Studies on Sex in the Hermaphrodite Mollusc Crepidula Plana. III

Resumen por el autor, Harley Nathan Gould. Universidad de Pittsburgh.

Estudios sobre el sex0 en el molusco hermafrodita Crepidula plana. 111, Transmisi6n del estimulo productor de machos por el agