraction of one side would most likely be due to a contraction of the longitudinal muscles of that side. Pearl (’03) is of the opinion that in planaria while turning away from mechan- ical stimulation, this turning is due to a contraction of the dorso- ventral muscles and, in consequence, to an elongation of the side away from the bending. In numerous experiments along this line, the author has been unable to satisfy himself as to which is true in orientation to light. The nerve fibers leading from the eye of a planarian must be connected indirectly with a rather complex system of muscles along either or both sides of the animal. In an animal under comparatively weak stimula- tion; the nerve impulse most likely is transmitted to a rather localized region of the musculature, viz., to a region near the cephalic lobes. The fact that the point of bending gradually moves posteriorly as the stimulation increases strongly suggests that in such cases there is a greater and greater spread of the nerve impulse along the musculature of a given side as each successive increase in the stimulation takes place. This assump- tion of course, would not explain the reaction of bending first toward and then away from the light under the influence of very intense illumination. It is likely that the latter involves a dif- ferent neuromotor mechanism, possibly analogous to protopathic stimulation in the vertebrate eye. In the human eye, for ex- ample, an intense illumination often involves a protopathic sen- sation entirely aside from the usual sensation of light. In this case the protopathic sensation involves a different mechanism than the usual sensation of light; it may, in fact, be invoked in a totally blind person (Sherrington, ’98, page 967). The reaction of bending the anterior end first toward and then away from the light is very similar to that described by REACTIONS TO LIGHT IN PLANARIA MACULATA 79 Pearl (’03, p. 580) as a result of continued strong mechanical stimulation. He believes that ‘‘It indicates the effect of the organism as a whole on its reflexes.”” Boring (’12) has observed similar reactions in Planaria torva after continued directive illumination. He (p. 241) believes that— It is quite conceivable that the abrupt reversal of directions for brief periods, the ‘wild jumps,’ are forms of a compensatory movement, which acts as a relief, not for the continued stimulation, but for the con- tinued movement away from the stimulus. . . . . Itis quite possible that these muscles (i.e., the ones which steer the animal to one side) after the continued contraction involved in prolonged movement to one side, become cramped, and there follows what is probably a natural physiological codrdination, when the muscles on the other side contract suddenly and strongly, stretching the fatigued muscles. Both of the investigators quoted above observed the bending of the animal first toward and then away from the stimulated side only after long-continued stimulation. While this reaction cer- tainly does follow long-continued stimulation, I am not certain, judging from the results of numerous observations, that it is necessarily preceded by continued stimulation. When an animal is very strongly illuminated this reaction will follow immediately even though the animal has been previously subjected to very little photic stimulation. Such reactions unquestionably occur in earthworms and fly larvae immediately after stimulation. It is hoped that this matter can be taken up later in more detail. It would raise some interesting questions if short strong stimula- tion produced the same effect as long-continued weak stimulation. FUNCTION OF THE EYES A. Reactions to light in specimens with both eyes removed The majority of investigators who have worked on the question of the function of the eyes in planarians have reached the con- clusion that these organs play very little part in the character of the responses to light. Thus, as previously stated, Loeb (94), Hesse (97), and Parker and Burnett (’00) maintain that decapitated planarians react essentially the same as normal specimens, but that all reactions require considerably more time. 80 W. H. TALIAFERRO In experiments on decapitated and normal Planaria torva, Parker and Burnett concluded (p. 385): Planarians without eyes react to the directive influence of light in much the same way as those with eyes, in that they have a tendency to turn away from the course when directed toward the source of light and to keep in it when directed away from the source, though with less precision and often to less extent than planarians with eyes. Planarians with eyes move more rapidly (1.12 mm. to 1.04 mm. per sec.) than those without eyes (0.89 to 0.82 mm. per sec.) and those moving away from the light (1.12 mm. and 0.89 mm. per sec.) than those moving toward it (1.04 mm. and 0.82 mm. per sec.) Opposed to these results we find that Lillie (01) maintains that posterior headless pieces of Dendrocoelum lacteum do not exhibit the usual responses to light. Also, Mast (10) main- tains that an undetermined marine turbellarian with the eyes removed fails to orient to a horizontal beam of light, while nor- mal specimens orient fairly precisely. The present experiments on the reactions of specimens with both eyes removed were designed to answer two questions: 1. Does removal of the eyes affect the character of the responses to light? 2. Does removalof the eyesaffect the rate of locomotion? The animals used in these experiments were anesthetized and their eyes cut out in the manner described in the section on methods. After each animal was operated on, it was allowed approximately twenty-four hours to recuperate. If, after this time, the animal showed any distortion about the head it was discarded. Then, as a further check, each animal was, after the experiment, fixed, sectioned, and stained. These sections were carefully examined, and the records of any animal were discarded if the sections contained any trace of the eye which was removed or if there was any deep cut into the ‘brain.’ By using this technique it is surprising how neatly a small organ like the eye can be removed. In successful operations, the only noticeable difference from normal specimens besides the absence of the eyes is a slight dislocation of the pigment of the body surface in this region (fig. 7). In some cases even this cannot be detected. The sections, in the majority of cases, revealed the fact that the ‘brain’ had not been cut at all in the REACTIONS TO LIGHT IN PLANARIA MACULATA 81 removal of the eyes. Throughout this series of experiments it was found essential never to use specimens which showed any distortion of the head. Such distortions are often followed by abnormal motor activities. 1. Character of reactions to light. Specimens with both eyes removed move about essentially like normal specimens. They exhibit the twisting reflex (p. 75) and occasional ‘wigwagging movements’ (p. 72) just as do normal specimens. When observed in non-directive light, no difference can be observed between them and normal specimens in either the rate or nature Fig. 7 Photographs fromunretouched negatives of normal and operated speci- mens. All magnified 30 diameters. A, normal specimen; FL, eyes; B, specimen with both eyes removed; C, specimen with left eye removed; EH, eye. Note the lack of distortion in those specimens which have been operated on. of locomotion. This is in marked contrast to the behavior in decapitated worms. Although no difference can be detected between the reactions of normal specimens and specimens with both eyes removed when in non-directive light, this is not the case when observed in directive light. Specimens with both eyes removed do not orient to a horizontal beam of light as do normal specimens. This is well illustrated in the following detailed description of one of the twenty experiments made, the results of which are essentially the same, 82 W. H. TALIAFERRO The specimen used in this experiment was first tested in a horizontal beam of light in which it was found to orient very precisely. The animal was then anesthetized with CO, and both eyes removed. ‘Twenty-four hours after the operation, the animal was again tested and its movements traced with a camera lucida. This tracing is reproduced in figure 8. At the beginning of the experiment, the animal was laterally illuminated and it proceeded for a short distance at right angles to the rays of light and then turned directly toward the light. Movement in this direction continued for only a short distance, when it turned again and proceeded in a diagonal path away from the light. This path soon led out of the beam of light into the shadow. Twice as the animal attempted to proceed from the shadow back into the light it hesitated, and after a sort of ‘avoiding reaction’ proceeded back into the shadow (fig. 8, 7 and 2). The third time it came to the margin between the light and the shadow it passed into the light without any perceptible reaction. The direction of the hight was now changed through an angle of 90° (fig. 8, P). The animal described a very irregular course away from the second light source. While proceeding away from this source it again moved from the illuminated region into the shadow and vice versa on two separate occasions, with no apparent reaction (fig. 8, 3 and 4.). After these observations were made, the animal was fixed, sectioned, and stained. A study of these sections revealed that the eyes had been entirely removed and that there was no apparent injury to the ‘brain’ or other organs. If, now, the reactions of this specimen in a horizontal beam of light are compared with those of normal specimens in similar illumination, it becomes evident that orientation is dependent upon the eyes. This conclusion is, moreover, strongly supported by the fact that blind specimens again orient precisely after the eyes regenerate. Although the eyes are clearly functional in orientation, the evidence at hand indicates that there is in eyeless specimens at times some indication of a slight orientation to the rays of light. The question then arises as to what factors are involved in the slight tendency toward orientation in these specimens. The an- REACTIONS TO LIGHT IN PLANARIA MACULATA 83 swer to this, I think, lies in the fact that, while the posierior end of the animal is sensitive to light, the anterior end, exclusive of the eyes, is more so. In regard to the anterior end, regardless of the eyes, being more sensitive to light than the posterior, Walter (’07, p.123) says: a Fig. 8 Camera-lucida tracing of the path in a horizontal beam of light of a specimen with both eyes removed. Arrows a and 6 indicate the direction of the rays of light. C-H, path of animal. When the animal reached the point P, light a was turned off and light b was turned on. At points 7 and 2 the animal gave a kind of ‘avoiding reaction.’ This did not occur at 3 and 4. Again, when a small beam of sunlight passing through a pinhole in an opaque screen was directed locally to different parts of a gliding Planaria maculata, it was found that tropic response would occur in case one side of the anterior end was illuminated, and that it was not necessary for the eye itself to be included in the illuminated area to obtain such responses. However, when the middle of the body or the posterior end was similarly stimulated the worm could not be made to turn. 84 W. H. TALIAFERRO As it is very improbable that the anterior end of a planarian can be illuminated without allowing a certain amount of light to enter the eyes, Walter’s experiments were repeated, using, however, specimens the eyes of which had been carefully cut out, and a horizontal beam of light instead of the localized point as in the above experiments. It was found that if a beam of light was thrown !aterally on the anterior end of such a worm, it turned away from the source of light in the majority of cases. It is important to notice, however, that this turning was not nearly so definite or precise as in the case of animals with eyes. A given specimen very often turned toward the light, swerving all the way around and thus proceeded away from the source of stimulation. The turning of such eyeless specimens was much more indefinite if the entire animal was illuminated instead of the anterior end. As the anterior end is more sensitive to light than the posterior, the animal would most likely maintain a position in regard to the light source such that the posterior end would shade the more sensitive anterior end. Such a position would tend to keep the animal directed away from the light and would explain the slight tendency to orientation observed in such specimens. It must not be supposed, however, that all of the reactions of speci- mens without eyes can be ascribed to this differential sensitivity of the anterior and posterior regions because a decapitated worm still proceeds, in general, away from the light. Although this is true, a decapitated worm does not show the slight tendency toward orientation such as is found in animals with both eyes removed. 2. Rate of locomotion. As pointed out above, no difference in the rate of locomotion can be observed under ordinary conditions between normal specimens and those which have had both eyes removed. In order to test this accurately, however, the follow- ing experiments were devised, using both directive and non-di- rective illumination. The non-directive illumination was fur- nished by placing a 125-watt gas-filled lamp 30 c.m. above a circular aquarium in which the animals were moving. The di- rective light was furnished by the same apparatus used to test REACTIONS TO LIGHT IN PLANARIA MACULATA 85 the reactions of specimens in a horizontal beam of light. In both cases the rate of locomotion was determined by means of a pantograph previously described in the section on methods. Ten normal specimens were placed in a dark room for twenty- four hours and then their rate of locomotion both in directive and non-directive illumination was ascertained. Throughout the experiment the animals were kept in the dark when not being observed, so as to keep the preliminary light environment as nearly constant as possible. After the rate of locomotion for the ten normal specimens had been ascertained, both eyes were TABLE 1 The effect on the rate of locomotion of removing only the eyes as compared with the effect of removing the entire anterior end AVERAGE RATE OF LOCOMOTION IN MILLIMETERS PER SECOND NUMBER OF N TRIALS Directi SNOEE TeHi CEN men Norn Specimens.) oo. ss. Hse sas. see eet eek 1.18 Halal 40 The same with both eyes removed: 1) Two hours after removal of eyes............... 1.25 39 2) Twenty-four hours after removal of eyes....... 1.10 1.08 55 The same with anterior end removed: ‘ Twenty-four hours after operation................ 0.62 0.71 45 removed from each specimen and, after twenty-iour hours, their rates of locomotion were again measured. In the case of non- directive illumination, the rate of locomotion was also obtained two hours after the removal of the eyes. Then, in order to see if the removal of the anterior end had the same effect as removing only the eyes, thesame specimens were decapitated, and after twenty-four hours their rates of locomotion were again ascertained. The results of these experiments are tabulated in table 1. This table shows that, in directive illumination, the average rate of locomotion for the ten normal specimens was 1.18 mm. per second; that after the eyes were removed, the rate in the same 86 W. H. TALIAFERRO specimens in the same illumination was 1.10 mm. per second; and that, after the removal of the anterior end, the rate was only 0.62 mm. per second. The table also shows that essentially the same results were obtained in the case of non-directive illum- ination. It shows, moreover, that in specimens tested two hours after the removal of the eyes the rate of locomotion actually increased. This is, no doubt, due to the mechanical stimulation of removing the eyes which has not yet had time to wear off, for, in normal specimens, shortly after small incisions are made in the dorsal surface, the rate of locomotion is similarly increased. These results show that the rate of locomotion is not appre- ciably affected by the removal of the eyes, whereas it is greatly affected by the removal of the anterior end, and they indicate very clearly that the photoreceptors which receive the orienting stimulus are not the ones which control the rate of locomotion. It has been shown by Walter (’07, p. 57) that, in general, plan- arians move faster in higher intensities than in lower. The photoreceptors involved in this increase of the rate of locomo- tion under increased intensity of illumination as well as in the experiments just described are other than the eyes—very probably the general body surface. 3. Discussion of experiments in relation to former investigations. The results of the preceding experiments are in accord with those of Lillie (01) and Mast (710) in regard to the character of the response to light, but are at variance with the results of Loeb (94), Hesse (97), and Parker and Burnett (’00) both in regard to the nature of the responses and the rate of locomotion. The question immediately arises as to what causes the disparity be- tween these results and those of the latter investigators. In regard to the nature of the response in specimens with both eyes removed, the answer probably can be found in the fact that the species used by the former investigators did not normally orient with any great degree of precision to the directive influence of light. There is no doubt that many planarians do not orient to light, and, of course, one would not expect to find any great change brought about by the removal of the eyes, if the eyes did not function in the normal animal. REACTIONS TO LIGHT IN PLANARIA MACULATA 87 In regard to the rate of locomotion in specimens with both eyes removed, the disparity between the results of this paper and those of the former investigators undoubtedly lies in the fact that they drew conclusions regarding the effect of removing the eyes from the behavior of decapitated specimens. From the results given in this paper, it is evident that such conclusions are not valid, because removal of the anterior end itself, regard- less of the eyes, has a profound effect on the rate of locomotion. If the entire ventral surface of a planarian is functional as an organ of locomotion, a very simple explanation of the decrease of the rate in decapitated worms suggests itself. Removal of the anterior end would remove a part of the organ of locomotion and hence would undoubtedly decrease the rate of movement. It is very improbable, however, that this can explain such a great decrease. The general effect of the operation and possibly the loss of the ‘brain’ act upon the general physiological tone of the animal, causing locomotion, among other physiological activities, to be retarded. B. Reactions to light in specumens with one eye removed In regard to the reactions of planarians with one eye removed, Mast (710, p. 132), in a paper already referred to, makes the fol- lowing statement: Planaria with one eye removed, either by gouging it out or by cutting off one side of the anterior end obliquely, turn continuously from the wounded side for some time, evidently owing to the stimulation of the wound, since after this is healed they tend to turn in the opposite direction. After regeneration is nearly complete they orient practi- eally as accurately as normal specimens. Unfortunately, from the standpoint of the present investiga- tion, no note was made of just how far the eye itself was allowed to regenerate in such specimens before accurate orientation to light was observed. The present experiments were designed to ascertain how ac- curately specimens with one eye orient to the directive stimula- tion of light and to find out, if possible, the mechanism of this orientation. 88 W. H. TALIAFERRO In these experiments, exactly the same technique was employed and the same precautions were observed as in the experiments of the preceding sections. The animals were anesthetized with CO, and one eye removed with a fine knife, as has been described (fig. 7). After the removal of an eye, the animals were given eight to twenty-four hours to recover from the operation. At this point any specimen which showed any distortion of the head or any loss of the bilateral symmetry of the contour of the anterior end was discarded. It is to be noted that in removing one eye there is more tendency to disturb the bilaterally symmetri- cal contour of the anterior end than in the case where both eyes are removed. B a ; <—__ A\ ee C\ —_——_—_ : D Fig. 16 Illumination of eye with light coming from different directions. A and B, animal possessing entire right eye; C and D, animal possessing anterior half of right eye. Arrows indicate the direction of rays of light; broken lines indicate path of specimen; x, position of rhabdomes which are illuminated. Animal D proceeds directly away from the light although the same rhabdomes are being illuminated as in C when it turned sharply to the left. The idea suggested itself to the author that the above con- clusion might not be valid because of the possibility that in removing the posterior half of the eye the pigment might be REACTIONS TO LIGHT IN PLANARIA MACULATA 105 carried by the knife and spread over the posterior surface, and that this might make an effective light screen just as in the case of the normal animal. Histological examination of such speci- mens showed this idea to be erroneous. A few granules are always displaced in making an incision, but these are very much scattered and obstruct the passage of light very little, if at all. Again, the body of the animal might act as a shading mechanism. This, however, was shown not to be true as the same results can be obtained when the source of illumination is lifted slightly above the horizontal plane, in which case the light does not pass through any more of the tissue of the animal’s body than in normal lateral illumination. We are consequently forced to accept the conclusion that light entering the eye from behind does not stimulate the rhabdomes as it does when it enters from the side. That is, that light striking certain rhabdomes from the direction indicated by the arrow a (fig. 17) is followed by a definite turning of the animal, whereas no such turning results when light strikes the same rhabdomes from the direction indi- cated by the arrow b. None of the experimental work has offered any explanation of this phenomenon. The structure of the retinula and its relation to the pigment-cup, however, offer two possible explanations. In the living condition in Prorhynchus applanatus, according to Kepner and Taliaferro (16), the middle region of the retinula or the region which corresponds to the ellipsoid of the vertebrates is the most refractive portion of the retinula. The same holds true for Planaria maculata after fixation. This region, because of its position, contour, and refractive index, must have some effect on the rays of light as they pass down the longitudinal axis of the retinula to strike the rhabdome (fig. 17, a). It occurs to one that possibly this region serves as a crude lens to concen- trate the rays of light upon the sensitive rhabdome and that photic stimulation depends upon this. If this is true, photic stimulation could not be set up in a given rhabdome unless the light struck the rhabdome approximately parallel to its longi- tudinal axis. Not only would the light have to strike the rhab- dome approximately parallel with its longitudinal axis, but it 106 W. H. TALIAFERRO would have to pass through the retinula in a distal direction. Otherwise the light would not be affected by the middle region. If the above suggestion is correct, the pigment would play no part in the localization of photic stimulation in the individual retinulae. We might, however, conceive that the middle region has no effect on photic stimulation, but that the rhabdome is itself so constructed that only light passing along its longitudinal Fig. 17. Diagram representing the relation between structure and photic stimulation in the individual retinula. A, one of the accessory cells which form the pigment-cup; 1, middle region of the retinula; N, nucleus of retinula; k, sensory rhabdome. Light striking the rhabdome parallel to the axis a results in stimulation. Light from the direction b or c does not result in stimulation. axis sets up stimulation. If this is true, light from either direction as long as it is parallel to the longitudinal axis might cause stimu- lation. The pigment, then, might serve to localize photic stimu- lation in that it would prevent light passing through the rhabdome in a proximal direction and allow light from the opposite direction to strike the rhabdome. Thus, in figure 17, the pigment is so placed that light along the arrow c cannot strike the rhabdome. While this suggestion ascribes a certain limited localizing function REACTIONS TO LIGHT IN PLANARIA MACULATA 107 to the pigment, it is evident that it does not ascribe to the pigment the entire function of the localization of photic stimulation as is done by Hesse (97). 2. Localization of photic stimulation in relation to the structure of the eye. If we are correct in the assumption that light must penetrate a given rhabdome along its longitudinal axis in order to stimulate it, then the structure of the eye should be such that whenever light enters it, under normal conditions, the longi- tudinal axes of some of the illuminated rhabdomes will be parallel to the stimulating rays of light. Let us consider this problem. The best way to describe the relation of the axis of the light rays entering the eye to the axes of the rhabdomes is to consider the actual condition of affairs when the eye is illuminated from each of several different directions. This is represented in figure 18. By referring to this figure the following may be seen: Light which strikes the eye from directly in front of the animal illumi- nates those rhabdomes which lie on the outer posterior margin of the pigment-cup, and the longitudinal axes of all of these rhabdomes are directed approximately parallel to the rays of light (fig. 18, A, a, § and 9). The same holds true for light entering the eye from an oblique posterior direction (fig. 18, B, d, 1), and, approximately, for lateral illumination (fig. 18, A, b, 2-6). When the light comes obliquely from in front of the animal, however, the light rays strike certain rhabdomes parallel with their longitudinal axes (fig. 18, B, c, 7) and other rhabdomes at various angles to their longitudinal axes (fig. 18, B, c, 5,6, 8, 9). The same is true for the rhabdomes when the light enters the eye from different points in the transverse vertical plane. Light which enters the eye from directly above (fig. 18, C, a, 8) and obliquely below (fig. 18, D, d, 1) strikes all of the rhabdomes in both cases parallel to their longitudinal axes. Light from obliquely above (fig. 18, D, c, 4-8) and, to a less extent, light from the side (fig. 18, C, b, 2-4) passes along the longitudinal axis of certain rhabdomes and not of others. This seems to indicate that the structure of the eye is such that if ight from any given direction enters the eye, it illuminates THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 31, NO. 1 108 W. H. TALIAFERRO the rhabdomes confined to a definite area; that, in such areas, there is always a large proportion of the rhabdomes which have their longitudinal axes parallel with the rays of light; and that, in a number of cases, the longitudinal axes of all of the rhabdomes in the illuminated area are nearly parallel with the rays of light. Fig. 18 Diagram representing the relation of the axis of photic stimulation to the axes of the various rhabdomes when the eye is illuminated from different directions. The diagrams are made from camera-lucida drawings of sections. A and B, frontal sections of the eye; C and D, transverse sections of the eye; A-P, antero-posterior line; D-V, dorsoventral line;a, b, c, d, arrows indicating the dif- ferent beams of light; ac, accessory cells; p, pigment-cup; 1-9, rhabdomes. REACTIONS TO LIGHT IN PLANARIA MACULATA 109 These observations strongly support the assumption that if light strikes a rhabdome parallel with its longitudinal axis stimu- lation results; whereas, if light does not strike a rhabdome along this axis stimulation does not follow. This will explain why it is not necessary for the pigment-cup to act as a localizer of photic stimulation. The latter is localized not by the pigment- cup, but by the position of the longitudinal axes of the various rhabdomes. NATURE OF THE STIMULUS DURING ORIENTATION At the present time there are two chief theories advanced to account for the orientation of organisms to light—the continuous- action theory and the change-of-intensity theory. The literature bearing on these two theories is so extensive that we shall limit the present review to a very brief outline. Loeb, who is the chief upholder of the continuous-action theory, maintains that an organism orients to light because of unequal — chemical changes induced by the light in symmetrically placed photoreceptors; that effects of these chemical changes are trans- mitted eventually to the locomotor organs, thus producing un- equal ‘tension or energy production’ in the musculature of the two sides, and that this results in a turning of the organism. According to him, after the organism has become oriented, the light produces equal chemical changes in the photoreceptors and the organism proceeds directly toward or away from the light owing to continuous and equal action of the light on sym- metrically located photoreceptors. In reference to his theory, he says (’16, pp. 258-259): The reader will perceive that according to the writer’s theory two agencies are to be considered in these reactions: first, the symmetrical arrangement of the photosensitive and the contractile organs, and, second, the relative masses of the photo-chemical reaction products produced in both retinae or photosensitive organs at the same time. If a positively heliotropic animal is struck by light from one side, the effect on tension or energy production of muscles connected with the eye will be such that an automatic turning of the head and the whole animal towards the source of light takes place; as soon as both eyes are illuminated equally the photochemical reaction velocity will be the 110 ; W. H. TALIAFERRO same in both eyes, the symmetrical muscles of the body will work equally, and the animal will continue to move in this direction. In the case of the negatively heliotropic animal the picture is the same except that if only one eye is illuminated the muscles connected with this eye will work less energetically Loeb holds, moreover, that the orienting stimulus in organisms, both animals and plants, is dependent upon the actual amount of stimulating energy received by the photoreceptors in accord with the Bunsen-Roscoe law. Opposed to the continuous-action theory, is the change-of- intensity theory, supported chiefly by the works of Jennings and Mast. According to this theory, the orienting stimulus is not dependent upon the actual amount of energy received by the photoreceptors, but to time-rate-of-change of the stimu- lating energy. Once an organism is oriented to light, it is sup- posed to receive no orienting stimulus until it leaves the path or axis of orientation. The great body of evidence, especially in the unicellular forms, tends to favor the change-of-intensity theory. Mast has dis- cussed this evidence fully in Light and the Behavior of Organ- isms (711) and in numerous recent papers (Mast, 716). In certain seedlings, however, Blaauw (’08), Fréschel (’08), Arisz (711), and Clark (13) have demonstrated that within certain limits orientation to light is dependent upon the actual amount of energy received. This is in accord with the continuous-action theory. Likewise, Mast (’11, p. 163) and Loeb and Ewald (14) have come to practically the same conclusion in regard to the orientation of the sessile polyp Eudendrium. The chief questions that are at issue between these two theories are: 1) Does stimulation during orientation depend upon the continuous action of light or to time-rate of change in the inten- sity? 2) Does the same stimulus that causes orientation continue to act after orientation? 3) Is it essential that the photoreceptors which receive the orienting stimulus be placed symmetrically? While this work was not taken up with any especial reference to these questions, some of the observations bear directly upon them. In regard to the first question, while some of the evidence favors the change-of-intensity theory, there is no direct evidence REACTIONS TO LIGHT IN PLANARIA MACULATA 111 that stimulation during orientation is due necessarily to either the actual amount of energy received or to the time-rate of change in intensity. In regard to the second and third questions, how- ever, we can draw definite conclusions. In the first place, as has been noted (p. 74), when an animal is proceeding away from the source of illumination, it tends to wander to the right or left (wandering reflex). When the animal has thus turned its head laterally to the extent that the rays of light enter the mouth of the pigment cup, it re-orients. This behavior strongly suggests that once the animal is oriented it receives no orienting stimulation until it leaves the path of orientation. In the second place, it was shown above (p. 95), under the experiments designed to map the regions of the eye, that no stimulation is received (or more exactly no reaction follows) as long as the pigment-cup is between the source of illumination and the rhabdomes. Now, an examination of the relative positions of the eyes shows very clearly that once an animal is oriented and is proceeding away from the light, no light can strike the rhabdomes unless it does pass through the pigment. From these observations we must conclude that when a planarian is moving away from the light the pigment-cup effectively shades the sensory portion of the eye. Therefore, as there is no continuous illumination of the sensory organs involved in orientation, there can be no continuous stimulation of these organs. The fact that specimens with one eye removed orient accurately to light (fig. 9) shows clearly that the symmetrical arrangement of the two eyes is not essential for orientation. The extent of turning is probably reflexly determined by the portion of the eye stimulated and is independent of the duration of the stimulation. It may, therefore, be concluded that, while our evidence is not conclusive in regard to the nature of the stimulus, orientation ~ in Planaria maculata is not in accord with the ‘continuous-action’ theory as defined by Loeb. 1D W. H. TALIAFERRO GENERAL SUMMARY 1. The eye of Planaria maculata is a typical turbellarian eye, consisting of two types of cells—the accessory cells forming the pigment-cup and the sensory cells or retinulae. _ 2. Each retinula consists of three regions—the nucleus-bearing region, the middle region, and the rhabdome, which show a striking resemblance to the three regions of the veretebrate retinula, viz., the myoid, the ellipsoid, and the rhabdome. 3. Planaria maculata is negative to light and orients accurately to a horizontal beam of light. 4, Orientation is, under certain conditions, direct; the animals may turn directly away from the source of light without pre- liminary trial movements. Trial movements are, however, at times functional in the process of orientation. 5. The location of the bending of the body, when the head is turned away from the light, depends upon the intensity of the light—the higher the intensity of illumination, the more posteri- orly the point of bending is located. It is, however, never located farther back than the pharynx. 6. If the intensity is high enough (or possibly continued long enough) to cause the bending to take place in the region of the pharynx, the animal no longer bends directly away from the light, but first toward and then away. 7. During the reactions of animals to a horizontal beam of light, two marked motor reflexes occur, viz., the twisting reflex and the wandering reflex. These reflexes are defined on pages 74 and 75. 8. Specimens with both eyes removed do not orient in directive illumination as do normal specimens. They move, however, in general, away from the light. 9. Removal of both eyes does not appreciably affect the rate of locomotion in either directive or non-directive illumination. 10. Removal of the anterior end, on the contrary, greatly retards the rate of locomotion in both directive and non-directive illumination. j 11. Specimens with one eye removed show no indication of circus movements or other abnormal motor activities. REACTIONS TO LIGHT IN PLANARIA MACULATA 113 12. Specimens with one eye removed orient accurately to . light, when illuminated on the normal side, by turning directly away from the source of light. 13. Such specimens do not orient to light when illuminated on the ‘blind’ side unless the head is moved so that light enters the remaining eye. If, however, the head is moved so that light enters the remaining eye (wandering and twisting reflex), accurate orientation may follow. 14. The rhabdomes in the eye are arranged in two localized sensory regions; illumination of the rhabdomes of the posterior and ventral edge of the pigment-cup is followed by the animal’s turning toward the side containing the eye, while illumination of the remaining rhabdomes is followed by the animal’s turning in the opposite direction. 15. Specimens possessing only the anterior portion of one eye, when illuminated from in front, do not turn sharply toward the side containing the eye, as do specimens possessing one entire eye. The loss of this reaction in such specimens is probably due to the loss of the rhabdomes situated on the posterior margin of the pigment-cup. 16. Removal of the posterior portion of the eye does not impair the capacity of the remainder of the eye to function in a normal manner. 17. Specimens possessing only the posterior portion of one eye react to light as do specimens with both eyes removed. Histological examination of such specimens shows, however, that removal of the anterior portion of the eye severs the con- nection between the remaining rhabdomes and the ‘brain.’ 18. The observed reactions in Planaria can be explained with- out assuming that the pigment-cup acts as a localizer of photic stimulation as suggested by Hesse (’97). It is possible, however, that the pigment has a limited localizing function in the individual retinula. 19. Light must strike a given rhabdome parallel with its longitudinal axis in order to cause stimulation of the rhabdome. Thus the position of the longitudinal axis of the rhabdomes results in a localization of photic stimulation. 114 W. H. TALIAFERRO 20. It is possible to explain this localization of photic stimu- lation in the individual retinula: 1) by supposing that the highly refractive middle region of the retinula acts as a crude lens to concentrate the rays of light on the rhabdome; or 2) by assuming a certain structure of the rhabdomes coupled with the shading action of the pigment-cup. This last assumption ascribes a limited localizing function to the pigment, but it does not ascribe to the pigment the entire function of the localization of photic stimulation as is done by Hesse (page 106). 21. Correlated with the assumption that light must penetrate a given rhabdome parallel with its longitudinal axis in order to cause stimulation, we find that light in entering the pigment- cup from any given direction illuminates the rhabdomes confined to a definite area and that a large proportion of the rhabdomes in such areas always have their longitudinal axes directed parallel to the stimulating rays of light and, in the case of light from certain directions, all of the rhabdomes which are illuminated have their longitudinal axes so directed. 22. Once an animal is oriented in a horizontal beam of light, it receives no orienting stimulation until it leaves the path or axis of orientation. 23. Orientation to light is not necessarily dependent upon the symmetrical arrangement of the photoreceptors. BIBLIOGRAPHY Arey, L.B 1916 Changes in the rod-visual cells of the frog due to the action of light. Jour. Comp. Neur. vol. 26, pp. 429-442. Arisz, W. H. 1911 On the connection between stimulus and effect in photo- tropic curvatures of seedlings of Avena sativa. Kon. Ak. Wet. Ams- terdam. Proc., pp. 1022-1031. BarDEEN, C. R. 1901 On the physiology of the Planaria maculata with especial reference to the phenomena of regeneration. Am. Jour. Physiol., vol. 5, pp. 1-55. Buiaauw, A. H. 1908 The intensity of light and the length of illumination in ' the phototropie curvature in seedlings of Arvena sativa. Kon. Ak. Wet. Amsterdam. Proc. Boumic, L. 1890 Untersuchungen iiber rhabdocéle Turbellarien. II. Plagi- ostomia and Cylindrostomia Graff. Zeit. f. wiss. Zool., Bd. 51, 8. 167- 479. Borne, E. G. 1912 Note on the negative reaction under light adaptation in the planarian. Jour. Animal Behav., vol. 2, pp. 229-248. REACTIONS TO LIGHT IN PLANARIA MACULATA 115 Cuark, O. L. 1913 Uber negativen Phototropismus bei Avena sativa. Botan. Zeitschr., Bd. 5, S. 737-770. Curtis, W. C. 1902 The life history, the normal fission and the reproductive organs of Planaria maculata. Proc. Boston Soc. Nat. Hist., vol. 30, pp. 515-559. Fr6scHEL, P. 1908 Untersuchungen iiber die heliotropische Prisentationszeit. Sitz.-Ber. math.-naturw. Kl. Akad. Wiss., Wien, Bd. 117, Abt. 1, S. 235-256. Hesse, R. 1897 Untersuchungen iiber die Organe der Lichtempfindung bei niederen Thieren. II. Die Augen der Plathelminthen, insonderheit der tricladen Turbellarien. Zeit. f. wiss. Zool., Bd. 62, S. 527-582. JANICHEN, E. 1896 Beitriige zur Kenntnis des Turbellarienauges. Zeit. f. wiss. Zool., Bd. 62, S. 250-288. Kepnmr, W. A., AND FosHen, A. M. 1917 Effects of light and darkness on the eye of Prorhynchus applanatus Kennel. Jour. Exp. Zodl., vol. 23, pp. 519-531. Kepner, W. A., AND LAwRENCE, J. S. 1918 The eye of Polycystis goettei (Bresslau). Jour. Exp. Zool., vol. 30, pp. 465-473. Kepner, W. A., AnD TALIAFERRO, W. H. 1916 Organs of special sense of Pro- rhynchus applanatus Kennel. Jour. Morph., vol. 27, pp. 163-177. Lerpy, J. 1847 Descriptions of two new species of Planaria. Proc. Acad. Nat. Sci. (Phila.), vol. 3, pp. 251-252. Liuuiz, F. R. 1901 Notes on regeneration and regulation in planarians. Am. Jour. Physiol., vol. 6, pp. 129-141. Loes, J. 1893 Uber kiinstliche Umwandlung positiv heliotropischer Thiere in negativ heliotropische und umgekehrt. Arch. f. d. ges. Physiol., Bd. 54, S. 81-107. 1894 Beitrige zur Gehirnphysiologie der Wiirmer. Arch. f. d. ges. Physiol., Bd. 56, S. 247-269. 1916 The organism as a whole. New York, x + 379 pp. Lors, J.. anpD Ewaup, W. F. 1914 Uber die Giiltigkeit des Bunsen-Roscoe- schen Gesetzes fiir die heliotropische Erscheinung bei Tieren. Zentb. f. Physiol., Bd. 27, S. 1165-1168. Mast, 8. O. 1910 Preliminary report on reactions to light in marine Turbel- laria. Carnegie Instit. Year Book, vol. 9, pp. 131-133. 1911 Light and the behavior of organisms. New York, xi + 410 pp. 1916 The process of orientation in the colonial organism, Gonium pec- torale, and a study of the structure and function of theeyespot. Jour. Exp. Zoél., vol. 20, pp. 1-17. Parker, G. H., anp Burnett, F. L. 1900 The reactions of planarians, with and without eyes, to light. Am. Jour. Physiol., vol. 4, pp. 373-385. Peart, R. 1903 The movements and reactions of fresh-water planarians: A study ofanimalbehaviour. Quart. Jour. Micro. Sci., vol. 46, pp. 509-714. Scumipt, A. T. 1902 Zur Kenntnis der Tricladenaugen und der Anatomie von Polycladus gayi. Zeit. f. wiss. Zool., Bd. 72, S. 545-564. SHerRineton, C. 8. 1898 Cutaneous sensations. Text-book of physiology edited by E. A. Schifer, vol. 2, pp. 920-1001. 116 W. H. TALIAFERRO TALIAFERRO, W. H. 1917 Orientation to light in Planaria n. sp. and the func- tion of the eyes. Anat. Rec., vol. 11, pp. 524-526. Water, H. E. 1907 The reactions of planarians to light. Jour. Exp. Zodl., vol. 5, pp. 35-162. WoopwortH, W. McM. 1897 Contributions to the morphology of the Tur- bellaria. II. On some Turbellaria from Illinois. Bull. Mus. Comp. 7061. Harvard Coll., vol. 31, pp. 1-16. e 4 * i r ae i hg ' ° “ 3 ‘ ae 7A ; 4 b 7 ‘. é m 1 +7 : soy bnenicy ) ae’ blo) ROG .. l ah ‘ie aly? Aly Sserseaey . ; ‘, ES 4 a - im lean ont ota yates , , See | | : ¥ Shea hole Wh Shp. whith . att Ss oe ; ' ind titi) ne Sins Pa ieee Ta’ lane —~ j Phy i f, th dni ea fy ns + ¥ pi my i dbs maa es A eto) Kays ual nb wali xabenh Piya’ ¥ atmmibardis iy th biota achat Cy WTP os vert LEE read “@ amaitirtnp ra HRT a Meyda pel Abit Beis Digit iii BF 7 ea bh Tar Cant a janis peide( Peitiaaliyi an yi) ingot te: Paid Stab A twin ae PEA WD AEN, phn at Babiani-s Cd RAM HOW? nth ifs 8 es ha alk Pein age Ai aii sil j i Page tart ~~ aad 8 1A Steen. abr a: va Le vs a il’ at Ts set aa 7 Ss) 94 Seb vobsnin rhea i a yi bey era vi Pat bn : es ve in dtr Aa ' Resumen por el autor, 8. R. Detwiler. Laboratorios Zoolégico y Anatémico, Escuela de Medicina, Universidad Yale. Experimentos sobre la transplantacién de los miembros en Amblystoma. La formacion de plexos nerviosos y la funcién de los miembros. El miembro anterior de Amblystoma transplantado en una parte del cuerpo situada en uno de los segmentos comprendidos entre el primero y el séptimo de estos, a partir de la posicién normal del 6rgano, presenta un decrecimiento normal en su capacidad para funcionar a medida que se transplanta mas lejos de su posicién normal. El] cambio de posicién del miembro. en un cierto numero de segmentos no implica un cambio corres- pondiente en la posicién de la inervacién segmentaria de su plexo, existiendo por consiguiente, una marcada tendencia en los miembros transplantados a recibir inervacién del nivel normal del miembro, en la médula espinal. Los miembros implantados tan posteriormente que es imposible que reciban inervacion desde el nivel correspondiente de la médula obtienen la mayor parte de su inervacién de los segmentos situados delante de ellos, en vez de recibirla de los segmentos correspondientes a la nueva posicién de dichos miembros. El decrecimiento gradual de la funcién en los miembros transplantados en un sitio cada vez mas lejano del punto en que aparecen normalmente parece estar directamente relacionado con la inervacién segmentaria, siendo mas perfecta la funcién cuando la inervacién se deriva del nivel normal del miembro en la médula. Esta pérdida gradual de funcién se atribuye a conexiones centrales defectuosas mas bien que a una disminucién en la inervaci6n efectiva peri- férica. Los resultados de los experimentos indican que el miembro transplantado ejerce una influencia que guia a los nervios segmentarios que contribuyen a su inervacién. La reaccion hacia esta influencia parece ser mayor en los nervios procedentes del nivel normal del miembro en la médula, puesto que estos nervios se alargan mas para ponerse en conexién con el miembro que los procedentes de los segmentos posteriores al nivel del miembro. Translation by José F. Nonidez Cornell University Medical College, N. Y. AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, APRIL 19 EXPERIMENTS ON THE TRANSPLANTATION OF LIMBS IN AMBLYSTOMA THE FORMATION OF NERVE PLEXUSES AND THE FUNCTION OF THE LIMBS S. R. DETWILER Osborn Zoological Laboratory and the Anatomical Laboratory, School of Medicine, Yale University TWENTY-TWO FIGURES INTRODUCTION The transplantation of limbs constitutes an experimental method which has been applied to the solution of a number of fundamental questions concerning the development of the pe- ripheral nervous system. Without doubt the most important applications of this method were made in connection with the question of the genesis of the nerve fiber in the work of Braus (04 and ’05), Banchi (’06), Gemelli (06), and Harrison (’07). Aside from their bearing upon the specific problem of the gen- esis of the nerve fiber, the transplantation experiments with the -exception of those of Banchi showed in general that if a limb bud is transplanted to an abnormal (heterotopic) position it will acquire a system of nerves which are connected with that part of the central nervous system of the host corresponding to the position occupied by the implanted limb rudiment. Although Braus (’05) claimed that aneurogenic limb buds (those taken from nerveless larvae) did not acquire nervous connection with the host, Harrison’s experiments showed that aneurogenic as well as euneurogenic limb buds (those taken from normal larvae) became supplied with peripheral nerves. That such nerves were partially functional was shown by slight volun- tary movements of the limbs as well as by movements in response to electrical stimulation. 117 118 S. R. DETWILER Apparently in all these cases the function of the limb was greatly restricted, and in no case cited was there any adaptive or codrdinated movements. Harrison (’07, p. 256), in describing his first experiment, says in regard to the function of an aneuro- genic limb, ‘‘No attempts were made to stimulate electrically, but spontaneous movements, though slight, were unmistakable.” Both Braus and Harrison observed that, regardless of the segmental nerve contribution, the architecture of the intrinsic nerve distribution is exactly the same as that in a normal limb. This is not an unusual phenomenon, for we know that in both normal and transplanted limbs, nerves reach the limb when it is still in the blastema stage. The union of the nerve with the differentiating limb system is made very early, so that in either case the final plan of nerve distribution is patterned according to skeletal-muscular differentiation and growth, probably in ac- cordance with Naussbaum’s law: that the course of the nerve within the muscle is an index of the direction in which the muscle has grown. The experiments to which reference has thus far been made were carried out on the anuran embryo, Bombinator being used by Braus, Bufo vulgaris by Banchi and Gemelli, and Rana sylvatica and Bufo lentiginosus by Harrison. In the majority of cases the limb buds were transplanted at a stage when the peripheral nerves were in part or completely developed. Ac- cordingly, when the wound in the host was made for the reception of the transplant, the terminal branches of the nerves of that region were severed and the implanted limb rudiment was placed in close apposition to the cut ends. Although these nerves so disturbed were originally intended to innervate other muscles, it was found that they would readily grow into the implanted embryonic rudiment and innervate the differentiating limb mus- cles as do the nerves in the normal situation. This fact would indicate that there is no specificity of a given motor neurone for any particular muscle fiber, so that the ultimate distribution and connection of the nerve fiber cannot be an intrinsic factor of the neurone. TRANSPLANTATION OF LIMBS IN AMBLYSTOMA 119 This leaves open the question of whether or not the limb rudi- ment, either in the normal or the heterotopic position, exerts any directive influence upon the segmental contribution of the spinal nerves or upon the final path taken by them in the inner- vation of the limb. The possibility of the limb’s exerting any such influence on the developing peripheral nerves could hardly be adequately tested by transplanting limb buds to larvae in which the peripheral nerve paths have already been formed, such as in the experiments hitherto described. As we have seen, limb buds in these cases are placed in the direct pathway of a number of already formed spinal nerves, the peripheral ends of which are severed in preparing the wound, and it is expected that these nerves should continue their growth into the rudi- ment so placed. Evidence as to whether or not the limb rudiment does exert any influence on the segmental contribution and on the path of the spinal nerves entering into the formation of the nerve plexus ought to be obtained by transplanting limb rudiments in embryos at a period before the spinal nerves have begun to develop. Then, any possible influence on the part of the end organ can exert itself on the nerve fibers at a time when initial outgrowth takes place. In the Urodele, Amblystoma punctatum, the an- terior limb rudiment can readily be transplanted at such a period. In the present paper, which contains a description of a series of both autoplastic and homoplastic transplantations on this form, certain findings are set forth which strongly suggest such an influence on the part of the limb. Another very important question on which the experimental results contained in this paper throw some light deals with the part played by functional activity of the limb on the differentia- tion of neuroblasts. The general problem of the effect of functional activity of an end organ on the differentiation of neuroblasts is one which has been tested but little by the experimental method, and the re- sults of different investigations as they stand to-day are not en- tirely compatible. 120 Ss. R. DETWILER The method of attacking this problem up to the present time has consisted in extirpating the end organ at a period either before or shortly after the peripheral nerves have begun to de- velop and of observing the effect of its absence on that part of the central nervous system ordinarily supplying it with nerve components. Braus (’06) extirpated the forelimb buds of Bombinator at a period prior to the outgrowth of the brachial plexus, with a view of determining the effect of the absence of the limb on the ventral horn region of the spinal cord corresponding to the limb level. It was found, from a study of larvae preserved ten days after the operation, that the brachial nerves had grown out to the limbless area and that they were as well formed as those onthe uninjured side. No reduction in size or number of the ventral horn cells could be detected. Observations, however, on operated larvae which were kept alive until just before metamorphosis showed that not only was the brachial plexus on the limbless side diminished in size when compared with its counterpart, but that, in addition, there was a distinct reduction in the size of the ventral horn area ordinarily supplying the limb. Asa corol- lary to the general developmental theory of Roux (’85), Braus concluded that the development of the ‘central nervous system,’ is readily divisible into two periods: the first, in which growth and differentiation are independent of functional activity, and, the second, in which further differentiation and growth continue only when under the influence of functional activity. Miss Shorey (’09), who performed a series of extirpation ex- periments on the limb buds of the chick and the amphibian embryos, came to the rather sweeping conclusion from her find- ings that no neuroblasts are self-differentiating and that all are alike dependent for differentiation on stimulation from end organs or from the products of the activities of end organs. Although the experiments of Harrison (10) left no doubt that the initial differentiation of the nerve fiber is a factor predeter- mined within the neuroblasts, they did not attempt to give any definite information on the part played by functional activity on the later differentiation of neuroblasts. TRANSPLANTATION OF LIMBS IN AMBLYSTOMA 2) It was suggested by Doctor Harrison that the best way of testing the influence of functional activity on the differentiation of neuroblasts would consist not solely of removing the end or- gan and noting the effects of its absence, but rather of transplant- ing the end organ so that it might function in a new environ- ment. In this way not only could the effects of removal be noted, but still better, the effect of continued function of the trans- planted end organ on that part of the central nervous system from which its innervation is derived. Accordingly, experiments were begun with this problem in mind, and while positive evidence has been attained from these experiments to show that the functional activity of the trans- planted limb will initiate a hyperplasia of the sensory neurones contributing innervation to the limb, the results of this phase of the experiments are taken up in a separate publication (Detwiler, 20). The present paper will consider questions mainly con- cerned in the formation of nerve plexuses and the function of the limbs. I wish to express here my thanks to Doctor Harrison for his suggestions and criticisms. ANATOMICAL -Even though a transplanted limb may be well innervated by spinal nerves of the host, it is obvious that the degree of function of the limb is conditioned by still other factors. Structural de- ficiencies of the shoulder-girdle or deficiencies in the shoulder musculature would greatly restrict its function, even though the limb were copiously supplied with nerves and the structures within it were perfectly developed. Although the developmental intimacy of the shoulder-girdle and limb led Wiedersheim (’92) to conclude that the shoulder- girdle can develop only when under the formative influence of the free extremity, the lack of interdependence of these two systems has been shown experimentally (Braus, ’09, and Det- wiler, 718). It has also been shown (Braus, op. cit., Harrison, ’18, and Detwiler, op. cit.) that in the transplantation of a typical £22 S. R. DETWILER limb bud, only a portion of the girdle rudiment is included. From his experiments on Bombinator, Braus (’09) claimed that from this fraction of the rudiment a complete girdle of reduced size 1s developed, and he concluded that the shoulder-girdle rudi- ment, like that of the limb, constitutes an equipotential restitu- tion system. Experiments hitherto reported (Detwiler, op. cit.) have shown that this rudiment in Amblystoma punctatum con- stitutes a mosaic and is incapable of qualitative restitution. When transplanted, only such components of the girdle develop as are represented in the corresponding portions of the implanted rudiment. Such girdles, however, although qualitatively in- complete, may become topographically complete by a process of hyperplasia which compensates for qualitative deficiencies. This is well illustrated in cases where in the absence of the supra- scapular rudiment the dorsal zone of the girdle becomes practically completed by hyperplastic development of the intact portion. The above is not always the case, however, and inasmuch as there is considerable variability in the degree of development of the shoulder-girdle in the heterotopic position, restricted function of the limb may be a result of marked deficiencies in the girdle. Intimately associated with the development of the shoulder- girdle is the development of the shoulder muscles. These, how- ever, may develop and differentiate independently of one another so that with a well-developed girdle there may be muscular deficiencies. The lack of interdependence of muscle and skeletal differen- tiation has previously been shown. Braus (’06 a) showed this to be true in the development of the pectoral fin of the Elasmo- branch embryo. Personal observations on the development of the shoulder-girdle of Amblystoma have shown that in the absence of skeletal differentiation, shoulder muscles may develop. It is obvious, therefore, that restricted function may result from muscular deficiencies in addition to those of the girdle. Even though skeletal and muscular differentiation may be com- plete, restricted function of the limb might be due to the failure of certain of the individual muscles to receive innervation, so that this offers a third factor conditioning the degree of function. TRANSPLANTATION OF LIMBS IN AMBLYSTOMA 123 With the above possibilities in mind it became necessary, for proper interpretation of the results, to make a study of the normal anatomy and the normal conditions of innervation. The brief description which is herewith presented is based upon a study of a series of cross-sections (10u) of a specimen preserved seventy days after the closure of the medullary folds. This has been augmented by a dissection of the shoulder region of an adult specimen. A. Shoulder-girdle A description of the cartilaginous girdle as found in a larva twenty days after the closure of the neural folds has previously been given (Detwiler, 718, p. 501, and fig. 23). The conditions found in a larva of seventy days do not show fundamental alter- ations in general topography. The suprascapula has extended its growth somewhat more dorsal and has lengthened out in an anteroposterior direction so as to form a flat plate of cartilage. The procoracoid has expanded in an anteroventral direction and the coracoid has undergone a ventral expansion so as to overlap its counterpart in the midventral line. The scapula has under- gone partial ossification and that portion of the procoracoid which enters into the formation of the glenoid cavity, although still cartilaginous, is about to undergo ossification. This is sug- gested by the greatly enlarged cartilage lacunae in that region. The adult shoulder-girdle is illustrated in figure 1. The entire scapula, together with those portions of the procoracoid and the coracoid which enter into the formation of the glenoid fossa, have become ossified. The greater part of the procoracoid and coracoid and also the entire suprascapula remain cartilaginous throughout life. B. Shoulder muscles So far as could be ascertained, no description of the shoulder muscle of Amblystoma appears in the literature. The muscu- lature, however, so far as has been studied, closely resembles that of Salamandra, a European tailed Amphibian described THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL, 31, No. 1 124. S. R. DETWILER by Furbringer (73). In referring to the musculature, Fiir- bringer’s nomenclature will be employed. The shoulder muscles may be separated into two groups, viz., the trunk muscles or those of myotomic derivation and the so- called limb muscles or those of somatopleural origin. The for- mer group contains the following: m. cucullaris (trapezius: Mivart), m. serratus magnus (serratus magnus anticus), m. levator scapulae, and the m. pectori-scapularis internus. None of these muscles derive their innervation from nerves entering into the formation of the brachial plexus. p.cor —-|-— Fig. 1 Outline drawing of the normal shoulder girdle of an adult specimen of Amblystoma. Stippled area represents bony portion. X 34. s.sc., supra- scapula; sc., scapula; p.cor., procoracoid; cor., coracoid; g.c., glenoid cavity. The second group contains the following: m. supracoracoideus, m. procoraco-humeralis, m. dorsalis scapulae (deltoid: Mivart; infraspinatus, deltoideus and teres minor: Humphrey), m. pec- toralis (pectoralis major and minor), m. subcoraco-seapularis (subscapular: Mivart), m. latissimus dorsi, m. coraco-brachialis brevis, m. coraco-brachialis longus, m. anconaeus scapularis medialis. This last muscle is undoubtedly the homologue of the long or scapular head of the triceps brachii of man. All of the muscles in the second group receive their innervation from branches of the brachial plexus. TRANSPLANTATION OF LIMBS IN AMBLYSTOMA 125 Since the rudiments of the muscles in the first group never become implanted along with the limb rudiment in a typical transplantation, it will not be necessary to consider the normal anatomy of these muscles. However, the rudiments of the muscles of the second group are involved in the transplantation, and for this reason a brief description of these muscles as they occur in their normal situation becomes necessary. 1. M. supracoracoideus. This is a broad, flat, fan-shaped muscle which takes its origin from the entire external surface of the coracoid with the exception of the medial and posterior margins. Its fibers converge to be inserted on the proximal part of the processus lateralis humeri. Innervation: n. supracoracoideus (fig. 2, n. sp. cor.), a branch of the third spinal nerve. 2. M. procoraco-humeralis. This is a somewhat longer and narrower muscle than the m. supracoracoideus. Its fibers take origin from the external surface of the procoracoid with the ex- ception of the distal anteromedial and anterolateral margins. The fibers converge proximally and become inserted on the pro- cessus lateralis humeri between the insertion of the m. supra- coracoideus and the m. dorsalis scapulae. Innervation: through a branch of the third spinal nerve (fig. Deities Ds COr.) 3. M. dorsalis scapulae. This is a somewhat thicker muscle than the m. supracoracoideus and the m. procoraco-humeralis and takes origin from the entire external surface of the cartilag- inous suprascapula with the exception of the dorsal, anterior, and posterior margins, the latter two of which serve for respective insertions of the m. levator scapulae and a portion of the m. serratus magnus. The muscle passes down over the external surface of the scapula, crosses over the tendon of the m. latis- simus dorsi, and inserts by means of a flat tendon on the processus lateralis humeri, medial to the insertion of the m. procoraco- humeralis. Innervation: by several short branches from the third spinal nerve (fig. 2, n. ds. c). 126 S. R. DETWILER 4. M. pectoralis. This constitutes a triangular-shaped muscle taking origin from the aponeurosis between it and the m. obliquus externus abdominis, the linea alba, and from a cartilaginous ster- num. Its fibers converge into a short tendon which inserts on the distal part of the processus lateralis humeri. Innervation: n. pectoralis, a branch of the common trunk formed by the union of the fourth and fifth spinal nerves (fig. 2, pC.) 5. M. subcoraco-scapularis. This is a short, thick muscle which takes origin from the medial side of the contiguous portions of the scapula and the procoracoid. The fibers pass out across the inner aspect of the shoulder joint and insert on the medial surface of the proximal part of the humerus. Innervation: n. subscapularis, a branch of the third spinal nerve (fig. 2, n. sb. sc.). 6. M. latissimus dorsi. This is a broad triangular muscle which arises from an aponeurosis between it and the longitudinal dorsal trunk muscles. The fibers converge into a long tendon, a part of which unites with the tendon of origin of the m. anconaeus scapularis medialis; the remainder passes across the lateral as- pect of the shoulder capsule and inserts on the proximal dorso- lateral surface of the humerus close to the processus lateralis humeri. Innervation: n. latissimus dorsi, a branch of the fourth spinal nerve (fig. 2, n. lt. dor.). 7. M. coraco-brachialis brevis. This is a short, rather thick muscle which takes origin from the posterior margin and from the outer surface of the posterior part of the coracoid, internal to the m. supracoracoideus. The muscle passes to the medio- ventral surface of the humerus and inserts on its proximal two- fifths and under cover of the fibers of the m. brachialis inferior (m. biceps brachii). Innervation: by the n. coraco-brachialis, a branch of the fourth and fifth spinal nerves (fig 2. 7. cor. br.). 8. M. coraco-brachialis longus. This is a long, rather thick muscle which takes origin from the internal posterior margin of the coracoid close to the glenoid cavity. It passes out along the TRANSPLANTATION OF LIMBS IN AMBLYSTOMA 127 ventral surface of the humerus, ventral to the m. anconaeus scapularis medialis and medial to the m. brachialis inferior. It inserts on the ventral surface of the distal two-fifths of the humerus. Innervation: by the n. coraco-brachialis (fig. 2, n. cor. br.). 9. M. anconaeus scapularis medialis. This muscle takes origin by means of a long, flat tendon from the glenoid margin of the scapula and from the capsule of the shoulder joint. X ‘uorjye1edo 94} 10}Jye skup sUIU-A}XIS poAdosoid [BVUIUY “UOT}ISod [vULIOU oY} 0} IOT10}S0d s}UdUBOS XIS Jo s9UBSIP OY} pop UL[dsuBs, QUIT] LOTIOZUV YYSIY “S2QQW 9SBO FO MOTA [BIZUBA Z ‘XX ‘uorjesedo oy} 104J8 SAvp XIS-AYXIS poAtosoid [vuUUy *UoT}ISod [VUILOU ay} 0} LOI10}80d s}uaUIBes very} JO s0UBISIP oY} poyUL[dsuvs} QUIT] LOIIO}UB YYSIY “EGY osvO Jo MOIA [RUBIA 6 SduYO00Ia JO NOILVNVIdxXa@ 6 ALVId 168 CBA 4 fat WTO’ TQ ITrraorrrw HaTIMLAd “W'S ory C tato dri dap Wik ATCA TO GW bok wer roku Te. 169 ae: en i * ad m \ Lei 1 z Wlawg me ay, | THE JOURNAL OF XPERIME , BD odtoey, vor. 31, No. 2 ', 1920 t? Resumen por los autores, M. F. Guyer y E. A. Smith. Universidad de Wisconsin. Estudios sobre las citolisinas If. Transmisi6n de defectos oculares inducidos. En los fetos uterinos de conejos de distintos troncos nan in- ducido los autores defectos oculares (principalmente defectos en el cristalino) mediante el suero de gallina sensibilizado para el cristalino de conejo. Aparentemente el efecto es especifico, puesto que los jévenes contenidos en conejas prefadas inyecta- das consuero de gallina puro o sensibilizado para los testiculos de conejo no presentan sus ojos afectados. Una vez establecido, el defecto puede transmitirse a generaciones ulteriores. Es un ejemplo de verdadera herencia (no simplemente transmisi6n pla- centaria) puesto que puede extraerse de la linea masculina. Translation by José F. Nonidez Cornell University Medical College, N. Y. AUTHORS’ ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICH, JULY 26 STUDIES ON CYTOLYSINS II. TRANSMISSION OF INDUCED EYE-DEFECTS M. F. GUYER AND E. A. SMITH Zoological Laboratory, University of Wisconsin SEVEN FIGURES AND FOUR PLATES INTRODUCTION In a former paper (Guyer and Smith, 718) we recorded the results obtained during the year 1916-17 from experiments in which pregnant rabbits and pregnant mice were treated with fowl serum sensitized, respectively, to the crystalline lens of the rabbit and of the mouse. It was found that antenatal defects in the lenses of the young could be secured in this way. Thus, in rabbits treated during pregnancy with fowl serum sensitized to rabbit lens, some of the young showed eye defects, such as opacity of the lens and partial or, less frequently, complete lique- faction of the lens. Similar results were obtained with mice of the genus Peromyscus. The present paper deals with the continuation and repetition of these experiments in rabbits, and includes an account of the transmission through successive generations of eye defects orig- inally induced by means of lens-sensitized fowl serum. To do away with the possibility of accident or coincidence, it was obviously desirable to secure other well-established cases This is particularly true for the genetical aspects of the experiments. Since, in our opinion, the fact of transmissiblity is by far the most significant one established, in repeating the original experi- ment we have taken pains to secure wholly unrelated stock so that we may be sure that we are not simply perpetuating a chance inheritable defect which has sprung up by some strange coincidence just at the time of our work. To safeguard the 171 2 M. F. GUYER AND E. A. SMITH experiments in this respect, we have imported rabbits from other States (Minnesota, Iowa, Illinois, and Indiana) and tested them genetically before treating them with serum or crossing them with our original strain. We have often been asked why we chose fowls and rabbits rather than some other forms for our experiments. Fowls were used as the source of the sensitized serum mainly because of the ease with which they may be kept and handled, and because they are not easily infected in surgical operations. Furthermore, it was thought that serum from an animal as far removed in rela- tionship from the rabbit as the fowl is would perhaps yield a more powerful serum than that from a mammal. This opinion, it should be noted, is based on statements we have found in books on immunity and not on our own experience. In his book enti- tled ‘Immunity,’ for example, Citron (Garbat translation) speci- fies (p. 144) chickens as among the best animals adapted to supply hemolytic sera, and also remarks that “an animal produces a better hemolysin the remoter its relationship to the animal from which the erythrocytes for injection are taken.”’ The use of so foreign a serum as that of the fowl, however, doubtless has its disadvantages, since the poisonous, hemolytic or general, shock effects arising from the introduction of such a widely different serum into the veins -of a rabbit cannot but be more severe than if serum from a more closely related form like the guinea-pig were used. The frequent severe illness and the occasional death which occurred in treated rabbits was probably in no small measure due to this factor. Nevertheless, the ad- vantages in using fowls seemed to outweigh the disadvantages so far that we have continued to use them. The availability of the large marginal vein in the ear for intra- venous injections is one reason for the use of the rabbit in experi- ments such as ours. Also, a doe usually bears from five to eight young in a litter and may have several litters in one year. The young, furthermore, will begin to breed at from six to eight months of age, though this advantage of early maturity is offset somewhat by the fact that the litters of very young females usually number only three or four individuals. While these TRANSMISSION OF INDUCED EYE-DEFECTS 173 facts all show that among mammals, rabbits are fairly favorable for breeding experiments, the fact that influenced us more than any other in choosing them is that in our experience they are physiologically well stabilized and rarely if ever in the course of ordinary breeding produce abnormal young. It is well known, for instance, that guinea-pigs of supposedly normal origin occa- sionally throw defective young. Sometimes it is a missing toe or toes, or perhaps there are extra toes. Again it may be some nerv- ous defect, such as congenital palsy or epileptic-like seizures, or perhaps, though less frequently, the defect is an eye anomaly. In breeding experiments carried on with guinea-pigs in the de- partment of genetics in the University of Wisconsin during the last few years several examples of the abnormalities just men- tioned have been encountered. In rats, also, the writers have seen two cases in which one eye was smaller than the other and was otherwise imperfect. As regards rabbits, however, we have never seen nor heard of such sporadic eye defects. The senior author has bred many rabbits for laboratory purposes and he has also conferred with a number of rabbit breeders, but has found no record of congeni- tal eye defects. In this connection some two years ago he made special inquiry of Dr. Orren Lloyd-Jones, of Ames, Iowa, a trained geneticist, who stated that in the four hundred and some odd rab- bits he had just been using in genetical problems he had observed nothing similar to the eye defects induced in our serum-treated stock. He also said that as a result of his various experiments in breeding rabbits, extending over a number of years, he had come to regard them as exceptionally stable forms. Likewise Dr. E. C. Rosenow, of the Mayo Clinic, who is constantly work- ing with rabbits and has done so for years, after looking over our experiments told us that he had never seen such eye defects in any of his rabbits. All testimony that we have received, therefore, coincides with our own experience that rabbits are stable forms whoily unlikely to develop eye defects unless, as in our work, these have been de- liberately induced by the experimenter. We dwell upon this point because when unusual results are obtained, the first thought 174 M. F. GUYER AND E. A. SMITH is always of coincidence and chance, and at the outset it is im- portant to realize how improbable it is that just the right chance variation would spring up at exactly the right time—that is, coincidentally with our treatments—to mislead us into believing that we had produced something that was destined to appear anyway in our different stocks of rabbits. Fig. 1 Profile view of rabbits to show natural bulging of the eyeballs of a normal rabbit (1) compared with one (4A5) in which the left eyeball is reduced in size, and one (6A4) in which it has practically disappeared. THE NORMAL EYE In order to understand the eye defects that have been induced in our stock, it is necessary to know the chief characteristics of the normal eye of the rabbit and something about its develop- ment. The eyeball of the rabbit, typically large and globular, measures about 16 mm. in diameter and protrudes beyond the contour of the head (fig. 1) so far as to be conspicuous. The outer or sclerotic coat is glistening white and contains no cartilage. The iris is also white with fine transparent radiations which TRANSMISSION OF INDUCED EYE-DEFECTS 15 extend from the outer edge toward the pupil. The lens, which is large, transparent and spherical, occupies about one-half of the posterior chamber. In albinos such as we used the blood-vessels that supply the retina give to the eye a rich red color easily seen through the pupil and iris (pl. 1, fig. 1). In the embryo the optic vesicles, which arise as outgrowths of the ventral lateral walls of the forebrain, are well developed be- fore the end of the ninth day. Between the tenth and four- teenth days several important changes take place. ‘The ectoderm opposite the vesicle thickens into a disk closely connected to the outer wall of the latter. As the outer wall is folded in to form a two-layered optic cup, this disk is pulled in, eventually separat- ing from the ectoderm as the hollow lens vesicle which les within the optic cup. The cavity of the lens vesicle is gradually oblit- erated by the thickening of the inner wall and the lens increases in size by the addition of successive layers to the outside. The process of folding is not confined to that part of the vesicle in contact with the lens. The ventral wall of the vesicle and a part of the optic stalk are pushed in, producing a cleft or choroid fissure in the bottom of the cup continuous with a groove in the stalk. A vascular loop which later becomes the central artery of the retina and its hyaloid branch enters the cup through the choroid fissure. Some of the loose mesenchyme cells surrounding the optic cup extend around the lens to form a membrane, the posterior part of which is supplied by the hyaloid artery. The anterior sur- face receives branches of the anterior ciliary arteries. Thus, by the thirteenth day, the lens is relatively large and is surrounded by a membrane richly supplied with blood-vessels. This vas- cular membrane is an embryonic structure which serves for the nutrition of the lens during its growth. It disappears before birth. THE DEFECTIVE EYES . The chief defect, common to all the eyes where there is suf- ficient eyeball left to permit of internal examination, centers in the lens. It is always opaque, in whole or in part, and it may be 176 M. F. GUYER AND E. A. SMITH greatly reduced in size. In some rabbits the opacity is evident without the use of the ophthalmoscope; in others, an examination with this instrument is necessary to disclose it. In several in- stances eyes which in all external respects appeared normal were found to have milky lenses when examined ophthalmoscopically. The defective lenses, however, are frequently accompanied by other characteristic anomalies (pls. 1 to 4). Often the abnormal eye has a staring look because the iris does not exhibit its normal reflexes and is usually more translucent than the iris of the nor- mal eye. The color of such an eye is peculiar; it is lavender at some angles and silvery at others (pl. 1). Apparently the ab- sence of the normal red is due in part, at least, to the clouded lens which keeps the reflection from the retinal blood-vessels from shining through. How much, if any, the retinal blood- vessels themselves are changed is now under investigation. Oc- casionally the hyaloid artery persists and a fine network of blood- vessels surrounds the opaque lens. While the whole lens sub- stance is usually clouded, in some lenses only spots are opaque. Not only may the lens be opaque but, as already mentioned, it may also be reduced in size. In such cases the eyeball, iris, and pupil are correspondingly small. The eyeball, for example, may be only one-half, one-third, or even one-fourth normal size (pl. 1 to 4) and sunken until the eye does not extend beyond the level of the head (fig. 1). Again, the ball may be rotated downward or inward until the cornea and parts visible behind it are nearly out of sight. The extreme is reached in those eyes in which the ball collapses, leaving no trace of pupil or iris. Accompanying these defects are frequently a cleft iris and less often, a persistent hyaloid artery, due to suppression of develop- ment. To understand these conditions one must remember that in the embryo the optic cup, instead of being a complete ring, is interrupted on the ventral side by the choroid fissure. If this fissure remains open instead of closing, as it should do normally, the anomalies just described result. Incomplete or cleft iris is known as coloboma when it occurs in man. It is obvious that during the developmental period, especially from the tenth to fourteenth day when the optic cup is forming TRANSMISSION OF INDUCED EYE-DEFECTS U7 and when the lens has a rich plexus of blood-vessels surrounding it, any lytic or toxic substance in the blood specific for lens material would have a good opportunity to attack it with maximal effect. For instance, such a substance could directly hinder increase in size through solution of one or more of the lens proteins. Or it is not unlikely that the sensitized fowl serum would form a precipi- tin with some of the lens protein. This may be the means by which the opacity of the lens is produced. A stunting of the lens would in all probability result in the production of a smaller eye- ball, inasmuch as the parts are so mutually related in develop- ment. The eye defect, once secured, does not always remain at a standstill in the affected individual, but may progress; or it may, at least, have associated with it conditions which lead to further changes in the eye. For example, individual 3A1, a male secured in one of the earliest experiments, had the lens of the left eye opaque, although the eyeball was but slightly less than normal size at the time the eyes opened some twelve days after birth. This eye not only did not keep pace in size with the other eye as the young individual grew larger, but actually retrogressed as if being acted upon by some kind of solvent. It became gradu- ally smaller, the ball collapsed and almost disappeared. At the present time, about three years later, there is practically no trace of an eyeball (pl. 1, fig. 83A1; pl. 2). The condition indicates that a solvent effect of some kind is in operation. Such post- natal degeneration occurred in several rabbits. LATER EXPERIMENT ON THE PRODUCTION OF EYE DEFECTS WITH LENS-SENSITIZED SERA In our later experiments much the same methods of procedure were followed as in those recorded in our first study (Guyer and Smith, 718). Fowls were sensitized with rabbit lens from four to six times at intervals of approximately a week, and were then left about ten days before killing. In most experiments the original method of injecting the pulped lens intraperitoneally was followed, though in a few cases the more difficult method of injecting the material directly into the femoral vein of the fowl 178 M. F. GUYER AND E. A. SMITH was practiced. It seems worth while to call attention to this intravenous method, inasmuch as some of the most pronounced effects produced in the uterine young were secured with serum from fowls which had been thus intravenously treated and had - later been further sensitized or resensitized by the intraperitoneal method. The rabbits were generally so mated as to have the young in utero somewhere near the ten-day stage of development at the time of the first injection of fowl serum. Only albino rabbits were used, as it was thought that the unpigmented iris would be of advantage in examining the lens and the interior of the eye. In the following detailed account of our later series of experi- ments, the first experiment of the new series is recorded as ‘Ex- periment 10’ in order to keep the designations the same as in our office records, and also to avoid confusion with the experiments discussed in our former paper (718). All injections into the rab- bits were intravenous unless. otherwise specified. Unless the eyes were visibly defective, they were recorded as normal. Experiment 10 In this experiment five fowls and three rabbits were used as specified in table 1. The chief purpose of the experiment was to find if lens-defects could be induced in the young of rabbits far advanced in pregnancy. Only two injections were given. It will be observed that the fetuses of two of the rabbits, numbers 13 and B, were within about nine days of birth when the first serum was introduced. Apparently: the dose, 8 cc., was too large. The evidence indicates that the young were killed in utero in all three of the mothers. Each doe became very ill, and B died two days after the second treatment. Dissection showed that she was carrying five young. Judging from their somewhat macerated condition, they had been dead some days. The other two does acted as if crippled. They did not recover from their stiffness and lethargy for several weeks. Our inference was that the young were gradually resorbed, or perhaps in the case of the one with young far advanced, aborted. The latter idea is based TRANSMISSION OF INDUCED EYE-DEFECTS 179 on the fact that under somewhat similar circumstances we have sometimes found in the hutches of other does chunks that seemed to be placentas, although all trace of the fetuses had disappeared. Since, in one case at least, we found the doe eating this material, it is possible that other abortions have occurred and gone unre- corded because the aborted young were eaten. TABLE 1 Experiment 10 I. Sensitization of fowls IDENTIFICATION pars—1017 | wonnens orsowus [PMDERQFZApDI| Nonuasaur | possoe res cc. cc. October 31 5atO) 12) 1 ed7, 6 20 3 November 7 | 5, 10, 12, 13, 17 16 25 4 November 14 | 5, 10, 12, 13, 17 6 20 3 November 21 | 5, 10, 12, 13, 17 6 45 8 November 28 | 5, 10, 12, 13, 17 6 20 3 December 8 5, Os I, i ales 10 25 4 II. Treatment of rabbits IDENTIFICATION DAYS DATE OF INJECTION beer easing PREGNANT DOSE OF SERUM REMARKS December 14 I 11 8 Mating,1 9 X2¢ 13 21 8 Mating, 13 2-x2¢ 8 Mating, B 9 X20 December 16 1 13 9 Very ill; no young 13 23 8 Very ill; no young B 2a 8 Died Dec. 18; had mac- erated young in uterus It is possible also that the severity of the treatment in experi- ment 10 was in some measure due to a more rapid entrance of the serum into the fetus because of greater permeability of the placenta in late fetal life, though we have no direct evidence on this point. But even if this were a sufficient explanation for the result in the case of rabbit 13 and rabbit B, it would hardly ac- count for it in rabbit 1, since she had been pregnant only eleven days when the serum was first injected. 180 M. F. GUYER AND E. A. SMITH Experiment 11 Four fowls and four rabbits were used as recorded in table 2. The rabbits ranged from ten to sixteen days in pregnancy when TABLE 2 Experiment 11 I. Sensitization of fowls DATE—1918 speeyie S NUMBER eee LENSES a araetonie cae scr ts cc. cc January 16 4 10 20 4 January 21 4 10 20 4 January 28 4 6 (half-grown) 15 4 February 6 4 4 adult, 6 newborn 15 4 February 14 4 4 19 4 II. Treatment of rabbits IDENTIFI- CATION DAYS DOSE OF DATE OF INJECTION NUMBERS | PREGNANT SERUM REMARKS ? OF RABBITS February 22 22 16 5 Mating, 222 X2¢ 17 10 5 Mating, 179 X20 C 14 5 Mating, C 9 X2¢ February 26 22 20 6 Ih 14 6 C 18 6 February 28 22 22 6 17 16 6 C 20 6 March 2 22 24 5 5 young; March 7; eyes normal Ike 18 5 C 22 5 5 young, March 9; some eye de- fect; see text March 5 17 21 5 3 young, March 16; eyes normal A 13 5 March 7 A 15 6 March 9 A 17 6 March 14 A 22 4 5 young, March 22; 1 with eyes normal, others chilled to death TRANSMISSION OF INDUCED EYE-DEFECTS 181 the first injection of the sensitized serum was given. It will be observed that the dosage was considerably smaller than that given in experiment 10. On March 7th rabbit 22 bore five young, all with eyes apparently normal. Rabbit C had five young March 9th. The eyes of these young did not open as soon as those of young do normally, and when finally open the eyeballs appeared to be somewhat flattened. In three of these young the characteristic reddish color of the albino’s eye was no- ticeably less intense. The mother died April 13th and three of the young some days later. The lenses of one of these, examined immediately after death on May 11th were found to be pasty and milky-looking. Rabbit 17 bore three young March 16th, all with normal eyes. Rabbit A gave birth to five young on March 22nd. She had not made a suitable nest for them, however, and when found next morning all were badly chilled. Although placed in an incubator, all died except one. The eyes of this survivor were found to be normal when the lids finally opened. Experiment 15 Three fowls and two rabbits were used, As shown in table 3, no young were secured. Rabbit A after four doses of serum de- veloped paralysis of the hind legs and died June 22nd. Rabbit 20 was obviously pregnant, but the young were killed in utero apparently by the later injections. She was ill for some time and although used in later experiments she proved. infertile. We have found infertility to be a common experience following deaths of young in utero. Apparently the resorption is so pro- longed that the uteri remain blocked for a long time or else changes are set up in the uteri which either obstruct them or otherwise prevent conception or placentation. In rabbits which abort their young, on the other hand, we have had little difficulty in securing young within a short time afterward. For example, rabbit 17 (tables 4 and 5) apparently aborted some of her young on January 5th, but following another mating she had five young April 5th. 182 M. F. GUYER AND E. A. SMITH Experiment 20 Twelve fowls and five rabbits were used as set forth in table 4. Although all of the rabbits had been observed to mate, only two of the five bore young. It was not determined whether the fail- TABLE 3 Experiment 15 I. Sensitization of fowls pami1018) | rrcaimcgmpamy |) ear a Grmemoem) |< FOR Gia ll: (nOneeens i, 0] Se Sec Un pan)” huge oil aia en een April 20 3 6 (half-grown) 10 3.0 April 27 3 6 10 3.0 May 4 3 6 10 oe0 May 11 3 6 10 3.5 May 18 3 6 10 335 Il. Treatment of rabbits IDENTIFI- < CATION DAYS DOSE OF DATE OF INJECTION NUMBERS | PREGNANT SERUM REMARKS OF RABBITS May 30 20 9 5 Mating, 20 X 2 A ? 5 Mating, A X 2 June 1 20 11 5 A ? 5 June 6 20 16 5 A I 5 June 10 20 19 5 20—obviously pregnant; young died in utero A i 5 A—developed paralysis of hind- legs; died, June 22 ure of nos. 11 and 20 to have young was due to lack of impreg- nation at mating or to death of the young in utero. After the fifth injection no. 22 became very ill, and on December 19th she was killed. Dissection showed the uteri to be filled with pus. There was no trace of young. If any had been present at first TABLE 4 Experiment 20 I. Sensitization of fowls pare—i0i9 | rowss secre [YOMBEROFRADOT| onan curt | poshon pen cc. cc. November 7 12 6 20 15 November 11 2 6 20 1.5 November 21 2, 6 20 15) November 30 2 6 20 11.5 II. Treatment of rabbits IDENTI- FICA- I DAYS DATE OF eLeN) a NUM- | PREG- DOSE OF SERUM REMARKS INJECTION BERS NANT OF RAB- BITS ——————————————————————— December 10 |} 11 iG 6 Mating, 11 29 x 44¢ 17 7 6 Mating, 17 9 X 16A2 7 7 6 20 Mating, 202 X20 December 12 : ; 5+3 (normal salt 20 9 solution) 16A1 5 6 Mating, 16Al 9 x 16A2¢ 22 5 6 Mating, 229 x 440 December 14 | 11 11 Le a 5+3 (normal salt 2 ne solution) 16Al 7 22 7 December 17 | 11 14 6 il7/ 14 6 20 14 6 16A1} 10 6 po 10 6 December 19 | 11 16 6 17 16 6 20 16 6 16A1| 12 6 22 22 6 No. 22, ill; killed; ovi- ducts filled with pus December 21 | 11 18 6 No. 17 had 1 young and 2 17 18 6 placentas; see text 20 18 6 No. 16A1 bore 4 young, 16Al| 14 6 January 7; eyes normal Nos. 11 and 20, no young 183 184 M. F. GUYER AND E. A. SMITH they must have entirely disintegrated. No. 16A1 had four young January 7th, all with normal eyes apparently. In the case of no. 17 one live young and what appeared to be two good- sized placentas without any trace of young attached to them were found on the morning of January 5th. The mother was gnawing at one of the placentas, and it was possible that young had been attached to the placentas originally, but had been eaten. The single live young one was not taken care of by the mother and soon died. The doe, although a good mother on previous occasions, had plucked out none of her hair nor other- wise prepared a nest for the coming of this litter. She appar- ently had no milk or at least made no attempt to suckle the young one that survived, although it was very active and insistent for a few hours. It is an interesting incident that this same doe in preparing a nest for her next litter which was born April 5th, denuded herself entirely of hair as far as she could reach along her belly and sides so that she looked almost like a hairless rab- bit. The nest was a huge mass of fur. Experiment 21 Six fowls and five rabbits were used (table 5). It was thought that possibly a more active serum might be obtained if the anti- gen were introduced intravenously into the fowl instead of intra- peritoneally. Accordingly, two of the injections of pulped lens, the second and third, were made into the femoral vein. The first and fourth were subcutaneous and the fifth was intraperi- toneal. Two of the rabbits, nos. 14A5 and 1444, bore no young. No. 18A2 aborted five young April 1st and died April 9th. No. 17 bore five young April 5th, the‘eyes of which were normal, at least to all outward appearances. No. 16A1 had four young April 6th. Both eyes of one of these (fig. 2) were markedly abnormal. Unfortunately, it died May 11th. The eyes of the other young in the litter appeared to be normal. No. 16A1 had been mated to a brother, 16A2. They were from a Minneapolis strain of rabbits. TRANSMISSION OF INDUCED EYE-DEFECTS 185 TABLE 5 Experiment 21 I. Sensitization of fowls DATE—1919 | ea a BABEII LENSES sGueroRT DOSAGE PER FOWL cc. February 8 6 6 20 2.0 ec. subcutaneously February 15 6 8 20 1.0 ec. intravenously February 22 6 6 15 1.0 ce. intravenously March 1 6 8 15 1.5 ec. subcutaneously March 19 2 4 8 3.0 cc. intraperitoneally II. Treatment of rabbits IDENTIFI- insecrion | Numpens |PReGNant| seRuM REMARKS OF RABBITS fig Ae apes cc. March 17 14A5 9 4.0 Mating, 14A5 9 xX 440. 18A2 13 4.0 Mating, 18A2 9 X 16A2 9 17 13 4.0 Mating, 179X442 16A1 12 4.0 Mating, 16Al 9 X 16A2 ¢% 14A4 9 4.0 Mating, 1444 2 X 16A2 0 March 19 14A5 zh 4.0 18A2 15 4.0 17 15 4.0 16A1 14 4.0 14A4 11 4.0 March 21 14A5 13 3.0 18A2 17 3.0 17 17 Bane) 16A1 16 3.0 16A4 13 3.0 March 25 14A5 17 2.5 No. 16Al1 bore 4 young April 6; 1 18A2 21 2.5 had both eyes defective; it died 17 21 2.5 SeMay, 11 16A1 20 2.5 No. 17 bore 5 young April 5; eyes 14A4 17 2.5 normal No. 18A2 aborted 5 young April 1 and died April 9 Nos. 14A5 and 14A4 had no young THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 31, No. 2 186 M. F. GUYER AND E. A. SMITH Experiment 22 Tnasmuch as it was desirable to get new defective-eyed indi- viduals from a stock wholly unrelated to the line in which we had originally produced eye defects, and since 16A1, after treat- ment with lens-sensitized serum, had already given us an indi- vidual with conspicuously abnormal eyes (opaque lenses and re- duced size), it was determined to try her again. The fowls used were two which had been left over from the earlier experiment in which 16A1 had been used (table 5), and as they had had their 70A/ TOAR 3 10A3 Fig. 2 Female 16A1 was mated to her brother, 16A2, shortly after which she was treated (table 5) with lens-sensitized fowl serum; one of her young, a male, had both eyes abnormal. See figure 5 for explanation of symbols. Fig. 3 Female 16A1 was mated to male 50 and subsequently treated with lens-sensitized fowl serum (table 6); two of the resulting young, 70A1 and 70A2, each had both eyes defective. For explanation of symbols see figure 5. last injection of pulped rabbit lens on March 26th it was thought advisable to resensitize them. They were given three addi- tional doses of lens intraperitoneally on June 12th, 14th, and 20th, respectively (table 6). They had in the previous sensitiza- tion (table 5) been given two of the injections of lens intrave- nously. Rabbit 16A1 was the daughter of a female shipped from Min- neapolis and was, therefore, unrelated to our Madison stock. She was mated to no. 50, a male obtained in Chicago. On the eighth, tenth, fifteenth, and seventeenth days of pregnancy, re- spectively, she was injected with 4 cc. of the lens-sensitized fowl serum through the marginal vein of the ear. On July 21st five TRANSMISSION OF INDUCED EYE-DEFECTS 187 young were born. One of these died two days later. Two of the remaining four had eyes normal in appearance and two had eyes markedly abnormal (fig. 3). One of the normal-eyed ones died when about three months old. TABLE 6 Experiment 22 I. Sensitization of fowls NUMBER OF RAB- NORMAL SALT DOSAGE PER paTE—1919 FOWLS REINJECTED! 507 LENSES USED SOLUTION FOWL ce. ce. June 12 2, 4 12 3 June 14 2 4 12 3 June 20 2 4 12 5 II. Treatment of rabbit IDENTIFI- ROECHION ieasons are Gare Sao ES OF RABBITS ~ cc. June 28| 16A1 8 4 Five young born July 21. 1 soon died; June 30| 16A1 10 4 2 had very defctive eyes; 2 had nor- July 5 16Al1 15 4 mal eyes July 7 16A1 17 4 16A1 2 was mated to 50 & CONTROLS To determine whether eye defects induced by lens-sensitized fowl serum as just described are attributable to the specific action of the antibodies or merely to a general poisonous or asthenic -effect of the fowl serum, it is obvious that careful controls must be instituted. Before the effect can be pronounced specific, it is also necessary to establish the fact that fowl serum sensitized to other tissues of the rabbit than crystalline lens will not induce the lens defects in question. To secure such controls we in- jected a number of pregnant does with pure (that is, unsensi- sitized) fowl serum, and still others with fowl serum which had been sensitized to rabbit testis. The experiments follow. 188 M. F. GUYER AND E. A. SMITH Experiments 12 and 18 In these two experiments four rabbits were used as specified in table 7. From 4 to 6 ce. of fresh normal fowl serum was used for each injection. Rabbit no. 15 received three treatments; nos. 20 and 24 five treatments, and no. 19, eight treatments. Num- DATE OF IN- JECTION—1918 | NUMBERS | PREGNANT March 9 March 12 March 14 March 16 March 19 May 4 May 7 May 11 IDENTIFI- CATION OF RABBITS TABLE 7 Experiments 12 and 13 . Control: Treatment of rabbits with normal serum DAYS DOSE OF SERUM REMARKS Mating, 249 X2¢ Mating, 199 X2¢ Mating, 209 X¥2¢ No. 20 bore 3 young April 8; eyes normal Mating, 159 X2¢ Nos. 24, 19 and 15 bore no young; 24 evidently had young killed in utero TRANSMISSION OF INDUCED EYE-DEFECTS 189 ber 20 bore three young, April 8th, all of which had normal eyes. The other three does became very ill and bore no young. No. 24, at least, had every evidence of having had the young killed in utero in a relatively advanced stage of development. As hap- TABLE 8 . Experiment 14 Control: Treatment of rabbits with normal serum IDENTIFI- DATE OF IN- CATION DAYS ° DOSE OF SERUM EMARKS JECTION—1918 | NUMBERS | PREGNANT SSD) NTA a OF RABBITS Mating, 199 X20 May 21 19 3 5 17 11 5 Mating, 17.9 X2¢ 22 8 5 Mating, 222 X2¢ 13 10 5 Mating, 13 9 X2o¢ May 25 19 7 17 15 342 22 12 (normal salt) 13 14 May 29 19 11 5 17 19 5 22 16 5 13 18 5 June 1 19 13 5 17 21 5 22 18 5 13 20 5 June 6 19 18 4 All apparently aborted or re- 1%: 26 4 sorbed young 22 5 2B) 4 No. 13 had hind legs para- 13 PAB) 4 lyzed for a time pens under such circumstances, although later mated repeatedly she remained infertile. Experiment 14 Four rabbits were used and five treatments with fresh normal fowl serum were given (table 8). Each rabbit apparently aborted 190 M. F. GUYER AND E. A. SMITH or resorbed her young. No. 13 had her hind legs paralyzed for some weeks, but ultimately recovered. Experiment 16 A single doe was used in this experiment (table 9). From the ninth to the twentieth days of pregnancy, inclusive, she was given six injections of fresh normal fowl serum. Two of the doses consisted of 5 cc. each; four of them of 6 ec. each. On August 22nd she bore five young, all with normal eyes. TABLE 9 Experiment 16 Control: Treatment of rabbits with normal serum eae GIS Usounen OF if eee DOSE OF SERUM REMARKS cc. July 31 22 9 6 Mating, 229 X2¢ August 2 22 11 5 August 4 22 13 5 August 7 22 16 6 August 22, 5 young born; August 8 22 17 6 eyes normal August 11 22 20 6 Experiment 17 Two rabbits were used and each dose of fresh normal fowl serum measured 5 cc. No young were secured (table 10). Experiment 18 Three rabbits were used (table 11). Inasmuch as all three be- came ill after the first injection of 6 cc. of fresh normal serum, the second and third doses were reduced to 5 cc. diluted with 3 ce. of normal saline solution. The remaining doses were each 6 cc. of undiluted serum. Nos. 24 and 15 proved infertile. No. 20 bore four young October 12th, all with normal eyes. TABLE lu Experiment 17 Control: Treatment of rabbits with normal serum ; -. |IDENTIFICX'TION sEcrroN—1918 Se August 6 13 19 August 8 13 19 August 10 13 19 August 12 19 August 15 19 DAYS PREGNANT ig 20 Ex DOSE OF SERUM or on REMARKS Mating, 13 9 X20 Mating, 199 X2¢ 5 No young born TABLE 11 periment 18 Control: Treatment of rabbits with normal serum DATE OF INJECTION— 1918 September 19 September 21 September 23 September 26 September 27 September 30 IDENTIFI- CATION NUMBERS OF RABBITS DOSE OF SERUM 5 + 3 (normal salt) 5 + 3 (normal salt) 5 + 3 (normal salt) 5 + 3 (normal salt) 5 + 3 (normal salt) 5 + 3 (normal salt) 6 6 6 6 6 6 191 REMARKS Mating, 15 9 X 44c¢% Mating, 202? X2 ¢ Mating, 24 9 X 44c¢ Nos 24 and 15 bore no young October 12, no. 20 had 4 young; eyes nor- mal] 192 M. F. GUYER AND E. A. SMITH Experiment 19 Inasmuch as only one rabbit was used and no young were born, the experiment (table 12) is not significant beyond helping to complete our record and also to demonstrate some of the dif- ficulties and discouragements which attend this kind of work. Experiment 23 With this experiment (table 13) the use of fowl serum sensi- tized to rabbit testis was begun. Four fowls and four rabbits were used. The fowls were given four injections of pulped testis TABLE 12 Experiment 19 Control: Treatment of rabbit with normal serum IDENTIFI- DATE OF pe NUMBER bear sige DOSE OF SERUM REMARKS RABBIT cc. September 26 13 9 6 Mating, 138 2 xX 44c¢ September 28 13 11 5 + 3 (normal salt) October 1 13 13 5 + 3 (normal salt) October 2 13 14 6 No young at intervals of about a week. To prepare the injection mass the testes of two adult rabbits were pulped by grinding in a mortar, normal saline solution being poured in from time to time until a total of 18 ec. had been added The mass was then pressed through two layers of cheese-cloth to strain out the larger par- ticles which would occlude the cannula of the syringe. Unlike lens emulsions, such emulsions of testis are always tinged more or less with blood. This would lead one to expect a more severe hemolytic reaction from antiserum produced from such emul- sions than from normal serum or fowl serum sensitized to such relatively bloodless tissues as the lens or the humors of the eye. Whether, in fact, an increased intravenous hemolysis occurred in the rabbits treated with serum sensitized to testis we did not DATE—1919 June 7 June 14 June 20 June 26 TRANSMISSION OF INDUCED EYE-DEFECTS 193 TABLE 13 Experiment 23 I. Sensitization of fowls to testis of rabbit phe MATERIAL USED 4 Testes of two adults 4 Testes of two adults 4 Testes of two adults 4 Testes of two adults NORMAL SALT | DOSAGE PER SOLUTION FOWL ce. ce. 18 4 18 + 18 4 18 4 II. Treatment of rabbits with serum sensitized to testis IDENTIFI- CATION DATE OF INJEC- DAYS TION—1919 hie PREGNANT DOSE OF SERUM RABBITS cc. July 5 17 8 6 34 6 6 35 7 6 33 7 6 July 7 17 10 |5 +3 (normal salt) 34 8 5 + 3 (normal salt) 35 9 5 + 3 (normal salt) 33 9 5 + 3 (normal salt) July 9 17 12 5 + 3 (normal salt) 34 10 5+ 3 (normal salt) 35 1 5 + 3 (normal salt) 33 11 5 + 3 (normal salt) July"12 17 15 6. 34 13 6 35 14 6 33 14 6 July,14 17 17 6 34 15 6 35 16 6 33 16 6 July 17 17 20 6 34 18 caG 35 19 6 33 19 6 REMARKS Mating, 1729 X 44¢ Mating, 349 xX 440 Mating, 35 9 X 50¢ Mating, 33 9 X 50¢ No. 17 had 7 young July 26; eyes normal No. 34 had 8 young July 29; one died; eyes of all nor- mal No. 35 had 7 young July 28; one died; eyes of all nor- mal No. 33 had 6 young July 28; eyes normal 194 M. F. GUYER AND E. A. SMITH determine, as the matter seemed to have little or no direct bear- ing upon the experiments in hand, but it is a fact that the rab-. bits showed more symptoms of illness after injection with such serum than with the serum used in earlier experiments, and in one instance, in a later set of controls, one rabbit died in convul-. sions about four hours after being injected. On the other hand, more of the does finally bore young than in any other set of experiments. The details of dosage, number of injections, dates, ete., are set forth in table 13. A total of twenty-eight young were obtained from the four does under treatment. Two of the young died before their eyes were open, leaving twenty-six to be examined for eye defects. The entire twenty-six were found to have nor- mal eyes. Experiment 24 Three fowls and four rabbits were used as shown in table 14. The fowls were each given 5 cc. of an emulsion of pulped rabbit testis in normal saline solution on five different occasions at in- tervals of about a week. Three of the rabbits received five injec- tions of the testis-sensitized serum, one of them only four. The latter, no. 16A1, died in convulsions about four hours after the fourth injection, having been pregnant twenty days. An au- topsy showed that she was carrying eight young. No. 17 bore seven young, all normal-eyed; no. 36, three young, all normal- eyed. No. 37 bore three young, but as she had made no nest and did not care for them in any way, they died. Thus the ex- periment yielded ten young which survived, all with normal eyes. IS THE REACTION SPECIFIC? Before entering upon the question of specificity, it seems ad- visible to say a word further about the nature of the defects. In our opinion, practically all of the eye defects obtained, both in the immediate young of treated mothers and in subsequent genera- tions, are of such a nature that they may reasonably be inter- preted as due primarily to suppressed or abnormal development. TRANSMISSION OF INDUCED EYE-DEFECTS 195 TABLE 14 Experiment 24 I. Sensitization of fowls to testis of rabbit DATE—1919 Re erent MATERIAL USED poe onl DOES eo ae cc. cc. September 16 3 Testes of two adults 12 4 September 23 3 Testes of two adults 12 4 October 1 3 Testes of two adults 12 4 October 9 3 Testes of two adults 12 4 October 16 3 Testes of two adults 12 4 Il. Treatment of rabbits with serum sensitized to testis IDENTIFI eaters ee er) ee mEMARKS OF RABBITS eee, + October 28 17 13 4.0 Mating, 17 9 X 16A2¢ 16A1 13 4.0 | Mating, 16A1 2 X 23A2 o" 36 13 4.0 | Mating, 36 9 X23Alc 37 10 4.0 Mating, 37 2 xX 16A2¢ October 30 i? 15 4.0 16A1 15 4.0 36 15 4.0 37 12 4.0 November 1 17 aly, 5.0 16A1 17 5.0 36 17 5.0 37 14 5.0 November 4 17 20 3.0 16A1 died in convulsions 4 hours 16A1 20 3.0 after 4th injection; 8 uterine 36 20 3.0 young 37 17 3.0 November 6 IL? 22 2a5 17 bore 7 normal young, Novem- 36 22 2.5 ber 15; 36 bore 3 normal 37 19 2.5 young; November 15; 37 bore 3 young, November 19, which died next day 196 M. F. GUYER AND E. A. SMITH of the crystalline lens. Inasmuch as the lens is relatively large in the eye of the rabbit, it seems legitimate to infer that the small size of the affected eyes, recorded in a number of cases, is due primarily to total or partial inhibition of growth of the lens. And since in its origin the lens is concerned so intimately, both mechanically and probably also chemically, with changes in the optic cup, it is not unreasonable to attribute such malformations as open choroid fissure resulting in cleft iris (coloboma), irregular- ities in distribution of retinal blood-vessels and of blood-vessels of the eyeball, abnormal postures of the eyeball, and flattening of the eyeball, as likewise due primarily to initial abnormali- ties in the lens. The occasional persistence of the hyaloid artery obviously points to an arrest of development of the whole lens apparatus at a well-recognized stage of its formation, since this artery together with a plexus of blood-vessels which invests the lens, though normal structures at one period of lens development, should atrophy and disappear once it is constructed. One other fact should be mentioned, namely, that in pulping and injecting the lenses, undoubtedly small amounts of the aqueous and of the vitreous humors were carried over also since no special effort was made to eliminate every trace of them. It is probable, therefore, that the sensitized fowl sera included to some extent antibodies for these humors. ‘This opens up the possibility that they, too, may have played some part in the eye deformations, although we have had no visible evidence that such was the case. The one central phenomenon in all the eye defects is the opacity of the lens—sometimes homogeneous, some- times pebbly, sometimes flaky in appearance—together with its diminution in size. The first thought that occurs to the embryologist, of course, is that perhaps the abnormal condition is due to just a general poisonous or inhibitive effect of a foreign serum upon the devel- oping fetus or upon a specially sensitive part of it, theeye. From the well-known work of Stockard (’09, ’14) and others, the eye, at its inception at least, is known to be particularly susceptible to deleterious chemical influences. In the present instance, how- ever, if the effect is a general one, then it should be as readily TRANSMISSION OF INDUCED EYE-DEFECTS 197 obtained through the use of unsensitized fowl serum or of fowl serum sensitized to other rabbit tissues than crystalline lens, as by means of fowl serum containing rabbit lens antibodies. But when our experiments with fowl serum sensitized to rab- bit crystalline lens are compared with our controls, it will be noted that eye defect appeared only in the young of mothers injected with the serum sensitized to rabbit lens. Leaving out of account the uterine dead, aborted young, and young which died RR 2 23AL LIAR LIAB RIA4 =—RBAS | 23A6 defective d d EVE Of right side diéd Fig.4 Female 22, after mating with male 2, was treated with lens-sensitized fowl serum; all of the resulting young (23A series) were normal. When 23A5 and 23A6 were bred together, however, an individual with a defective right eye was born. See figure 5 for explanation of symbols. before the eyelids separated, but counting the young obtained in the experiments recorded in our earlier paper (718), 61 young were obtained from mothers treated during pregnancy with lens- sensitized serum. Of these, 4 had conspicuously defective eyes and 5 others had eyes sufficiently different from normal eyes to be recorded as abnormal, and therefore probably due to the effects of the sensitized serum. In one instance, although the immediate young of a mother treated with lens-sensitized serum seemed unaffected, they begot one young one with a defective right eye (fig. 4). From mothers treated with pure fowl serum 198 M. F. GUYER AND E. A. SMITH 12 living young were obtained, and from others treated with serum sensitized to rabbit testis, 36 were secured, or a total of 48 young which survived long enough to show the condition of their eyes. In not a single one of these 48 controls was there evidence of eye defect. As far as our experiments go, therefore, the interpretation is that the effect of the lens-sensitized serum is specific. As to why the lenses of the mothers into which the lens-sensi- tized serum was originally injected were not attacked, we have no further explanation to offer than the suggestion in our earlier paper that the lack of circulation of blood through the lenses of adults prevents the sensitized serum from reaching them in suf- ficient quantity to produce visible change. In the fetus the con- dition is very different. There, after the tenth day of develop- ment, the lens capsule is highly vascular, receiving blood from a specially developed branch (hyaloid artery) of the retinal ar- tery. This hyaloid artery, together with its plexus of blood- vessels surrounding the lens, normally disappears shortly before birth. PLACENTAL PENETRATION Yet another problem that requires attention is that of how the lens antibodies penetrate the placenta. From the study of F. R. Lillie (17) on the free-martin, it appears that in the case of two-sexed twins in cattle if the sex hormones of the male circulate in the female, the latter is transformed into a sterile free-martin. This happens only when secondary fusions of the chorions of the two individuals occurs, permitting direct anastomosis of the fetal circulation so that the blood of each may flow through the veins of the other. By implication therefore, without this direct connec- tion of blood-vessels the sex hormones of the male would presum- ably not reach the blood of the female fetus. It seems reason- able to suppose that as regards penetration of the placenta, sex hormones and ordinary antibodies or cytolysins would come in the same category. This may or may not be true. But it is possible that under normal conditions small quantities of sex hormones from the male do reach the blood of the female in the TRANSMISSION OF INDUCED EYE-DEFECTS 199 case of two-sexed twins, but that they are there neutralized by appropriate antibodies generated in the female fetus, and that it is only when the latter is overwhelmed by blood from the male through direct continuity of the blood-vessels that the antibodies are insufficient to accomplish neutralization. The condition known to exist in the blood of pregnant mothers for neutralizing, or better perhaps digesting, any placental fragments which may escape into the maternal blood-stream renders this hypothesis less far fetched than at first sight it might appear to be. However this may be, the situation as described by Lillie has ‘suggested to the authors the necessity of knowing more about the manner in which such substances as antibodies get through the placenta from mother to young. The junior author is already engaged in researches looking toward a solution of this problem. Whatever the means, it is obvious that, in general, antibodies can penetrate the placenta, since it is well known that induced immunity to various forms of bacterial infection are transmitted through the placenta to the uterine young. Also it has been ‘shown that such a foreign substance as madder when fed to preg- nant mothers will pass through the placenta and color the bones -of the fetal young. It is well established, moreover, that certain pathogenic agents may traverse the placenta and produce ante- natal infections. In our experiments, that all the young were not invaded, or that they were unequally invaded, or that being invaded some were more resistant than others to the influence of the antibodies, is evident from the fact that a very substantial majority of the total number of young obtained showed no specific effect of the treatments. It is not impossible that in the struggles of the mothers, slight breaking down of the walls of the placental blood- vessels occurred in some cases, permitting some direct flow of the maternal blood into the fetus. And it may be that only such fetuses got a sufficient amount of the lens antibodies to have their own lenses affected. But whatever the means, the important fact is that penetration was accomplished in some way, with the result that defective-eyed offspring were occasionally produced. SMITH GUYER AND E. A. M. F. 200 *pozdAyered ‘q fpeurtou ‘wu fporp ‘p faaroajop ofa VJO] ‘YoRlq J[ey yo] :eArjoosop oA9 ysis ‘YoRlq JlBy yYysIa faaAtoejop soko YJoq ‘Joquids Yyouyq [[B {savuUTa] *sazo -I10 [SO[VUI 9}BOIPUI SoIBNHgG “UINIOS [MOF POZI}ISUIS-SU9] OY} YIIM po}BaT} SBM TOIYM [VUII] OY} So}VOIpUI 10}UID SY Ul USIS + OY} YIIA ofot10 oY, ‘“WMOYS Orv JABYO dy} UI pojuosordod syeNpIAIpUL oy) Jo sBurpeur ay} Jo [je OJON “S[TBNPIATPUI poAd-9ATJOoJop UOIYV10UES YYXIS puv YYFY oy} Jo 9UlOS Jo voISIpod oy} SUIMOYS JABYO G BLT PALL IASID SIKI Y{O SYBE bY8E YB 2VEF oe EIA Go Pp PAP //0 SS SS OLDS OF 1¥Y82 #¥B?S 2VE2\EVEA wyg9 4VO/ DSi SUSt WV GROWS EWS Ib-S /VOn DVO YO troy _U 109 209 VbV9| ZkOl OVO GYOl 1VO/ zvo/ CYO/\FYO/ Pp aq IALLIIfJapP USAC a a ae a oe See Hed L G0. Ut VU waaay ig EUIZ oUuII 1U29 TRANSMISSION OF INDUCED EYE-DEFECTS 201 TRANSMISSION OF THE DEFECTS THROUGH BREEDING Perhaps the most interesting and important result of our ex- periments is the establishment of the fact that the defects, once secured, may be transmitted to subsequent generations through breeding. So far, we have succeeded in passing the condition to the sixth generation, and there seems to be no reason why it will not go on indefinitely, since the imperfection tends to become worse in succeeding generations and also to occur in a proportionally greater number of the young. At present we have thirty-seven living individuals with markedly abnormal eyes. Many more could have been secured if all the defective-eyed animals had been mated as frequently as possible. Up to the present, however, our chief aim has been to pass the defect through as many successive generations as possible. As an example of increased intensity of the defect in later generations, the case of a male (3A1, fig. 5; pls. 1 and 2) with a bad left eye may be cited. Many of his grandchildren had both eyes abnormal, culminating in two which never opened their eyes. Subsequent dissection of the latter showed that minute eyeballs were present under the closed lids. This progressive intensification of the defect was to be expected, perhaps, up to a certain limit, since close inbreeding was practiced. A glance at figure 5 shows that sometimes one, sometimes the other, and not infrequently both eyes were affected. This irregu- lar unilateral and bilateral transmission recalls the somewhat similar genetical histories of such deformities as polydactyly and brachydactyly. Little effort has been made so far to find out just what genetical factors are involved in the transmission of the defect. The abnormal condition has in general the characteristics of a Men- delian recessive. When defective-eyed males or females are bred to normal-eyed individuals from other stock, for instance, only normal-eyed. progeny result in the ensuing generation. But the defect may be made to reappear in subsequent generations if appropriate matings are made. A good example of this is found THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 31, NO. 2 202 M. F. GUYER AND E. A. SMITH in the male-line experiments shown in figures 6 and 7. Again, two apparently normal-eyed individuals from the defective line have had bad-eyed offspring. But on the other hand, two 123 12B2, killed LL ih ee Len VO Ae sex undelermmed HWA WAS. HAaKAAYAHAS HAN\ 2EA4 43Al Y3SAL4Y3AS 43A4 Fig.6 Inheritance of the defect through the male line. Symbols same as in figure 5. defective-eyed individuals (fig. 5) may have what appear to be normal-eyed young, so that on a strictly Mendelian interpreta- tion we should have to suppose that heterozygotes sometimes TRANSMISSION OF INDUCED EYE-DEFECTS 203 show the defects or, in other words, that normality is not always dominant. Such ‘reversed’ dominance, however, is by no means unknown in the annals of Mendelism. We are entering upon a series of matings to clear up this and other doubtful points con- nected with the exact mode of inheritance. For starting our investigation into the inheritance of the de- fect, the offspring of a female designated in our pedigree charts as no. | and a male, no. 2, were selected (fig. 5). This pair had already yielded a normal litter of five before they were used in the serum work. After being mated to no. 2, November 30, 1916, 39 JA/ J2EI B2B2 32B3 32BY SLB5 3ZAI RAZ 3B2A3 32A4 3LAS 32 AG Ms A A 46Al 4OAR HAS 4644 YOA5S 46A6 4OAT Fig. 7 Inheritance of the defect through the male line. Symbols same as in figure 5. the female had been injected with fowl serum sensitized with rab- bit lens. The details of this sensitization and the schedule of injections are given in our 1918 paper, page 73, table 2, in which this same female is designated as rabbit B. In the ensuing litter, born December 30, was a male with a markedly defective left eye which in time almost entirely disappeared. In order to find out whether or not this defect could be transmitted to the next generation, this male, numbered 3A1, was mated to his sister 3A2, whose eyes were normal as far as could be ascertained from an external examination (fig. 5). The offspring, known as the 4A series, born November, 1917, showed surprising results, for three females from the litter of eight young had abnormal eyes. In 204 M. F. GUYER AND E. A. SMITH two (4A4, 4A5) the defect was on the left side as it had been in the father, while in the third (4A1) it was the right eye that showed the abnormality. In the right eye of 4A1 the iris, al- most transparent, was interrupted below (coloboma) and did not expand or contract (pl. 1, 4A1). The eye as a whole was smaller than the left eye. The lens likewise was smaller and was opaque, . causing the peculiar silvery hue already described. ‘These same defects appeared in the left eye of 4A4 and of 4A5 (pl. 1, 4A5). Another litter (fig. 5), 4B series, from the same parentage was born March 14, 1918. One female, 4B1, had a left eye like her father with no trace of iris or pupil (pl. 1, 4B1). The eyeball was so small and collapsed that the condition of the lens could not be determined. The eyes of the remaining five (four males and one female) were normal in size and appearance. The female, 3A2, was next bred to a male from normal stock, and on July 26, 1918, gave birth to six normal-eyed young, the 4C series. When bred to another normal male, she again, on December 24, 1918, produced six young in which the eyes showed no abnormalities (4D series). Finally she was again mated to her brother 3A1, and on May 5, 1919, brought forth eight young. One in this litter, known as 4E1, had both eyes defective, but it died before the sex was determined. It will be noted that each of the three separate times 3A1 and 3A2 were bred together some young with abnormal eyes were obtained. In all, from this pair, a total of twenty-two offspring were secured. Of these, seventeen had eyes which appeared to be normal and five had eyes which were defective. This is about as near to the 3:1 Mendelian ratio, obtained through the breeding of two heterozygotes, as can be approximated in twenty- - two individuals. The female parent, however, showed no evi- dence of eye defect. When she was bred into normal strains the immediate young were always normal-eyed. We have not as yet tried to extract the defect from her normal-eyed progeny. It may be mentioned in this connection, although the details are not discussed till later, that the male of this pair, 3A1, was re- peatedly bred into normal strains and always yielded normal- eyed young, but we have extracted the defect again from this TRANSMISSION OF INDUCED EYE-DEFECTS 205 line, both by mating back to 3A1 and by mating to another de- fective-eyed male (figs. 6 and 7). To return to the 4A series, some of the females of which had meanwhile been bred back to 3A1 or to their brothers. On April 26, 1918, 4A5 (left eye defective) produced six young sired by 4A8, a normal-eyed brother (fig. 5). Two died immediately. ' Of the remaining four (6A series) three were females; 6A2 (pl. 1) had colobomaand opaque lensesin both eyes; 6A3 had coloboma in the right eye only and the lens had an opaque rim; in 6A4 (pl. 1) the left eye was much smaller with the eyeball rotated toward the front leaving only a small part of the pupil visible through which an opaque lens could be seen. In 6A1, (pl. 1), the male, each eye had coloboma and an opaque lens. Next, 445 was bred back to her father 3A1. From this mating four young were born December 28, 1918. In this litter (fig. 5) one female, 6B (pl. 4), had a right eye about one-fourth normal size and the iris, incomplete toward the corner, did not expand or contract; the lens was clouded. The left eye had a normal pupillary reflex, but the iris was more translucent than normal and the margin of the lens was milky. The eyes of the other three were apparently normal. Another female, 4A1 (pl. 1), when mated to 3A1 (fig. 5) gave birth to five young June 21, 1918. Of this litter, 10A1 (pl. 1), a female, had coloboma and an opaque lens in the left eye, while the right eye outwardly normal contained a slight flaw in the lens; 10A2 (pl. 1), a male, had the left eye similar to 10A1 except for a larger pupil, and the right eye very small and peculiar in color; 10A3 (pl. 1), a female, had both eyes small with irises in- complete and lenses clouded; 10A4 (pl. 1), a male, had eyes like 10A3 until he was half grown, but later the left eye collapsed so that no detail could be made out in it; 10A5, a male, with eyes apparently normal had the hind legs completely paralyzed and died on August 19, 1918. However, it should be noted that the eyes of 10A5 were never examined with the ophthalmoscope and such examination has revealed cloudy or flawed lenses in some eyes which outwardly appeared normal. 206 M. F. GUYER AND E. A. SMITH Next, 4A1, was bred to her brother 4A8 (this mating not shown in fig. 5) and her progeny of six born December 27, 1918, were allnormal. Lastly, she was mated again to 3A1, and on July 17, 1919, brought forth seven young, the 10C series. One died im- mediately. Of the six remaining, five with normal eyes lived and one with both eyes defective died. To continue the history of the 4A series, 442, a normal-eyed | female, was first bred to 4A7 (eyes normal) and gave birth to five normal young on May 7, 1918. Then she was mated to 4 A8 (eyes normal), and on December 11, 1918, produced five young which were likewise normal. After being bred to 4A7 on June 3, 1919, she aborted one young. None of these matings are shown in the chart (fig. 5). Another normal-eyed female, 446, when mated to 3A1, had a litter of eight normal young on June 9, 1918. However, when she was mated to her brother 4A7, a normal-eyed male, one male, 8B, from the litter of five born December 27, 1918, had the left eye normal in color and structure but small and a right eye with a translucent iris and cloudy lens (fig. 5). A female, 444, with a defective left eye also was mated to 3A1 and gave birth to eight normal young on June 10, 1918. Previously (April 4, 1918) she had also borne two normal-eyed young, although they had been fathered by 6A1, a male with both eyes defective. The defective-eyed offspring from the 4A series consists of eleven individuals: males 6A1, 10A4, 10A2, and 8B; females 6A2, 6A3, 6A4, 6B, 10A1, and 10A3; 10C with sex undetermined. Passing now to the 4B series, one female, 4B1 (fig. 5) with the left eye collapsed was mated to 3A1 and produced four young on December 12, 1918. In one female, 27A, the right eye was exceedingly small, while the cornea and lens were both opaque. Only one of the 4E series had defective eyes and it died before it was old enough to leave progeny. Altogether, then, twelve defective-eyed rabbits were produced in the fourth generation; four males, one with sex undetermined, and seven females. In two females, four males, and the one in which the sex was unde- termined, both eyes were affected, while in two females the left eye was abnorml and in three females, the right eye. TRANSMISSION OF INDUCED EYE-DEFECTS 207. Offspring from the fourth generation are chiefly from matings between the 10A and 6A series, hence each series cannot be con- sidered separately. The female 6A2 in which both eyes were defective was mated to 10A4 (both eyes abnormal), and on April 5, 1919, bore seven young, all of which died between July 20 and July 28, 1919. However, four of the lot had eye defects (fig. 5). One female had a bad left eye and two males and one female had both eyes defective. Of the males one never opened his eyes, but a postmortem held after his death on May 7th revealed small eyeballs under the shut lids. a TRANSMISSION OF INDUCED EYE-DEFECTS PLATE 2 M. F. GUYER AND E. A. SMITH PLATE 3 EXPLANATION OF FIGURES Show ing the left (top picture) and right. eyes of individual O8A 4 (text pie 5). ‘The left eye is slightly smaller than normal size; its iris is complete and is fue tional; the lens, however, is so opaque as to be seen easily without the aid of an — ‘Ophenalnoscope. The right eye, noticeably reduced in size, has an opaque lens — and a cleft iris. ais ) 220 TRANSMISSION OF INDUCED EYE-DEFECTS PLATE 3 M. F. GUYER AND E. A. SMITH 221 PLATE 4 “ EXPLANATION OF FIGURES: It is markedly reduced _ Showing the. right eye of female 6B (text figure 5). Sits in size (compare with the normal eye of 3A1, pl. 2) and is otherwise defective. the lower picture the lids are being separated to 0 show the iris which is incom- plete below. Ct te * * a 222 TRANSMISSION OF INDUCED EYE-DEFECTS PLATE 4 M. F. GUYER AND E. A. SMITH Resumen por el autor, George H. Bishop. Universidad de Wisconsin. La fecundaci6on en la abeja. I. Los 6rganos sexuales masculinos, su estructura histolégica y funcionamiento. Los cambios que tienen lugar en la estructura histol6gica del aparato sexual mesodérmico en el zingano recién salido del huevo, indican que los zinganos jOvenes no pueden fecundar a las reinas a causa del estado no maduro de dichos 6rganos sexuales durante un periodo de nueve dias por lo menos. Los espermatozoides y el mucus permanecen en la vesfcula seminal y el reservorio gland- ular mucoso, respectivamente, hasta el momento de la eyacula- cidn. Los espermatozoides se insertan por sus cabezas en la pared de la vesicula seminal, cuya drea superficial aumenta a causa de la formacién de surcos y pliegues alternados que se dis- ponen en espiral alrededor de su cavidad. La cavidad del con- ducto eyaculador ectodérmico, que se forma por invaginacién del extremo anterior del pene, no se abre en la de la porcién meso- dérmica del aparato hasta que su extremo ciego quitinoso re- vienta al pasar los fluidos espermaticos. La musculatura de la base de la glandula mucosa estd dispuesta de tal modo que su contraccién bajo la accién del estfmulo eyaculador separa esta region basal del reservorio mucoso distal, permitiendo el paso de los espermatozoides procedentes de la vesicula seminal a tra- vés de la base de la glindula y desde aqui al exterior por el con- ducto eyaculador. Durante la relajacién que sigue a la primera contraccién espasmédica, el mucus sigue al esperma, a causa de la presién ejercida por la contraccién abdominal, de tal modo que obliga a penetrar a todo el esperma en los 6rganos femeni- nos. Después se coagula en contacto con el aire, cuando el pene se desprende del zingano. Varios estimulos artificiales causan una eyaculacién normal en apariencia, y los mas seguros son la inyeccién de un dcido débil en el térax y la decapitacién durante la huida. La estructura de los 6rganos, naturaleza de los li- quidos, y funcionamiento del aparato bajo la influencia de estti- mulos artificiales indican un papel diferente de los espermato- zoides y mucus durante la copulaci6n. Translation by José F. Nonidez Cornell University Medical College, N. Y. AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, APRIL 19 FERTILIZATION IN THE HONEY-BEE I. THE MALE SEXUAL ORGANS: THEIR HISTOLOGICAL STRUCTURE AND PHYSIOLOGICAL FUNCTIONING GEO. H. BISHOP Zoological Laboratories of the University of Wisconsin THREE TEXT FIGURES AND THREE PLATES CONTENTS ire BE GOCAULC COM cate ar tte arch oie aie o.fue ie < ceois, snofee oiaids as « 0/60 abe ebay ofeain ae 225 A StOLIC alleen tte Oa Tals sree spacers ir e's aitiorovalaiaths, efesis our oo nie eiclavonctehete serene 227 TPESCEIP TOM Oe eMAle ONe AMS 5/7) LEP ic RID Wo at led owes cies ain be + shaunaipienn aes 228 rcaeraiy Athi sil MUO 147. .odcrscrhiridk,s aches ize fiete bla atel dada etw oa d'os. o's olde gees 233 Pree Mins 1) Coy rea TTC TRNAS oy ole nr oioyig Sahay ia) Rc eyetes chase =i-tb in Ge) o ah snel6 eke. Zs ever eyeNegn gt owe 234. ene RAT UCIIE MEIN oioeranavs crise stiniaa dt aie eee «5.0 8 2 was» se erie 234 ZaMuselevliayers!. <2 4502 oe ete NF e SEEM Raabe arene tals Oi ae ast onde Milela ulate 239 Me SCTE LORY. CUCL :-thre Be tA DISET » Sie AE ple Clete dete bac ONS A Ne, Sale Dale ane ate 245 1D, IPInOlOpay om bos nap ate ObCb HO rome oon e ROneL DO OTC COO DO toonmioona oc 246 i CCE VLOG aiatelat 5 ot ciate eo Hore lel lcinroyelorn oe eigen Mae lnitskel din cnn ale Saunier 246 2. Correlation between age and functioning................+..0+- 249 By Ii Grav onillenaoranrornohyovectan Guna acedaoues sapoagnabeuasien 4 bnaoonte .. 250 (Cone icine ity Tyee Be 3 See io ot Ot ete CO TIaC CEM EMO no acne narrates ack 256 STOTT 9 ESS Ais OURS D COEDS OO Coe COD DUCA O OOD nH EIR ratio Orne Mipiche aa cee 257 INTRODUCTORY The reproductive mechanism of the male honey-bee has been so often and so variously studied that one feels called upon to state at once the occasion for its further investigation. The following study developed out of a series of unsuccessful attempts at the artificial fertilization of queen bees. Through a number of seasons this had been attempted by the methods which have been described by others as well as with newly devised apparatus. Two general classes of attempts were made. In the first, queen and drone were held in juxtaposition and the extrusion of the drone’s organ brought about by pressure on the abdomen. In the second class of experiments, the seminal fluid of the drone was dissected out and injected with a pipette into 225 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 31, NO. 2 226 GEO. H. BISHOP the organs of the queen. From the first experiments (juxta- position of queen and drone) it became more and more evident that extrusion of the drone’s organ caused by artificial means did not necessarily, nor generally, duplicate the natural act of copulation, even when it seemed to do so. For the second (injection) experiments, it became necessary to know both what the character and functions of the components of the male spermatic fluid were and what disposal was made of them during and after the normal act of copulation. Finally, copulation in the bee has been witnessed so rarely and can be observed directly with such difficulty that a study of the structure and functioning of the reproductive organs is the most hopeful avenue of approach to the problem of fertilization in the honey-bee. Thus, while the anatomy of these organs has been worked over repeatedly, the physiological and functional aspect of fertilization in the bee has received inadequate attention. To. obtain the further data, regarding the functioning of the drone’s organs, which seem a prerequisite to success in artificial matings, and to investigate the physiology and the mechanics of fertilization in the bee, work has been conducted along the following lines: Histological and anatomical study of the drone organs and their respective secretions, by means of dissections, paraffin sections, and stained whole mounts and hemisections, mounted in balsam. Manipulation of drones to cause extrusion of the penis, with ejaculation of the spermatic fluid; in the attempt to produce artificially a complete physiological duplication of the results of normal copulation. The histological work was commenced in 1915 under Prof. Trevor Kincaid at the University of the State of Washington; after several years of independent and unsystematic attempts to induce controlled copulation between queens and drones by mechanical stimuli and technique. It was continued during the next two years in the zoological laboratories of the University of Wisconsin. Doctor Marshall of that laboratory has given particularly valuable help, not only by way of advice, but by assisting in those operations that required the attention of more FERTILIZATION IN THE HONEY-BEE D7 than one person. Preparation for publication has been delayed by absence occasioned by the war. HISTORICAL Three papers! deal specifically with the development and histology of the drone sexual apparatus, and a larger number treat more or less comprehensively the gross anatomy, especially of the copulatory organ itself. The work of Bresslau‘ on the spermatheca of the queen and the accompanying mechanism has been checked by Zander® in a general account of the develop- ment of the organs in both male and female forms. Mating flights have been but rarely observed, and only incidentally, by bee-keepers, etc., and reported in their professional journals.® 7 Mating experiments have been reported frequently, but the very few cases of artificial or controlled matings reported as successful have not been sufficiently checked. ‘There seems in this work’ to have been little attempt to take into consideration more than the superficial morphology with which the anatomical studies referred to have made us familiar.® 1 Koschevnikov, G. Zur Anatomie der mainnlichen Geschlechtsorgane der Honigbiene. Zool. Anzeiger, Bd. 14, 1891. * Michaelis, Geo. Bau und Entwickelung des mainnlichen Begattungsapparat der Honigbiene. Zeit. fur wissen. Zool., Bd. 67, 1900. 3 Snodgrass, R. E. Anatomy of the honey-bee. U.S. Dept. of Agriculture, Bureau of Entomology, Technical Series 18, 1910. 4 Bresslau, Ernst. Der Samenblasengang der Bienenkiénigin. Zool. Anzeiger, Bd. 29, 1905. 5 Zander, Enoch. Die Ausbildung des Geschlechts bei der Honigbiene, Zeit. der angewandten Entomologie, Bd. 3, 1916. § Shuck, 8. A. Note in American Bee Journal, 1882, p. 789. 7 Pratt, E. A. Note in A BC and X Y Z of bee culture. A. I. Root Co. 8 Shafer, Geo. D. A study of the factors which govern mating in the honey- bee. Michigan Agr. College Exp. Sta., Div. of Entomology, Technical Bulletin 34, 1917. This bulletin furnishes an exception to the above statement. Concerned pri- marily with artificial fertilization experiments, it describes the superficial ap- pearance of the queen’s organs (oviducts) after normal copulation and of the drone’s organs after extrusion of the penis has been brought about by pressure; there is also speculation on the nature of the stimulus that causes extrusion of the drone’s organ in natural and in artificial conditions. He includes as well a valuable bibliography of experiments on artificial and controlled matings of drone and queen bees. ' 228 GEO. H. BISHOP A detailed consideration of this literature, even of that part of it which deals specifically with the organ of copulation, will not be undertaken here. The morphology of the organs and the complex adaptations for mating have been adequately descr.bed, and in general there is no serious disagreement as to the position of the insects or the relation of their organs in copulation. How- ever, the physiology of the process has been almost entirely ignored. The functioning of the ‘mucous gland’ has received little more than speculative attention; the disposal of the sperm in the queen’s organs has scarcely aroused curiosity, and the intricacies of functioning of the internal sexual organs of the drone seem to have escaped notice for the most part. The present paper is rather an attempt to supplement the morpho- logical data with physiological, than to controvert the facts established. The papers above cited are therefore not of im- ‘mediate bearing on the work under consideration, other than as a point of departure. For a detailed description of the anatomy of the sexual apparatus the reader may be referred to any of the more recent papers (as Snodgrass). A brief and general summary will suffice to present the anatomical picture necessary to an understanding of the work which follows. DESCRIPTION OF THE MALE ORGANS The mating flight of the queen bee takes place at least five days after the emergence of the imago, and probably ten or more days after the emergence of the drone. The rapidly flying insects meet inthe air, the drones in pursuit. According to the reports of eye witnesses and to the evidence from examination of the drone organ left in the queen’s vagina after copulation, they clasp face to face and drop at once to the ground. The drone is stunned and soon dies. The queen twists the drone organ in two, by flying or crawling in a circle around the drone, retaining the portion broken off. This gradually dries up, and is pulled away by the bees in the hive some hours after the queen has returned thither. | FERTILIZATION IN THE HONEY-BEE 229 The penis of the drone (text fig. 1, a) is elaborately adapted to this manner of mating. It is a hollow tube, ectodermal in origin, non-muscular, growing by invagination from the ninth segment of the abdomen. Three main functional regions can be identified; the penis tube proper (a), the enlarged bulb at its anterior end (6), and the ejaculatory duct (c), leading from the bulb to the mesodermal sexual organs. The penis tube, proximal to the external opening, is of relatively large diameter; Text fig. 1 Diagram of one half of drone sexual apparatus, viewed from the medial side, showing the unpaired penis and ejaculatory duct, and the right members of the paired mucous glands, seminal vesicles, and testes of a mature drone. Internal anatomy of base of gland and vesicle shown as in optical sec- tion. The parts have been slightly displaced in mounting on the slide, in order to view them all in the horizontal plane; i.e., the anterior portion of the ejacu- latory duct lies normally between the two glands, not below one of them; the tip of the other gland, joining the one figured at 7’, extends dorsally at about right angles to this one, and not in the horizontal plane with it, and the vas deferens at h bends around the base of the gland so as to bring the seminal vesicle lateral to the gland rather than dorsal. a, penis proper; b, bulb of penis; c, ejaculatory duct; d, body of gland; e, seminal vesicle; f, vas deferens; g, testis; h, lower vas deferens leading to basal transverse pocket of gland, 7, to which is applied the base of the cone-shaped end of the ejaculatory duct, 7; k, slender muscle attaching the gland to the posterior abdominal wall; 1, valve of muscle covered with glandular epithelium, which guards and partially surrounds the orifice of the vas deferens and partially divides the lumen of the gland proper from the basal pocket into which opens the vas deferens, and which upon contraction of the gland’s muscu- lature divides these regions completely; m, that portion of the gland’s lumen which pushes out into the angle of the gland dorsal to the opening of the vas deferens; n, blind end of the ejaculatory duct. 230 GEO. H. BISHOP it has a fairly stiff but elastic chitinized wall, and bears a series of complexly modified plates, bristles, and protrusions which appear to facilitate its entrance into, and secure its retention within, the vagina of the female. ‘The medial portion, the bulb, is merely an enlarged and rounded part of this tube; it is on either side partially enclosed by a lateral shell-like plate, formed by the chitinous thickening of the wall of the bulb. This bulb tapers off into the third portion, the ejaculatory duct, a thin-walled, elastic, narrow-lumened tube leading to the seminal vesicles and the accessory glands (text fig. 1, d and e). . In copulation, this apparatus is everted from the drone’s body into the vagina of the female. Since the penis itself has no muscles attached, its eversion is due to pressure from the muscular contraction of the abdominal walls. Starting at the region proximal to the genital aperture, the penis is gradually forced out from within, as one might force out a glove finger that had been turned inside out in stripping off, by blowing into the wrist of the glove. The eversion extends, according to Zander,’ back to the median bulb, which, acting as a sper- matophore, is kept from everting by its two lateral plates above mentioned (text fig. 2, B). Schafer’ finds that the bulb also everts and concludes that it does not act as a spermatophore, but that its size merely enables these lateral plates, whose definite function is to hold the penis within the queen’s organs, to turn inside out and lodge in their appropriate position like the gates of a canal lock (text fig. 2, C). The entrance of the ejaculatory duct into the bulb, according to this scheme, is thus brought through the everted bulb, and becomes the end of the everted penis. Either condition (B or C) may be produced artificially by greater or less pressure applied to the drone’s abdomen. The mesodermal portion of the sexual apparatus (text fig. 1, d-h) consists of three elements: 1) Paired testes, at the time of emergence of the imago, occupy a large part of the dorsal portion of the abdominal cavity; they undergo gradual diminution as the sperms are discharged, until at maturity only small triangular remnants remain (text fig. 1, g). 2) Passing posteriorly from FERTILIZATION IN THE HONEY-BEE 231 each of these, leads a vas deferens, proximal to the testis sharply coiled (f), and distally expanded into a seminal vesicle (e). 3) Distal (posterior) to the vesicle again, the vas deferens curves sharply (h) to enter the third element, the accessory gland (d). This organ extends anteriorly to a region slightly beyond the testis, the rudiment of which in the mature drone is usually applied dorsally to the tip of the gland (text fig. 1, g). Text fig. 2 Diagrams of drone’s organs, showing uneverted, partially everted, and completely everted relationships of the various portions, in A, B, and C, respectively. a, posterior or proximal tubular portion of the penis with modi- fications for facilitating copulation, e, f, g; b, bulb portion of penis, with lateral chitinous plates shown in black; c’, proximal end of ejaculatory duct c, expanded where it joins the bulb of the penis; d, the mucous glands. For further explana- tions see text. In development, each vas deferens grows back from the testis sheath until it meets, ventrally, a cup-like invagination of the ninth segment of the abdomen which is to form the penis and the ejaculatory duct. The vas deferens fundament then curves back on itself to form a hook like the letter J, later, becoming a U. The recurved tip of the J forms the mucous gland; the stem the seminal vesicle. A branch of the ejaculatory duct penetrates 232 GEO. H. BISHOP the lower portion of the U, at the base of the gland, thus uniting the ectodermal and mesodermal parts. Zander notes that the lumen of the ejaculatory duct does not become continuous with that of the gland until the contained fluids burst through the thin partition at the time of emptying of the secretions (see diagrams, pl. 1, and pl. 3, figs. 7-10). (‘Die Beriihrungstelle, an der beide Kanalsysteme [of duct and gland], anscheinend bis zur Samenentleerung blind aneinander stossen,”’ etc.) He leads one to infer that at the maturity of the drone (text fig. 2, A, b) the bulb of the penis, acting as a spermatophore, receives this secre- tion. Shafer, without noting this partition, infers that the sperms remain in the vesicle or the base of the gland until copu- lation, and do not pass into the penis bulb, but at the time of copulation are carried through the bulb in the ejaculatory duct (text fig. 2, C). The accessory or mucous gland (text fig. 1, d, and pl. 1), developing from the blind recurved end of the vas deferens fundament, enlarges into a gourd-shaped body, lined with columnar glandular epithelium and enclosed by three muscle layers. These layers are an external longitudinal, a medial circular, and an inner layer which consists of three longitudinal bundles of fibers, extending from the base of the gland more than half way to its tip. The musculature is heaviest at the base, 1e., around the entrance of the ejaculatory duct, and attenuates toward the distal end. The whole is enveloped by a thin structureless membrane well supplied with tracheae. As the gland’s lumen becomes filled with the secreted mucus, its distal end assumes a bulbous contour. The three muscle tracts of the inner layer cause an infolding of the glandular lining of the organ into three corresponding ridges, giving a cross-section of the lumen the shape of a clover leaf (pl. 1, fig. 3). The seminal vesicle (text fig. 1, e, and pl. 1, e), like the gland, is lined with glandular epithelium, here thrown into ridges (KXoschevnikov, “in Ringwalzen eingereiht”’). There are two muscle layers surrounding it, an outer longitudinal and an inner circular layer. These correspond to the outer two of the three layers of muscle of the mucous gland. A membranous envelope FERTILIZATION IN THE HONEY-BEE Das covers the vesicle; this is continuous with the envelope of the gland on one side and of the testis on the other. Sperms, passing into the vesicle, tend to arrange themselves radially in its lumen, their heads attached to the wall, their free filaments toward the center (pl. 2, figs. 5, 5b). The testes, which mature their sperm some days before the emergence of the drone, and at this time occupy most of the abdominal cavity, rapidly de- generate thereafter; in old drones they are noticeable only as small greenish-yellow remnants applied dorsally to the accessory glands. PRESENT INVESTIGATION Attempts to obtain motile sperms from drones, by dissection or otherwise, demonstrate that they are not available in all drones. This fact has been variously interpreted. McLain? inferred that there were three classes of drones. One class yielded no spermatic fluid when extrusion of the penis was brought about by compressing the abdomen. A second class yielded only mucus from the accessory gland. A third yielded a seminal fluid containing sperm. Shafer,’ in discussing McLain’s work, agrees with him that the food which drones receive at mating time is important as a stimulant to the copulatory impulse. The writer sought to correlate the observed facts of McLain with the known fact that young drones (younger than an age variously stated to be from ten to twenty-one days) will not mate with queens. Drones of different ages were selected for study, ranging from pupae whose eyes were just becoming pigmented to mature insects three weeks after emergence. These stages are designated in the present paper by the letters A to H (table, p. 252). The fact became immediately apparent that a definite and complicated histological development and growth of the organs, rather than a special food stimulant, was involved in the differ- ence of functioning observed. This development takes place ®McLain. Description of experiments on artificial insemination of queen bees, in report of the entomologist, on Experiments in Apiculture, U. S. Com- missioner of Agriculture’s Report for 1885. 234 GEO. H. BISHOP after the drone is of superficially mature appearance, and con- tinues to the fifth day of imaginal life at least though the drone is probably not functional until later (table, p. 252) The contents of the organs, their mode of responding to such stimuli as cause extrusion of the penis, and the motility of the sperm are all correlated specifically with the age of the drone. The age can be determined by superficial examination of the degree of degener- ation of the testis. This undergoes a gradual shrinkage, and changes from a creamy-yellow color and bean-like shape, of 5 mm. length, to greenish-yellow color, flat triangular shape, and 1.5 mm. length. This change in the testis is a convenient measure of the histological development, size, and maturity of the other organs. A. Histology and anatomy of male organs The vas deferens, seminal vesicle, and mucous gland are derived from a common fundament; when mature they are histologically similar; they perform their functions of secretion and contraction in essentially the same manner. A detailed description of the histological elements and of their physiological characteristics may apply, therefore, to all the parts derived from this fundament taken together. The two histological elements chiefly concerned here are the columnar cells of the glandular epithelium which forms the continuous wall of the lumen of these organs and the muscle layers that continuously envelop them. Concerning the glandu- lar cells, the important considerations are the manner and nature of their secretion and the qualities of the product; concerning the muscle layers, their arrangement and the effect of their contraction upon the contents of the organs at different stages of development. 1. The glandular epitheium. The glandular cells (pl. 2, figs. 5a, 6a) are extremely long and narrow, twenty or thirty times as long as thick, with oval nuclei, one to each cell, scattered through the basal half of the cell layer. The nuclei are so large relatively to the diameter of the cells that the oval form may be assigned to compression by the cell walls. Their scattered FERTILIZATION IN THE HONEY-BEE 2a arrangement seems to be due to the fact that the nuclei bulge the walls slightly outward, and force nuclei of adjacent cells alternately upward and downward. The chromatin of the nuclei is mostly in three to five granules, the rest faintly scattering through the clear plasma. The chromatin stains more densely — while secretion is taking place, and shrinks and takes the stain less densely when it ceases, but its disposition in the nucleus does not appear to alter. The cytoplasm is very finely granular (after fixation), slightly more densely staining around the nuclei, especially when the cells are functionally active after growth is complete. The distal ends of the cells contain larger and denser staining granules that give, in (cross-) section of the epi- thelium, the appearance of a dense granular band (pl. 2, figs. 5a, and 6a). The cells of the glandular epithelium are modified according to the region of the lumen which they line. The cells lining the vas deferens between testis and seminal vesicle (text fig. 1, f) . and those between the vesicle and the mucous gland (h) are more cubical than columnar. Here the nuclei are placed more evenly side by side and have a more founded outline, but the character- istic structure and mode of secretion of the.cells is identical. The cells of the seminal vesicle are about a half shorter than those of the gland, their nuclei are smaller and similarly disposed. Secretion takes place by strangulation, with dissolution of the cell substance (pl. 2, figs. 5 and 6b). The dense granular area at the tip of the cell widens, the granules increase in size, in refractiveness, and in density of staining, and finally vacuoles may appear among them. The end of the cell rounds up into a globule of secretion, which sloughs off into the lumen of the organ. ‘This process is most pronounced in the gland, where the secretion retains its coarse granular character. These granules are transformed to highly refractive globules of some- what larger size, as if by absorption of some of the fluid; the mass of the secretion at the same time becomes more viscous. In the seminal vesicle the granules are smaller and soon dissolve to a pale plasma (pl. 2, figs. 5 and 5b). In the narrow portions of the vas deferens, at either end of the vesicle, neither the granu- 236 GEO. H. BISHOP lation nor the strangulation are apparent. This is possibly owing to the slowness of secretion and the small degree of dissolution of these short cells. In the distal region of the vas deferens adjacent to the gland, the lumen does not always, as elsewhere, become clearly defined, but may remain loosely stopped with a network of strands and membranes which appear to be remnants of the walls of cells that filled this space (pl. 3, fig. 10, and text fig. 1, h). These cells become shortened inside their former membranes, to form a thin epithelium against the muscular layer enclosing the vas deferens. The cytoplasm is dense, the nuclei shrunken. The picture closely resembles the final ap- pearance of the basal portion of the gland into which this portion of the vas deferens serves to conduct the sperm (pl. 3, fig. 10). As the drone approaches sexual maturity, this process of secretion and reduction of the glandular epithelium commences in the tightly coiled epididymis-like portion of the vas deferens leading from the testis (text fig. 1, f). It progresses from the tips of the cells back to the bases (pl. 2, figs. 5, 5 a, 5 b), and in the vas deferens as a whole, from the testis posteriorly through the seiminal vesicle. Shortly after the stage at which the cells lining the seminal vesicle start secreting, the cells lining the mucous gland commence to break down into secretion in the anterior end of the gland. The change progresses posteriorly again. Thus the cavities of these organs are enlarged through dissolution of their walls. This occurs earliest anteriorly, affecting last the posterior regions where the contents of both organs are to be evacuated into the ejaculatory duct (text fig. 1, h and 7). When this process has reached an advanced stage, it leaves the walls of the organs characteristically sculptured. In the gland (pl. 1, figs. C, D, EZ) the cells entirely disappear anteriorly, leaving a very thin membranous bulb-like sac which expands with mucous secretion. Posteriorly, the cell nuclei recede toward the basal region of the cells, the chromatin shrinks, the cytoplasm becomes heavily vacuolated, and the ends of the cells protrude into the gland’s lumen in fringed and ragged patches (pl. 3, fig. 10, m). Vacuolization at the bases of the cells often appears to push whole areas of the cells out into the lumen, leaving their FERTILIZATION IN THE HONEY-BEE 237 bases attached to the muscle layer by attenuated remnants of the cell walls. There is evidence that the cells tend to break down unevenly; during the early stages this leaves elevated circular ridges running around the long axis of the gland; but these are neither regular nor do they persist except in vague outlines in the final stages.' In the vas deferens and seminal vesicle the effect is more elaborate (pl. 1, figs. C, D, E,e and pl. 2 figs. 5 and 5b). Com- mencing at the anterior end of the vas deferens the cells break down unevenly and in such a manner as to leave the surface of the epithelium in very definite ridges. This is much more clearly defined and regular here than in the gland. This condition is described in the mature insect by Koschevnikov as ‘‘in Ring- walzen eingereiht”’; but a close inspection of a cleared whole mount or hemisection reveals an arrangement as of a spiral screw with four successive threads. There are about seventy turns, each ‘thread’ making fifteen to twenty turns of the spiral, though occasionally one ridge ends and is replaced by a new one. As will appear later the function 1s apparently to increase the surface for attachment of the spermatozoa. The nuclei of the epi- thelial cells arrange themselves, not parallel to the basal mem- brane of the epithelium, but in a layer following the folded surface (fig. 5b). The nuclei retain appearances of activity and do not show shrunken chromatin and clear plasma as do the remnants of cells in the epithelium of the gland. The commencement of this secretory and erosive process in the vas deferens overlaps the period of spermiogenesis in the testis. As the lumen enlarges it becomes filled with fluid. The sperms pass into it and through it into the seminal vesicle; here as the sperms descend the cells also break down into a secretion. This process in the seminal vesicle serves three purposes: provides a medium for the spermatozoa by dissolution of the glandular elements, renders the rather firm glandular wall flexible and capable of considerable distention, and allows the enclosing muscles to act easily at the time of ejaculation of sperm. The sperms, still grouped in bundles as they left the cysts of the testicular tubules, attach themselves by the heads to the 238 GEO. H. BISHOP ribbed surface of the vesicle, and the tails project into the lumen. When spermiogenesis is complete and all the sperms have become attached, a cross-section of the organ (fig. 5b) shows, inside the muscular ring, first a ring of nuclei following the contour of the inner surfaces of the spiral ridges, then a distinct line of sperm heads at the surface of the epithelium, and finally the remainder of the lumen almost filled with the sperm filaments radially arranged, extending outward from a narrow central space. ‘The spermatozoa even after attachment show a grouping into bundles. Region of the ejaculatory duct. The development of the ejaculatory duct and its junction with the mucous gland-vas deferens fundament has been referred to above. The relation of the three parts, mucous gland (7), proximal part of vas deferens leading from the seminal vesicle (h), and the ejaculatory duct (j), requires a more detailed description (text fig. 1 and pls. 1 and 3). The paired mucous glands lie parallel in the posteroventral region of the abdomen; the bulbous ends containing the mucous accumulation point anteriorly. The basal portion of each gland, with which both vas deferens and ejaculatory duct connect, bends at an angle of about 45° in the medioventral direction (text fig. 1). The tips of the two glands meet medially. The ejaculatory duct divides as it enters the Junction of the two glands, and a branch penetrates the wall of each. The vas deferens (h) makes a sharp curve from the seminal vesicle and enters the gland on the medial side, dorsal to the entrance of the ejaculatory duct (j). Around and particularly above the entrance of the vas deferens, the muscular wall of the gland is greatly thickened, and projects into the gland’s cavity as a lip or valve guarding the entrance of the vas deferens ((). This valve partially cuts off from the body of the gland anterior to it the basal portion of the gland’s cavity (7) into which lead both ejaculatory duct and vas deferens. It thus divides the cavity of the gland into two regions at the bend of the gland described above. One region of the gland’s lumen becomes distally an elongated sac containing mucus, lying parallel to the FERTILIZATION IN THE HONEY-BEE 239 main axis of the abdomen. Its posterior margin is the valve projecting from the dorsomedial side of the lumen. Below this valve, the second region consists of a small flat pocket lying across the base of the gland (text fig. 1 and pl. 1, 7). Into this pocket and from the valve’s posterior surface opens the vas deferens (h). Applied ventrally to this pocket is the expanded end of the corresponding branch of the ejaculatory duct (J). This flattens out into the base of a cone, whose wall does not break through into the gland’s lumen, although the gland’s wall is penetrated by the blind end of this duct (pl. 3, figs. 9 and 10). The relation of the parts therefore admits of the following hypothesis as to its functioning. If the flat pocket is collapsed, the edge of the valve is pressed close against the opposite side of the gland’s lumen, shutting off completely the whole basal region of the gland from the sac full of mucus (pl. 1, fig. E, and pl. 3, fig. 10). The mouth of the vas deferens is applied at the same time exactly over the flattened blind end of the ejaculatory duct. If this be burst through, the result is a passageway through this system of organs extending through the vas deferens, seminal vesicle, lower vas deferens to the basal region of the gland, and out through the ejaculatory duct. It extends past the body of the gland as if the latter’s content were not to be discharged with the content of the seminal vesicle; although in develop- ment the gland and vas deferens form a continuous tube, a tube whose lumen is closed off from the lumen of the ejaculatory duct by a membrane of chitin over the blind end of the latter. A consideration of the musculature of the region further points to this manner of functioning. 2. The muscle layers. The ejaculatory duct has no muscles in its wall; it is a single-layered tube of ectodermal origin invagi- nated from the hypodermis and chitinized on the inside. The vas deferens, seminal vesicle, and gland have two muscle layers, outer longitudinal and an inner circular layer, forming a continu- ous envelope over the whole of these organs. Running from the base of the gland half or more its length distally, a third or inmost muscular layer, consisting of three separate tracts of fibers, has been described (pl. 1, figs. 1 to 4, x, y, z). A closer analysis of 240 GEO. H. BISHOP the course of these fibers indicates that they do not comprise a distinct third layer, but that they consist of a modification or distortion in the arrangement of certain bundles of the inner or circular layer in this region, and that this rearrangement is the method by which the otherwise simple musculature of the gland’s base is adapted to an involved and complicated manner of functioning. The change, during development, in the relation- ships of the gland to the vas deferens and seminal vesicle on the one hand and to the ejaculatory duct on the other gives a clue to the origin of this ‘third layer.’ Recalling that the vas deferens grows posteriorly from the testis sheath as a J- and finally a U-shaped fundament, one arm of which forms the mucous gland, the musculature may be de- seribed more carefully. It consists of a relatively heavy circular layer of fibers which is not distinctly separable into fascicles, lying next to the glandular epithelium, and a relatively thin longitudinal layer collected into distinct fascicles, between which a connective-tissue network allows for distention of the organ (pl. 2, figs. 5 and 5b). There are about thirty-five of these fascicles in a cross-section of the vesicle; in the gland they are not so distinct and the fibers are arranged in a less specific man- ner. Both layers are thinner over the narrow portions of the vas deferens adjoining either end of the seminal vesicle, and both taper off over the distal portion of the gland into a very thin and elastic connective-tissue membrane. It is at the base of the gland, where the musculature is heaviest and whence originate the three bundles of fibers comprising the inmost muscle layer of the gland, that the ejaculatory duct becomes adjoined to the mesodermal portion of the sexual ap- paratus. Only in the light of the significance of this junction can the elaborate conformation of this musculature be adequately interpreted. We may picture at the bend of the U-shaped gland-vesicle fundament one branch of the ejaculatory duct penetrating this muscle mass to reach the lumen of its respective gland. At this place the wall of the gland protrudes to meet the duct. This protruded portion becomes the basal end of the gland (text. FERTILIZATION IN THE HONEY-BEE 241 figs. 1 and 3, 7). One arm of the U, representing the gland (d), increases in size, while the other, that representing the vas deferens (h) remains relatively small. The result is, first, that the vas deferens becomes a fine duct leading into the massive gland and, second, that as the gland protrudes toward the ejac- Text fig. 3 Diagram to explain the derivation of the third or internal muscle layer of the mucous gland. Lettering as for text figure 1, with addition of the following: A-B-C-E, original direction of the lumen of the gland-vesicle funda- ment in development; A~B-—C-D, direction of lumen in mature gland, whose base has enveloped the blind end of the ejaculatory duct 7; XYZ, path of the circular muscle fibers around the end of the vas deferens in the wall of the gland; X’’"Y’"’Z"’, fibers around the body of the gland; X’Y’Z’, fibers distorted out of the cireum- ferential position by the protrusion of the gland’s base to meet the end of the ejaculatory duct at 7, and by the second protrusion into the elbow of the gland, m; X-X'’, Y-Y"’, Z-Z"’, three inner longitudinal muscle tracts derived from fibers of the circular layer by change in shape of the gland’s base ati and m. See text for further explanations, also text figure 1. ulatory duct to form a definite basal pocket (7), the entrance of the vas deferens into the gland is left distal to and at one side of this secondarily formed basal region; that is, it comes to enter not at the end of the gland, but at some distance up its side. From a U shape, the lumen of the two organs, mucous gland and THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 31, NO. 2 242 GEO. H. BISHOP (A\p vas deferens, takes the form of a square-root sign C E ? D text fig. 3), in which the perpendicular stem represents the gland, the horizontal arm the vas deferens, and the portion of the per- pendicular below the arm, the protrusion which meets the ejac- ulatory duct. This change in shape distorts the course of the fibers of the longitudinal and circular muscle layers. Their original course along and around the U-shaped axis of the original fundament is modified in conformity with the change from this axis to that of the mature gland. Where the base of the gland protrudes to meet the ejaculatory duct, the muscle layers are carried out in its wall and greatly thickened, investing the end of the duct and the base of the gland with a heavy and complexly arranged musculature (pl. 1 and pl. 3, fig. 10). Fibers of the outer longi- tudinal layer still pass longitudinally as before on one side, the dorsal and medial, as far as the vas deferens, and on the other side, the ventral, down to the basal tip of the gland whence leads the ejaculatory duct. From here they pass anteriorly again on the dorsal side to the vas deferens. On the lateral aspects of the base of the gland, the fibers of this layer must pass across in a transverse direction to reach the vas deferens. The fibers of the circular layer are correspondingly distorted. This layer still encircles the gland above the entrance of the vas deferens (text fig. 3, 2,’ y,” 2’) and encircles the end of the vas deferens itself (x, y, 2) at its juncture with the gland. But below the region of the vas deferens entrance, these circular fibers, whose fundamental course may be considered to have been in concentric rings about the end of the vas deferens, are distorted by the base of the gland having protruded toward the ejaculatory duct (2’, y’, 2’). They extend toward the base of the gland in the same general direction as the longitudinal fibers of the same region, but criss-crossing them diagonally (pl. 3, fig. 10). FERTILIZATION IN THE HONEY-BEE 243 The fibers forming the valve that partially closes off the anterior from the basal or posterior portion of the gland (/) may be derived from a thickening of the circular layer around the end of the vas deferens. ‘To form the valve these fibers protrude into the lumen of the gland anterior to the vas deferens and furnish posterior to it additional material for thickening the muscle mass about the end of the ejaculatory duct. Contraction of the muscles encircling this region would close off the basal pocket in the gland by compressing the valve (I) against the opposite wall; contraction of the muscles extending along the base of the gland would collapse the flat pocket (7) cut off by this valve. In this manner the aperture of the vas deferens would be forced against the blind end of the ejaculatory duct, as the anatomical relations of these parts, previously described, indicates to be possible (p. 239; pl. 1, E, and pl. 3, fig. 10). One more modification of the shape of the gland requires explanation. The vas deferens enters the mature gland on the median side; just anterior to its entrance the gland bends at an angle of 45° to meet the ejaculatory duct, so that its basal tip points ventrally and medially toward the tip of the other gland (text figs. 1 and 3). Thus the tips of the two glands come to point toward each other; in fact, are joined superficially. Anterior and dorsal to the entrance of the vas deferens and to the valve, the lateral portion of the gland’s lumen protrudes out to form the elbow or bend (text fig. 1, m). This valve, which guards the aperture of the vas deferens, forms the posterior boundary of the protruded portion, and the muscle fibers of the gland’s wall are carried out around the lumen (text fig. 3, y’-2’). This protrusion of the gland’s wall thus causes a distortion of the muscle layers somewhat in the same manner as does the pro- trusion toward the end of the ejaculatory duct on the ventral side. A cross-section of the gland just at this angle would show as a result of these changes a tripolar arrangement (pl. 1, figs. 1 and 2). At one pole is the entrance to the vas deferens (h), at a second, the base of the gland envelops the end of the ejac- 244 GEO. H. BISHOP ulatory duct (7), and at a third, the lumen of the gland swells out into the gland’s elbow (m). In text figure 1 these regions may be identified between x and y, v and z, and y and g, respectively. The three tracts of the inner ‘third layer’ may then be derived as follows (pl. 1, figs. 1 to 4, x, y, and 2, and text fig. 3, x’’—2, y’’—y, and 2’’—z): a. Fibers of the inner circular layer originating along the median side of the gland, from a region anterior to the opening of the vas deferens, and from the muscle mass in the valve guarding it, pass on the medial side of the gland toward its base, here penetrated by the ejaculatory duct (2’’—2). y. Fibers from the same region, but passing dorsal to the vas deferens, extend posteriorly around the protruding elbow of the gland (y’’-y). z. Both sets of fibers pass anteriorly on the opposite side of the gland’s base, between the ejaculatory duct and the gland’s elbow, to the region on the gland opposite to the end of the vas deferens (2’’-z). The anatomical findings suggest and bear out this derivation for these three muscle tracts, except that the muscles extend anteriorly further along the side of the gland than the mass of the circular fibers from which they are believed to have been derived. This may be considered a functional modification to afford that insertion of the fibers on the sides of the gland which would enable them to operate most effectively. Along these three tracts and extending for about the same distance, the glandular epithelium is elevated into the lumen of the gland in such a manner as to give its cross-section a trilobed shape (pl. 1, fig. 3). Qne channel so caused (between two adjac- ent tracts) extends distally from the expanded elbow of the gland; along the lateral aspect, between y and z; a second, along the medial side from the valve anterior to the opening of the vas deferens, between x and y, and a third, between x and z distal to the end of the ejaculatory duct and passing opposite to the valve, is continuous ventromedially with the basal transverse pocket of the gland which receives vas deferens and ejaculatory duct. Distally all three merge into the uniformly rounded bulbous sac in which the gland ends. FERTILIZATION IN THE HONEY-BEE 245 3. The ejaculatory duct. The ejaculatory duct and the glandu- lar lining of the basal portion of the gland with which it connects remain to be described (pl. 3, figs. 7 to 10). The blind end of the duct penetrates the gland’s muscular coat; here it expands into a cone whose base becomes applied, as described before, to that aspect of the gland’s basal region which is directly opposite the opening of the vas deferens. The hypodermal cells forming this cone become elongated around its base from cubical to a distinctly columnar form. ‘The base of the cone becomes heavily chitinized, especally that part lying over the cells which are most elongated (pl. 3, fig. 9). At the center of the base the cells are shorter, and here the chitin is laid down in two layers over a very small area (pl. 3, figs. 7 to 10, n). Between these two layers is a small mass of material staining more densely (with iron-alum-haematoxylin) than the chitin, which later disappears or else shrinks greatly, leaving the two layers separated by a space. The layer toward the lumen of the gland is con- siderably thinner than the other (pl. 3, fig. 10), n); both together they form a weakened area in the base of the cone which may be likened to a drum. The columnar cells of the base of the cone now recede laterally and decrease in length, finally leaving this double chitinized drum alone to close the end of the duct (pl. 3, figs. 9 and 10). At the same time the glandular layer of the mucous gland breaks down as described heretofore, and these gland cells over the center of the chitinized drum also withdraw laterally. This leaves the chitin drum exposed to the lumen of the gland, but unperforated, in which condition it can be demonstrated at all stages investigated, provided methods are used in killing which do not distort the organs to such an extent that the drum is burst. This fact, together with evidence to be submitted, indi- cates that both sperms and mucus remain in the seminal vesicle and gland, respectively, not only until maturity, but even until copulation. 246 GEO. H. BISHOP B. Physiology 1. The secretions. The content of the mucous gland is elabo- rated first in the distal end, and tends to collect there throughout the process of elaboration; the thinning of the glandular wall allows of considerable distention of this distal end to its character- istic bulbous shape, and nutriment is evidently absorbed actively by the organ, for it increases in size until the stage F (nine days). The secretion changes in character from fluid to viscous, and aquires increasingly the property of immediately coagulating to a tough, cheesy or doughy mass. This happens on contact with air, water, Ringer’s solution, alcohol, lymph from the drone’s abdomen, or any bland reagent in which an attempt was made to mix or dissolve it. It shrinks markedly in fixation and dehy- dration, and, especially when taken from an old drone or from the exposed organ removed from the female after copulation, it becomes so hard as to nick the microtome knife. It is slightly alkaline in reaction. Spermatic fluid removed from the seminal vesicle consists of very little lymph-like fluid densely packed with sperms. This is so dense that it will barely drop from a needle. The sperms being attached to the vesicle wall, it takes an appreciable time for them to loosen when the vesicle is freshly torn under a micro- scope. Up to the stage E (five days) they have to be squeezed loose; in later stages the stimulus of breaking open the vesicle causes them to release themselves readily, until at stage G (twelve days) they pour forth from the slightest cut of the vesicle in a writhing mass. Up to stage D or E the sperms, except for a very gentle beating of the filaments, are inactive when released. From this stage until apparent maturity (nine to twelve days) their activity when released increases, as well as the readiness with which they are expressed from the vesicle. It is concluded that in the vesicle the sperms are at all times at rest or nearly so, for if the vesicle is abruptly torn under the microscope the sperms attached along the torn edge appear quiescent for a moment. Also the spermatic fluid remains in the seminal vesicle until the time of copulation, not as Shafer suggests partly in the FERTILIZATION IN THE HONEY-BEE 247 base of the gland, nor as Zander leads one to infer, in the bulb of the penis (this acting as a spermatophore). Inspection of the drone’s abdomen, opened without fixing the organs, gives some appearance of support to these views, for then, due to disturbance ‘in dissecting, the sperms are frequently found in the base of the gland or even in the penis bulb. The spermatic fluid, in contrast to the mucus of the gland, mixes readily with any bland aqueous medium, salt, sugar, or lymph solution, but any dilution seems to decrease the activity of the sperms for a long time, though without necessarily killing them. Sperms on a slide under a cover-glass in salt solution were not killed by two hours’ contact with ice, and fertile females have been frozen to — 2°C. for fifteen minutes without rendering subsequently laid eggs infertile.1°. The spermatic fluid and the glandular secretion are miscible in the penis before exposure to the air, and the sperms are intensely activated by the secretion of the gland. Particles of the vesicular wall stimulate them similarly. Whether this action is mechanical, as giving the heads of the sperms a firm hold for the exertions of their filaments (they collect around droplets of the secretion) or whether the action is chemical, as a stimulant, is not apparent, Contact with the mucus will not activate sperms that are too young to release themselves from the vesicular wall, and in the oviduct of the queen after copulation the sperms separate out of the mass of mucus and enter the seminal receptacle alone. The evidence seems to point, therefore, to the stimulus being a mechanical one, expecially since sperms are activated by the mechanical act of being torn loose from the wall of the vesicle. The seminal vesicle when filled with spermatic fluid assumes a distinct yellow color, as contrasted with the pure white of the mucous gland. This contrast of color is noticeable whenever the transparent organs of the drone or queen are filled with these secretions. For instance, in freshly dissected drones the yellow 10 Dzierzon has stated that queens can be rendered infertile, and hence ‘drone layers,’ in this way; but though the statement is widely quoted, the writer has not found a single other recorded instance of its being done, experimentally or otherwise; he was unable to produce the expected result by any temperature, either prolonged or extreme, from the effects of which queens would recover. 248 GEO. H. BISHOP spermatic fluid can be seen passing through the base of the gland and the lumen of the ejaculatory duct, and the contents of these organs can be distinguished by the color. When the two fluids are loosely mixed in the bulb of the penis, the areas of yellow and white can be distinguished, and if the drone is stimulated to complete extrusion of the organ, with ejaculation, a rough deter- mination can be made by color as well as by consistency as to whether sperm or mucus has been emitted. When a queen has been newly fertilized, the penis attached in her organs can often be seen to be distended with clear white mucus, while the oviducts are distinctly yellow when dissected out. ‘This is found to be due to the fact that after copulation the sperms collect in a layer next the wall of the oviduct and conceal a central core of mucus. Sperms when densely crowded exhibit a tendency to lie parallel in masses, the filaments beating in unison, giving a characteristic undulatory appearance. This grouping approximates their ar- rangement when attached to the walls of the vesicle (pl. 2, fig. 5b). Free on the slide, the masses of sperms arrange themselves in whorls or undulating bands; after copulation a cross-section of the oviduct of a queen shows a wavy band next the oviduct wall, and in the spermatheca of a fertile queen the sperms again arrange themselves in whorls, with the densely staining heads massed and the lighter staining filaments extending parallel. When diluted on a slide or mixed (in the oviduct) with mucus, or when, in a newly fertilized queen, only a few sperms have entered the spermatheca, the arrangement is scattering and indiscriminate. As for functions consistent with these characteristic qualities and behavior of mucous and sperm, respectively, actual results of copulation afford the final data. To anticipate a forthcoming paper dealing with this subject in detail, the sperms are received into the spermatheca of the female before the mucus is disposed of, and the latter is dissolved gradually from the distended oviducts of the queen bee into which both sperm and mucus are injected at copulation. The penis with which the female returns from the mating flight is distended with mucus alone, which has so hardened on contact with the air as to effectually stop and seal off the torn end of the organ. Having followed FERTILIZATION IN THE HONEY-BEE 249 the sperm through the penis in ejaculation, the mucus has forced all the residual sperm out of the penis, so that whatever material is not injected into the female organs, and is thus to be lost when the penis is dropped, will not be the physiologically more valuable spermatic fluid. 2. Correlation of age with functioning. With the foregoing facts in mind, we may follow the differences in the response of the sexual mechanism, at different stages of development, to artificial or natural stimuli. In a young drone (up to four or five days) the chitinous blind end of the ejaculatory duct is still reenforced with layers of glandular and hypodermal cells; the walls of both mucous gland and seminal vesicle are stiffened with unresolved glandular epithelium; the sperms are either still in the testis tubules or are firmly attached to the vesicular wall, and are incapable of the activity which later characterizes them, ~ and it is doubtful whether the valve which eventually occludes the gland’s lumen is in the early life of the drone capable of doing so, for since the lining of the basal portion of the gland is the last to be resolved into secretion, this valve is still stiffened by a heavy glandular coat. The result of stimulating drones less than four or five days old is either no secretion at all when the organ is extruded or a secretion composed entirely or in large part of mucus, or if sperms are present, the glandular wall of the vesicle has pulled away with them, and the sperms are inert or vibrate their filaments but feebly. After the fifth or sixth day the reaction is markedly different. The reinforcing cells over the end of the ejaculatory duct have withdrawn; the mucus is more viscous; the sperms release them- selves more and more readily from the vesicle and are extremely active; the glandular walls of the organs are thin and pliable, and the sperm content of the vesicle is discharged through the ejaculatory duct ahead of the mucous content of the accessory gland. The whole reaction of the drone is also more violent and spasmodic. These conditions, while virtually established, as stated, at five or six days of age, seem to become accentuated up to the age of nine or ten days, although the morphological and histological changes after the sixth day are slight and although 250 GEO. H. BISHOP the variation in the physiological reactions concerned makes it difficult to measure accurately the degree of the response."! The following table will summarize the data correlating age of the drone with the histological and physiological findings. 3. Manipulation of drones. If a drone’s abdomen is pinched sharply between thumb and forefinger, the pressure will generally cause partial or complete extrusion of the copulatory organ. The penis tube may evert throughout its length, as described heretofore, everting the two lateral chitinized plates that enclose its bulb, and also drawing the ejaculatory duct through the everted bulb (text fig. 2, C);in this case whatever fluid is expressed forms a drop at the end of the penis. Extrusion may stop, however, before this bulb has turned inside out (text fig. 2, B). The fluid will then remain for the most part in the bulb of the penis (b) and in the elastic and expanded end of the adjoining ejaculatory duct (c). There may be little or no spermatic fluid expressed, or the fluid may consist entirely of mucus, or it may consist of both mucus and sperm, rarely of sperm alone. Selecting drones all of which were known to be old enough to function in normal copulation, experiments were undertaken to find what controlled the normal protrusion of the organ and the normal ejaculation of the secretions. It was found almost impossible at first to dissect these drones without disturbance of the sexual apparatus. Drones held in the hand, without mechanical pressure being applied by the fingers, will often extrude the penis with a sort of explosive contraction of the abdomen. Even when extrusion does not occur, the mucous glands of dissected mature drones will generally be found to have burst at the expanded distal end, or else sperm and mucus will have been forced into the base of the gland, ejaculatory duct, or penis. If the drone’s head is amputated a disturbance invariably occurs; frequently this goes as far as 11 Whether functional maturity and ability to effectually inseminate queens is attained at the time of apparent histological and physiological maturity of the organs and secretions described, is a matter which only mating experiments can determine. Mr. F. W. L. Sladen, apiarist of the Canadian Department of Agri- culture, informs me that queens mated to drones under two weeks of age pro- duced a large percentage of infertile eggs. (See his forthcoming report for data.) FERTILIZATION IN THE HONEY-BEE 2 complete extrusion of the penis with ejaculation of spermatic fluid. Removing the abdomen from the thorax before dissecting lessens the effect; reducing the temperature also renders old drones less irritable (but young ones, three days old, more so). Slow injection of all fixatives containing acid causes contraction of gland and vesicle, with bursting of the gland or extrusion of contents through the ejaculatory duct. More satisfactory results with Bouin’s fluid finally led to the use of picric acid for killing, and the best results were obtained by injecting cold saturated aqueous picric acid solution through a fine-drawn pipette into the side of the thorax just beneath the wing, forcing the fluid in very slowly until the abdomen became slightly distended. This seemed to be effective partly through inhibiting the stimulation of the sex organs by the ganglia in the thorax, since indications of stimulation by these ganglia were observed before the irritant that was being injected could have reached the abdomen. Chloroform, ether, and cyanide were not satisfactory as anaesthetics to prevent distortion. By rapidly opening a freshly cut-off abdomen under the low power of a binocular, the parts may occasionally be exposed quickly enough to allow of observation of the activity of the organs. ‘The abdominal pressure that might force the penis to extrude is in this case eliminated by opening the abdomen, so that muscular contraction of the walls of gland and vesicle is the effective agent of the activity that follows. The typical observation under these conditions is twofold. First, a peculiar twitching contraction of the base of the mucous gland tends to straighten out the angle or elbow of this gland (pl. 1, fig. E), and often, by forcing the contents toward the distal end, bursts this through and releases the mucus in the abdominal cavity. Second the yellow spermatic fluid can be seen passing through the trans- parent vas deferens, base of the mucous gland, and down the ejaculatory duct to the penis. Mucus and sperm are thus separated, and microscopical examination of the organs killed immediately in this condition (pl. 3, fig. 10) shows that in the base of the gland the mouth of the vas deferens has been forced against the blind end of the ejaculatory duct. Generally the ULIGM oUOIp PUB JUA[OTA ST SN[NUITy4S jl popnayzxo AT[Nj oq AvuI us -IQ/ ‘OUO]B SNonuUE JO WOT4VT -novle oy} YJIM 10 ‘UOT}A1I0S ou YIM yng ‘9 vseq4s 4B UBYY AjIpvet a1ow spuodser auoiq UO0TJ9IINAS JO UOT} -ejnovfa ynoyyIM puw ‘ABA yaed Ayuo Ayjensn ynq ‘uvs -10 JO uoIsnajzxo Aq I[NUIT}S SuoIjs 03 puodsad [][IM ouo0Id eoues1OUIO e1ojoq ysnf [YUN SuryepnuTys uo opnijxo you ][TM stueg ‘suoljoro0s o[qeviooidde oN puis jo unljeyyide re[n -pur[s 04 pesod -xo yonp jo pug suaxory} uy = “ATyB10 -JB] MBIpPy IM 0} Ulseq yonp jo puoe jo sje oyeredos saoAv] = UT4IYO OM}. ‘SuUExoryy OLN oe OOS OO Alec! SSo]LO][OO pue uryy uryyo ‘puv[s poiely -oued sey qouvig ‘uur SF ‘qysuey ‘in04 -u0d =snoq[ng ‘er0yy «= AToyoyd -Uul00 peAjos -a1 S[joo puis pue ‘ury3 ATA St [pear ATpeysTq ‘uu Ze ‘Yysua'T “OATPOS JLVY IG TSIp SOs S112) ‘uur ¢ ‘YYsUe'T “IB[NUBIS UOT} -a1008 ‘ayB[ns -UBIYS S][S9 [VISIC, “UU §-7 ‘YISue'T “AT[CISIP UWOoTZ -a1008 O[}4T] AIOA sumods YAIM SesIv[Ud spOTSaA @A[OS -SIp woT}o10 -98 JO sopnuRIy ‘puso aoddn 4v Iedvep ‘pespr4 aoBJINS JO SO] 418d Joddn ur pe} -VENIIOD SoWOd -oq [eM ‘oT -ISOA UT UOTJOLVIG Iv] -nUvIS “SOA JO S]T[9Q ‘suerejop SBA UI SoDUOUT -u100 = WOTJaINBG “WU & ‘SIJSOT, ‘poyovy -¥8 4sour ‘oody T19s suatods aWIOY *[[NJ opoIsa A. ST[VM 04 YIVIPV ‘9[Isaa ‘ues UMOp ABA yey sutredg Ren enny sisouosorusedg "SOA [BVUTUIOS JO gied aoddn 04 pusosep suuzeds ‘STSousSOrTUIedg stsouosotunseds JSII] “SISOUAG0} -vullods soseys yse'yT “uowopqe Ilg “wur ¢ seqsay, plo skep ¢ Ssos1ouls OU0IG MoT[OA MO] UTZIYO Apoq ‘yaep soAny ‘Q0UaS.1OW9 alojoq skvp Z poyueur “sid AT VYSTy soAy ‘“SUISIOWO a1 JOq ssep 7 52 Ss) SINGd AO NOISAYLXa DNISOVO ITOAWILS OL ASNOdSAY GOAd AVOLVTNOV LH aNV19 AUOSSHIOV @TIOISHA IVNINGS SISHUNGUDOLVYNUAdS aa appv GoOViLS ras fo supb.o ay) fo buyuoyounf ay, pun yuaudojanap fo abpjs ay) pun auoip fo abo oy7 waanjaq worn)a1.0/) pesn spoyjour YyIM a1qG¥vz904 -9p SBM 9SUBYD OU YY} 109} VW “ABp YYZI 94} 07 dn poonp -o1d Aj[Isva o10UL puB Ud] -OTA d10UI 8q 0} SUI9aS UOT}OV -o1 ysnoyy ‘Aep yyuru 104j;8 eimyeu APjUsIvdde snyearvddy eAT}OB uledG ‘snonut Aq PeMOT[OJ “Ysug sumeds jo uory -ejnoela YIM pue ‘aoue[OTA DATSO[AXO O14STIOJOVIVYD YIM AjIpvoer 910M papniyxo ueZIC OAT}OB AIOA YOU U9}JO 918 19}j}eT ‘sumzods puv snonut jo uonsinoefo YyIM opn7y -xo 0} posnvd oq ABUT URSIQ ee esuByo ON esuvyo ON esuvyo ON ein -sur Ayjuersddy asuByo ON arqeysmnsury -SIp osuvyo ON 9ZIS UI asuByo ON “UOLy -01098 JUSS [IS “UIU #9-G ‘Y3ua'T *AVLINY wu [euormouny yuo -Ivddy ‘opaoor yonp ‘ovla 1aA0 ST]2Q “SurAToOs -oI UOISII [eseg esuvyo ON esuByo ON IOpIM pouodo uoUr -ny ‘peyeponova puv[s 0} suaroj -9P SBA JO ST[AQ ‘asuvyo Ou ‘Q[DISOA [BUTULOG PeZtsa ey eter “SOA “poqqtt AT -dsop pue uryy [[@ arepnpuryy esuvyo ON dAOGB SB UOTMIpuod suIBG “UU FT S1}So], peounouoid o10T JUTY W981 ‘UOI}IPUOD saUIeG “Ulu Z ‘IO -[09 UI YstudeI3 SUIUIN} SI}SoJ, ‘staiods dar} MoT Plo skup 1Z plo skep ZT plo sAvp 6 Po skep ¢ 253 254 GEO. H. BISHOP chitinous drum will have been burst through; the valve guarding the entrance of the vas deferens will be pressed against the opposite side of the gland’s lumen so as to occlude the latter, and the mucous content will often have burst through the anterior end of the gland’s wall. If the organs are not killed at once, the musculature relaxes, and the gland assumes a some- what more normal contour. When the drone is opened more deliberately, especially after the injection of an acidified fixative or after stimuli that cause partial extrusion of the penis, the picture is different. The spermatic fluid will have passed down as before through the base of the gland and ejaculatory duct into the bulb of the penis (text figs. 1, b, and 2, A or B, b), but the mucous content of the gland will in this case have followed it down. The two are still unmixed. The sperm is invariably collected at the lower end, as if it had come down first, and the mucus lies behind it. From this it is concluded that after the first spasmodic contrac- tion of the gland and vesicle that expresses the spermatic fluid through the gland’s base, and shuts off the outlet of the mucus through the same channel, the muscles then relax enough to open the lumen again, and the mucus follows the sperm into the penis. The bursting of the end of the gland is in this case prevented by the increased and compensating pressure of the abdominal contraction, since the muscles of the abdomen contract in coordination with the sexual apparatus. Very rarely, by careful opening of the drone, the gland does not burst upon contraction, and after relaxation its mucous content can be seen to follow the sperm down as has been inferred to be the normal manner. With this manner of action in mind, various methods of stimu- lating the drones were tried, to find by what method could be brought about the most complete ejaculation of both mucus and sperm, together with complete extrusion of the penis, in the order above stated. The following treatments tested on mature drones are set down in order of increasing effectiveness in produc- ing the desired effect. FERTILIZATION IN THE HONEY-BEE 255 1. Bouin’s fluid, cold, or picric acid solution injected through the thorax caused no disturbance of the organs with careful handling. Cooling in general reduced irritability. 2. Slow pressure on the sides of the abdomen, especially if the drone was cold, often caused bursting of the abdomen between the sclerites, sometimes extrusion of the penis without ejacu- lation of fluid. 3. Allowing the drones to fly toward a bright window or artificial light and warming them to 40°C. often made pressure on the abdomen effective in causing ejaculation, but not always was the ejaculation complete. 4. Weak acid or fixative containing acid injected into the thorax, and especially into the abdomen, caused very violent contraction of the organs, but often killed the tissues without complete ejaculation of the fluids. Injection of these acidified reagents into the abdomen through the thorax caused ejaculation most frequently. 5. Sudden and slight pressure applied with the fingers to the sides of the abdomen of a warm excited drone generally caused a violent extrusion of the penis with complete ejaculation of both sperm and mucus. This was accompanied by an intense con- traction of the muscles of the abdomen. ‘This reaction of the drone was in close conformity with the result to be expected from anatomical considerations. The pressure is to be interpreted distinctly as a stimulus, and need by no means be sufficiently great to forcibly express the penis without a very pronounced reaction from the drone itself. 6. The most complete and most uniform results were obtained by holding a drone by the head, allowing him to use his wings as in flight until he was intensely excited, and the abdomen became distended as in rapid flight ; the head was then deliberately pulled from the thorax. The drone reacted with what is believed to be substantially a normal orgasm as far as concerns the state of the organs of sex. It is inferred that the violent stimulus of decapitation under these conditions in some way duplicated the stimulus of sexual excitement, as far as the sexual mechanism is concerned. 256 GEO. H. BISHOP Under this treatment, the penis everts throughout its whole length, including the bulb at its end (text fig. 2, C); the expanded end of the ejaculatory duct is brought through the bulb to form a cup-shaped disk at the extremity of the penis (fig. 2, C, c’), and from the central perforation of this cup (the ejaculatory duct) proceeds first a drop of yellow sperm, then a white mass of viscous mucus. The emptied and distorted sexual organs (mucous glands and seminal vesicles) are often forced into the base of the extruded penis by the violent contraction of the abdominal walls (text fig. 2, C, d). The drone is paralyzed or stunned, but sometimes recovers enough to crawl about feebly, and may live for several hours. ‘CONCLUSION It may be seen, therefore, that several lines of evidence point consistently to one specific manner of functioning of the sexual mechanism of the drone. First, the anatomical arrangement of the parts is such that the seminal vesicle is in more direct com- munication with the ejaculatory duct than is the mucous gland, and this connection is of such a nature as to suggest definitely a separate discharage, and therefore a distinctive separation of function, for sperm and mucus (p. 239 and pl. 1, E). Second, the arrangement of the musculature is such that its contraction brings about exactly that arrangement of ducts and apertures which will discharge first sperm, and then mucus, into and through the basal pocket of the gland and thence into the ejacu- latory duct and penis (p. 243 text fig. 3, and pl. 3, fig. 10). Third, the physiological behavior of sperm and mucus is so character- istically different as to suggest a difference of function and dis- posal (pp. 246 to 247). Fourth, by actual observation, a dis- posal of sperm and mucus, entirely consistent with the anatomical and physiological findings, is induced by stimuli that may be considered closely to simulate, or even actually to duplicate, those stimuli that cause the normal reactions of the sexual organs. Under suitable conditions, the action of these organs, and the passage of the secretions through them in the expected order, may be observed under the microscope (p. 250). Finally,. FERTILIZATION IN THE HONEY-BEE 25 as will be shown in a subsequent paper, these secretions dispose themselves in the organs of the female at the time of copulation, and are disposed of by the female’s reproductive mechanism after copulation, in a manner not only entirely consistent with the interpretation given above, but in an order that seems to preclude any interpretation which deviates materially from one herein set forth (p. 248). SUMMARY 1. The drone is not sexually mature at the time of emergence of the imago, but undergoes a further growth period of at least nine to twelve days. The progress of this development is described in this paper for the sexual organs. 2. The sperm and the mucous of the accessory lanl change both in character and in behavior as the process of development goes on, as does also the mode of functioning of the organs which elaborate and contain them. 3. Sperm and mucus each remain in their respective receptacles until copulation, and do not mingle before that time. 4. The partition closing these organs off from the ejaculatory duct, consisting of the chitinous lining of the blind end of that duct, does not break through until copulation. Then the secre- tions burst through it as they are forced out of their receptacles by contraction of the muscular walls of these organs. 5. The musculature of the whole base of the gland is so ar- ranged as to cause, on violent contraction, the shutting off of the distal portion of the gland from the proximal by a muscular valve. The mucous content is thus closed off from its outlet through the ejaculatory duct; at the same time sperm is allowed to pass through the vas deferens and basal portion of the gland into the ejaculatory duct. This spermatic fluid is thus the first to be ejaculated. 6. The mucous content of the gland, upon relaxation of the muscles of the base of the gland, is then free to pass after the sperm, and forces all the sperm out of the organs. It also ap- parently forms a plug by coagulating on exposure to the air (e.g., when the penis is torn from the drone at the time of copulation). THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 31, No. 2 258 GEO. H. BISHOP 7. The bulb and elastic end of the ejaculatory duct do not act as a spermatophore, although after copulation, and while still attached to the queen, they may serve to hold what mucus may not have been fully expressed into the oviducts. 8. The drone’s organs may be inspected in an undistorted state only by the most careful manipulation, as they are easily stimulated to contraction and explusion of their contents. This contraction may be watched in a freshly opened drone. It is inhibited by injection of picric-acid solution into the thorax and thence into the abdomen. It is stimulated by injection of acids or of fixatives containing acids. The use of acidulated fixatives may be responsible for erroneous views that have been put forward as to the normal quiescent condition of these organs. 9. A response apparently duplicating the results of the normal act of copulation may be produced with considerable certainty by various means, as enumerated. PLATES 259 PLATE 1 EXPLANATION OF FIGURES C, D, E, and H Stages of development of the sexual organs of the drone (table in text, p. 252). Camera-lucida drawings of whole mounts, to show, in opiteal section, changes in size and conformation of the sex organs from emer- gence of the imago until sexual maturity. > 18. Black shading, muscular envelope; lines and dots, glandular epithelium; broken lines, spermatozoa; dots, mucous secretion; cross-hatching, hypodermis of ejaculatory duct; lines, chitinous lining of same. c, ejaculatory duct; d, body of gland; e, seminal vesicle; f, vas deferens from testis; h, vas deferens leading from vesicle to gland; 7, basal region of gland; 7, cone-shaped end of ejaculatory duct, applied to 7; k, slender muscle attaching gland to posterior abdominal wall; l, muscular valve guarding vas deferens orifice, and partially separating d from 7; m, region of lumen of gland extending out into elbow of gland; n, chitinous drum over end of ejaculatory duct. Legend on figure D applies alike to all. Numbered lines on C and E locate cross-section drawings of subsequent figures (q.v.). C Drone at time of emergence. Secretion has just commenced in distal por- tions of gland and vesicle. D_ Drone three days old. The bulbous expansion of distal portion of gland as a mucous reservoir is noticeable, and the spiral serration of the vesicular wall has extended throughout the organ. E Drone five days old. Organs almost mature. Sperm attached to walls of vesicle; wall of gland largely resolved into mucus, chitinous end of ejaculatory duct attenuated. The fixative has stimulated the basal musculature of the gland to slight con- traction, without, however, discharging the content of either gland or vesicle. The transverse pocket, 7, into which open vas deferens, h, and ejaculatory duct, j, is closed off from the distal reservoir of secretion, d, by the valve, /, and the ends of the vas deferens and ejaculatory duct are brought almost into appo- sition (compare pl. 3, fig. 10). H Drone twenty-one days old, basal portion of gland and seminal vesicle. Later stages than five days show but slight further modification. The basal por- tion of the gland here is not distorted, as in E, but shows normal resting relation- ships of the parts. Even at this age the contents of the organs have not been discharged into the bulb of the copulatory organ. 1 to 4 Successive cross-sections of accessory mucous gland and seminal ves- icle of drone at stage A, four daysbefore emergence. X24. Located on figure C, by lines numbered 1 to 4, respectively. Legend same as for figure D, with addi- tion of X, Y; Z, regions of three inner muscle tracts. 1 Section through branched end of ejaculatory duct, and the basal portions of the two mucous glands. 2 Section through gland just above entrance of the vas deferens. Between the area representing the section of the distal portion of the gland’s lumen, m, and that representing the basal portion, 7, the black band, /, represents the edge of the muscular valve which, on contraction of the basal musculature of the gland, closes off the basal portion from the distal mucous reservoir (figs. C and E). 3 Section at middle of gland. Between X and Y, Y and Z, and Z and X, may be seen in cross-section the three channels into which the gland’s lumen is modeled by the three muscle tracts, X, Y, and Z. 4 Section of the gland just anterior to the end of the seminal vesicle, through the region of the testis in the abdomen. 260 [ALV1d dOHSId “H ‘OAD Aad-AGNOH AHL NI NOLLYZITILYGA 261 ‘ad Ye poyejonova ATLABay s]oo ‘YySuUey ur WSBI1D9p Aqo1oyy Gory ‘sT[oo ay} Jo SPU [BISIP ayy UlOd] HO Suyemsurvays oar ‘h ‘WOT}OIOS Jo Se[nqo[s ‘uouny S,puv]s ay} Ur sary ‘s ‘Uu0ly “1098 JO SSBUL WV ‘EFEX ‘UOT}OINOS Jo oBv4s9 0478] “Yq o8eys ‘aAoqe su suUBg qg ‘OPE X = "padusUuTUIO9 svy WOT}91098 a1OJoq ‘W o8v4s ‘pues jo Unipeyyides tpnpuvys JO WOT}IOS-ssoIg ug ‘stutods YIM peyord Ajasuap ‘Busey d rea]0 & 07 peAOsor oioy sey Gg aINSY JO WoTyoa908 TVINUBIS BY, *st4804 oy WOT pepudsdsap ysnf Sorpunq url surzeds 9S00] SUIRIUOD Uo] MoIEU B AT[Baqyuag ‘tunipeyyidsa ayy jo S[[99 09 poyouzyqe Speoy “AT[Vrpet poSuvare sulodg ‘gfe x ‘A 98e4s ‘wunIpeyyrdo Te[Npuvys “oporsaa [eururos FO [[BM JO WOT}DeS-ssor9 qg : ‘sso00id ayy jo uorydoour oy4 9}BOTPUL Toponu oy} punorwe SBoIv IIsuap ay} pure S[[99 94} JO spua oy ssoroR puvq IBynuess oy ynq ‘peouour “W109 Jou svYy UOTJaI00g ‘OPE X "Y O8vys ‘untpeyyrda TVNpurvys ‘aporsaa |vurutes JO wory10d jo UOTJIAS-SsOIDQ, -ouT¥S JO S][99 [eroyyidea jo MOIA posieluq we “SUIUTT Iv[npuvys ey} Jo UoTsora UaAoUN Aq poinydynos ST 9[OISeA ay} Jo [eM 9} YOY OUT sasprr [wards aatssooongs INOJ 9Y} JO suoryoes “p ‘9 ‘@ ‘o Sping Snosussouo0y v 03 poAjos ~9l Jo You ‘sTya0 Te[Npuvls oy} wor TOT}Z91008 Jo saynqo[s ‘6 TOA] oposnut Iepno “ATO “wa [rake] SPSNUL [BUIpNyIsuo] ye) DIT IN JO adoyaaua ONSST}-9AT} 29ND SNOUBIQUIBUI aso] ‘a ‘SEI X OD e848 ‘oporsoa [vurues jo WOT}OAS-ssoI’) Gg PT? “OL ITV “LT eqeyd ‘A e1n3y uo vg pus v¢ pu ‘5 o1nSy uo 9 pue G porequinu soul] Aq pozwooy ‘Q[DISOA pur pUuvl[s Jo suoryde9s-ssor9 JO sSurmerp Bplony]-v1owedg SHUODIa ao NOILVNV1dxa 6 HLVId 262 6 ULV Id dOHSI@ "H ‘OUD Had-AWNOH FHL NI NOILVZITILYRA ‘ATJOUTJSIP SIOAVT [BIDAOS BY} 9OVIZ OF F[NOUIP 1 SOYBUT JNO Wd SBY WOTJOOS OY} YOY 4 opSuv oy} Yonoyy ‘QInsy oy} WOIy pourey -qo oq AvUI osVq S,pUL[S oY} JO oINyR[NosnuT oy} Jo Ayixo[durod vy} Jo vopl UY ‘pues oy} JO UOTSaI [Bseq VY} jo Jo Zursopo oy} Aq pUL[S dy} JO ALOAIOSOI [vISIP 9Y} UI PoUTeJoI SUToq SNONUI dy} ‘Suede -Jop SBA oy} WoIJ Jonp A1oywNoelo oY} 10ZUe P[Nod 9UOTB PING otyeuttodg = ‘aovy[d soye} UoTyepNovla o10Joq Sururezqo A]LIByUSMOUL ‘uo1ye[ndoo Sutanp ‘WoTzIpuod ayy soyeordnp AT} UoIVdde a10Jo10y} PUBS oY} Jo UoTZLOd [esBq oY} JO WOIFIpUOD STYL, ‘qorqur yyoy st ‘C ‘gonp A10zR[Novlo oy} Jo pus oy} 19AO ‘Uw ‘U14Iyo JO WINIP 9Y} FVY} OS ‘poyoVIzUOD JOU SBY OpOISOA [VUTUIES YT, “WSMlOpqe oy} OFUL SHONU oy} Sursvoyer “ysinq svy (peInsy Jou) puvys oy} Jo pus [BISIP EYL ‘2 ‘gayood [eseq oy} Wody (Pp) SITY} SUIpIArp “uoUIN] §,puv[s oy} Jo opis oqtsoddo 944 ysurese ‘7 ATBA oY} aoL0F puv ‘(uw 4B) uoTpIsodde ur yonp AroyE[NoV fa ayy Jo pus dY} PUL 9OGILO SUdIOJoOp SBA OY} BUIIG 0} SV OS “OATPBXY olf} JO uorpoolut oy} JO osnBo -oq poyovrjyuod svy ‘plo sAup ou0-AjUAMy OUOIP B WOIJ ‘poinsy o1oy UBSIO OYJ, *) o1n3y uo sv Sut1e}}0T “NOL “LE X “H 99849 ‘ouvyd [ey4IBes oy} WOIj.o[suv JYSI[S B YB ‘pUBIS Jo osvq PUB SUaLoJap SBA Jo yivd I9MO]T YsSnoiy} uorzIIG OT ‘) ainSy uo sv BurseyyoT “WE “OST X “O ‘yuourdojeAep jo 93848 I9}V] 7B ynq ‘Bg oInSYy Ul UAMOYS UOT}Ipuod oY} jo UOIJVOyIUSVU IOYSIF, “6 “JONP 9} JO pUd 9} JO 10}U90 YSNOIY} UOTJIS B JO JVY} ST Bomstq “f-G Ul popnypourl Bore MOYS D UI SOUT] po}}od “L aInSy UO SB SUII0}}0'T ‘OL “Le X “gq e8eig “gonp Arozepnovls oy} Jo pues sy} IdAO uoty pied snouryrys 9[qNop oY} JO UOTZVULIOJ OY} MOYS 07 ‘puB]S Jo esBq JO SUOTIIOS [BIOS ‘BD § (CT ‘Sy 9x0} orvdui0g) ‘wu uorztod [e4sIp oy} WoL SIT} Surprarp Ayperjaed dA[BA ‘7 {puB]S Jo Joyxood Jesuq ‘2 ‘puLys oy} Suo]e AjLo11oyuv Zurssed yonp A107R] -novta Jo uorjy10d o[pprur yo uorzoes ‘9 Syonp Aroye[Nowls jo pus ‘C {suarejep SBA *Y ‘aUIVS JO SUIUTT SHoUTyIYO ‘sour, ‘yonp AroyepNovla jo umnrtpeyyide “Buryoyey Sso.90 ‘uunrpoyyide Iepnpueys ‘sjop puv soul] seposnu “ov “LE X “OD e8vjg ‘pues jo oseq pu yonp Aroyeynoela Jo puo YSNoIy} WOLpes-ssoro OIYVUIWIBABVICE 2 SHuUnNDIA AO NOILVNVI1dXo € ALVId 264 € ALV Id dOHSId ‘H ‘OaD dqaad-AUNOH HHL NI NOILVZITILY AA uw N Resumen por el autor, George H. Bishop. Universidad de Wisconsin. : La fecundacién en la abeja. II. El uso de los fluidos sexuales en los 6rganos de la hembra. El proceso copulatorio de la abeja tiene lugar al aire libre y su naturaleza debe inferirse, por consiguiente, del exdmen de los insectos antes y después del apareamiento. La configuraci6én del tracto vaginal en la reina es tal que el pene del zangano solo puede penetrar ligeramente en el orificio, quedando el bulbo del pene, de gran tamafno, en el vestibulo génito-anal, en posicién caudal a la de la vagina propiamente dicha. Las ‘“‘pneumop6- fisis’ del 6rgano del zAngano sirven aparentemente no como 6r- ganos de retencion, sino para abrir el orificio vaginal para la in- sercién del pene, inflando los diverticulos de la bolsa a cadal ado de la vagina. Los espermatozoides entran primero en los 6r- ganos y llenan los oviductos pares, penetrando después el mucus, mas viscoso, para formar un nucleo central, Ilenando la vagina caudalmente donde se endurece para formar un tapon vaginal cerca del orificio. El 6rgano del zingano puede desprenderse de la vagina de la reina al cabo de unas dos horas. Los esperma- tozoides y el mucus se mezclan solo parcialmente, tendiendo a separarse los primeros hacia las paredes de los oviductos y a pasar caudalmente, aparentemente por quimiotaxis, al conducto de la espermateca que se abre en la vagina. El mucus se absorbe porlos oviductos mas lentamente. Lamayorparte delosesperma- tozoides han entrado en la espermateca al cabo de unas seis horas después de la cépula, mientras que parte del mucus puede per- manecer en los oviductos durante dieciocho horas. Los esper- matozoides y el mucus se disponen de este modo separadamente en los 6rganos de la reina, de un modo ya anticipado por el examen de la estructura y funcionamiento de los 6rganos del zangano (deseritos en un trabajo anterior). Translation by José F. Nonidez Cornell University Medical College, N. Y. AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, APRIL 19 FERTILIZATION IN THE HONEY-BEE Il. DISPOSAL OF THE SEXUAL FLUIDS IN THE ORGANS OF THE FEMALE G. H. BISHOP Zoological Laboratories of the University of Wisconsin TWO TEXT FIGURES CONTENTS rat OCU ETO MMs Pepe et ATTEN Lae ie ere ee ae eds ees ole eis Sate hate ceeeekdes 267 ERESHORIC ANS Air tune ee te ae ALE AS de ee wale Se oie gehen 268 Amat OMY Ol SOXUa lap PALALUS ic < Scla cine elens) have Usos wa idw abides aged Hors Oeraes 269 DEseniptionzOf lent wliZeGe Queens! nec nis wie ow kyoes ,0.3 per cent), abolish the primary inhibi- tory effect of a current of the kind just mentioned. In animals treated with these poisons the closure of such a current produces at a certain stage, instead of inhibition, an acceleration of the ciliary motion. It is noteworthy that an acceleration is the nor- mal effect of closing a similar current in Bolina and Pleurobrachia. 5. The primary inhibition of the ciliary movements in Beroé cannot be explained without the assumption of formations which, at least from a physiological point of view, serve as cilio-inhibitory nerves. ‘These are paralyzed by chloral hydrate and atropin, probably by cocaine as well. 440 GUSTAF FR. GOTHLIN 6. The primary cilio-inhibitory mechanism probably consists of receptors at the surface of the body, which transfer their impulses to a net of nerves. The nerve net in its turn transmits them to end apparatuses which inhibit the vibrations of the swimming plates, probably by blocking the neuroid conduction between them. At any rate, there is present an inhibitory mech- anism acting at a distance in an animal that has no central ner- vous system. | 7. The mechanism for primary inhibition also functions in specimens whose statolith apparatuses have been removed by operation, if one only waits for the moment when the stimulatory effects of the operation have disappeared. 8. There is an intimate connection between primary and sec- ondary (i.e., muscular) inhibitory mechanism in Beroé. It is probable that they both use the same receptors, but the primary mechanism can be caused to function by impulses of weaker intensity than the secondary one. PRIMARY INHIBITION OF CILIARY MOVEMENT 44] LITERATURE Bauer, W. 1910 Uber die anscheinend nervése Regulierung der Flimmer- bewegung bei den Rippenquallen. Zeitschr. f. allg. Physiol., Bd. 10, S. 231-248. Betrue, A. 1895 Der subepitheliale Nervenplexus der Ctenophoren. Biolog. Zentralbl., Bd. 15, S. 140-145. Cuun, C. 1880 Die Ctenophoren des Golfes von Neapel. Monographie I der Fauna u. Flora des Golfes von Neapel. Leipzig. Ermer, To. 1873 Uber Beroé ovatus. Zoolog. Studien auf CaprilI. Leipzig. 1880 Versuche iiber kiinstliche Theilbarkeit von Beroé ovatus. Arch. f. mikrosk. Anat., Bd. 17, S. 213-240. ENGELMANN, TH. W. 1879 Die Flimmerbewegung. Hermanns Handbuch d. Physiol., Bd. I: 1, S. 380-408. 1887 Uber die Function der Otolithen.. Zool. Anzeig., Bd. 10. S. 439-444. Go6tTHuin, G. Fr. 1913 Die doppelbrechenden Eigenschaften des Nervenge- webes, ihre Ursachen und ihre biologischen Konsequenzen. Kungl. Svenska Vetenskapsakademiens Handl., vol. 51:1, 92 pp. ; 1917 Inverkan ay konstant elektrisk strém pA flimmerrérelsen hos kammaneter. Upsala Likareférenings Férhandlingar, Ny féljd, vol. 22, pp. 522-542. Hertwic, R. 1880 Uber den Bau der Ctenophoren. Jena. Kwnupsen, M. 1905 Havets Naturlaere Skrifter udgivne af Kommissionen for Havundersoegelser n:0 2/. WKoebenhavn. Krvuxkensera, C. F. W. 1880 Der Schlag der Schwingplittchen bei Beroé ovatus. Vergleichend-physiologische Studien zu Tunis, Mentone u. Palermo, Abt. III, S. 1-22. Linus, R.S8. 1908 The rdle of calcium salts in the mechanical inhibition of the etenophore swimming-plate. The Americ. Journ. of Physiol., vol. 21, pp. 200-220. MorrensEn, Tu. 1912 Ctenophora. The Danish Ingolf-Expedition, vol. 5, pt. 2, Koebenhayn. Naaeu, W., 1893 Versuche zur Sinnesphysiologie von Beroé ovata. Arch. f. d. ges. Physiol., Bd. 54, S. 165-188. Parker, G. H. 1905 The movements of the swimming-plates in ctenophores with reference to the theories of ciliary metachronism. Jour. Exp. Zool., vol. 2, pp. 407-423. Samassa, P. 1892 Zur Histologie der Ctenophoren. Arch. f. mikrosk. Anat., Bd. 40, S. 157-2388. Scuneiper, K. C. 1892 Einige histologische Befunde an Coelenteraten. Jenaische Zeitschr. f. Naturwissenschaft, Bd. 27, S. 379-454. Verworn, M. 1891 Studien zur Physiologie der Flimmerbewegung. Arch. f. d. ges. Physiol., Bd. 48, S. 149-180. 1891 Gleichgewicht und Otolithenorgan. Ibid., Bd. 50, S. 423-472. Resumen por el autor, H. J. Muller. Universidad Columbia, New York. Nuevos cambios en la serie de ojos blancos de Drosophila y su importancia sobre el modo de presentarse la mutaci6n. En el presente trabajo, el autor da a conocer tres nuevas muta- ciones del alelomorfo normal (W) del locus del ojo blanco: 1) éeru, que aparecié primeramente en un solo macho; 2) marfil, encontrada por Sturtevant en nueve hijos de una misma hembra; 3) blanea, encontrada un en ojo de un macho y transmitida a la progenie. Naranja, hallada en un macho y no transmisible es probablemente un alelomorfo adicional. Estas mutaciones, con las siete mutaciones previas de W de- muestran que las desviaciones considerables de este gene son tan aptas para ocurrir como las desviaciones pequefias, las cuales probablemente son muy poco frecuentes, y que la seleccién apli- cada a este locus no produciria efecto cumulativo. Los colores de los ojos producidos por estos alelomorfos en diferentes combinaciones prueban que no son simplemente vari- aciones en la cantidad del gene. Conforme ilustran los modos de origen de las mutaciones, estas pueden presentarse en diferentes estados del ciclo vital. El hallazgo de mutaciones que aparecen en individuos aislados, mas a menudo que en varios individuos de una familia, no indica que las mutaciones tienen lugar mas facil- mente durante la maduracién o la fecundacién, y se explica por la existencia de un nimero mayor de células en los estados tar- dios del ciclo vital. El autor d& a conocer un método mediante el cual puede estimarse la influencia de la multiplicaci6n celular sobre los nimeros relativos de las mutacions sencillas, multiples y en mosaico que se estan produciendo. Las pruebas obtenidas en las mutaciones en mosaico indican ademas que la mutacién tiene lugar en uno solo de los miembros de una pareja de alelo- morfos en un tiempo determinado, y que el acto que produce la mutacin esta excesivamente localizado. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, SEPTEMBER 13 FURTHER CHANGES IN THE WHITE-EYE SERIES OF DROSOPHILA AND THEIR BEARING ON THE MANNER OF OCCURRENCE OF MUTATION H. J. MULLER Department of Zoology, University of Texas THREE FIGURES CONTENTS ATA ROMUICELOHES Sey ramet vere ie he cic Se histak Slocastldlate eareick See MEP D re) coe 443 HGR CRUE 1 SERN EIR ted hoe os ce. sd ucheivat bee eg eae Lk 445 A. Manner of origin, and time of occurrence of the mutation....... 445 B. Locus, and mode of reaction with other members of the W series 446 ETE TENOR Z Se nse ek ee ee Mea Te NG EP 446 AS Nico nemtion Omigam ! Lins 26 )5 ff. eee ae et cece ale 1a ee eR 446 B. Its reactions with the W series of allelomorphs.:................ 448 PPO CUS OLE ROT Sao 5 5 acai eee ee Aa Ne TS Sette 6 fas lei SRE 2A OI 448 De einerat which the mutation, occurred...) oo... Jai. 3.1. See se 449 LUE, ANNIE SENSES bt Re Eo ed ee ee ME en 450 PATA elit ta STE Ud OT RATT coche ok kA opty eek Sed eons acre, wndtous. evant ete ‘ats bees 450 B. Genetic behavior......... he AERA OCR BI Su Ba kh 4, Se 452 Dee ae possible additional allelomorphss 612220, 2.0...04 260 .. J.n do eee 452 V. The mutations of W as quantitative fluctuations of the gene.......... 454 VI. The stage in the life-cycle at which mutation occurs.................. 459 VII. The degree of localization of the event which produces the mutation.. 469 SHUTOANIT ENA Pee INS ee Ps oie Sen Ney ete: D107, WAN sR rg «AA OL OE 470 Tinea iIbeNenbed Ma tenets fo cote coer arenes alas bee) stl | tPA eee oes 473 The locus designated as W has yielded: the largest series of multiple allelomorphs so far observed in Drosophila. It was at this locus that the factor for white eyes (w) was found by Mor- gan (’10), whence the designation W for the normal allelomorph; subsequently to this eosin (we) was found at the same locus by Morgan (712), cherry (w*) and buff (w») by Safir (13, 716), blood (w*!) and tinge (wt) by Hyde (’16), and coral (w) by Lancefield (18). The case of deficiency (Notch 8) found by Mohr (719) should also be listed here, as this included a muta- tion in the locus W; although flies homozygous for this mutant 443 444 H. J. MULLER factor could not be secured, on account of the lethal action of the deficiency, the dilution of eye color which it produces when in combination with the other allelomorphs of W shows that in its color effect it is an ‘ultra-white.’ Each of the mutant allelo- morphs of W arose by a single mutation from the normal gene, excepting eosin, which arose by a mutation of the gene white, and which is therefore removed from normal by two mutations. All of the allelomorphs affect the same character, eye color, and together they form a graded series.1 And, in addition to the mutations originally observed, certain of the factors—w and possibly we—have been found to arise more than once: white having arisen several times by ‘reverse mutation’ from eosin and the normal red eye color once reappearing by ‘reverse muta- tion’ from white (Morgan and Bridges, 719). The locus W therefore represents the nearest approach yet found in the fruit-fly to the supposititious condition of factor fluctuation which most selectionists have postulated, and the findings con- cerning it have in fact already been made use of by Jennings C17 a, b) as an argument in favor of such views. Four more mutations, one of which probably, and three of which certainly, belong in the same series may now be reported; one of the latter, ivory, was found by Sturtevant, whose own account of it he has kindly allowed to be incorporated in the present paper (section II); the other three mutations were found by the author. It will be of interest to examine these mutations with reference to the question of factor fluctuation raised above, and also to consider the case as a whole in its present relation to the prob- lem of mutation in general. The data concerning each of the new mutants will be given first. 1JTt had been thought (and mentioned in the literature) that these mutant allelomorphs also affected body color, inasmuch as flies containing them appear lighter than red-eyed flies after being killed and ‘extracted’ for several days in 50 per cent alcohol. I have found, however, that decapitated red- and white- eyed flies show no such difference after treatment with alcohol. The effect is obviously due to the red-eye color of the normal flies becoming partly dissolved by the alcohol and distributed through the body of the fly; flies with lighter eyes have less color to be thus distributed. THE MANNER OF OCCURRENCE OF MUTATION 445 POR U A. Manner of origin, and time of occurrence of the mutation This mutant gene causes the eye to be of a very light yellow color, perhaps most aptly characterized as ‘écru.’ This color may easily be mistaken for white if white-eyed flies are not present for comparison, but comparison readily shows that the ‘éerw’ eyes are distinctly yellower than the white. On the other hand, they are very slightly lighter than ‘tinge’ and ‘buff,’ which were heretofore the nearest to white in the series. Ecru males and females are alike in color. Eeru Paeey first among the descendants of a cross of a red-ey ed ! a by red-eyed ee o (j—jaunty wings, S’—star eye, ee: all these factors are in chromosome II). In a cross of this sort certain of the offspring have the same composition as their parents, and these may be used to make another cross of a type exactly like that by which they themselves were pro- duced. Thus, flies of the same heterogyzous composition may be maintained and crossed generation after generation. In the present instance this process had been carried on for about five months (in order to maintain a stock containing the lethal), and during this time no other eye color than red appeared; but suddenly, in about the tenth generation, a single male fly was found with écru-colored eyes. That this male was a product of the cross, and not due to contamination from outside, was proved by crossing it to a dons when it gave the count to be 4; 71 expected of a male of composition 3 *. The mutant itself had star eyes, owing to the dominant factor 8’. The écru color in this cross proved to be recessive, as all of the offspring were red- eyed except one sterile écru-eyed son, produced by primary non-disjunction. In the second generation, produced by breed- ing together the offspring, half of the males and none of the females were écru, and the rest were red; this proved that écru was due to a single sex-linked factor. 446 H. J. MULLER Since males receive all sex-linked factors from their mother, this factor must have arisen by mutation in an ovarian cell of the mother of the original écru male (either before or after its fertilization, but probably before its cleavage). Since none of the brothers of this male were likewise écru the mutant gene could not have existed in many of the egg cells of the mother, hence the mutation could not have occurred in an early oogonial stage, but probably took place in a late oogonium or in an oocyte. B. Locus, and mode of reaction with other members of the W series Ecru males were then crossed to white-eyed females, and pro- duced daughters intermediate in color between écru and white. This showed that écru was either an allelomorph of white, or that, when present, it caused white to be partially dominant. To decide between these two unequal possibilities, the F, from the cross of écru by white were then bred together, and it was found that no red-eyed crossovers were produced; écru therefore lay in the same locus as white, or was completely linked to it; in other words, the two mutants were allelomorphs. Crosses of just the same type were performed with écru and eosin, and with precisely similar results. ‘The F; females were intermediate in color between écru females and eosin females, and in subsequent generations no crossing over took place be- tween the two factors. ‘There is consequently no question that wee is a member of the W series of allelomorphs, and that it behaves like the other mutants of the series in being recessive to normal (W), and in giving intermediates when crossed with white, eosin, and, presumably, with the other mutant members of the series. II. LVORY? A. Manner of origin In an experiment designed to fix an increased value of crossing over in a part of the second chromosome, brother-sister pair 2 The results in section II were obtained by Dr. A. H. Sturtevant, and nearly all of this section was written by him. THE MANNER OF OCCURRENCE OF MUTATION 447 matings were made in successive generations. Each mating was Cr Cr Dr € purple eyes; ¢ = curved wings). More recent experiments have shown that a third chromosome mutant was present also, and was at least in part responsible for the increased crossing over referred to (Sturtevant, ’17). No evidence of the presence of any mutant genes in the X chromosome of this line has been found, except in the case now to be described. of the type oi bp. 69#\\(o0—) black body: ~p.\'— TABLE 1 SECOND CHRO- 22, 5 Jad, J, ; MOSOME CHARAC- parm on coun | 2R-EEp-9o10R | GZ. ern covon | Gt. nym couox | Mma on (+ = WILD TYPE) February 3........ 20 14 4 2+; 2be. WebWeaver) ee 14 10 0 Hebruary. iS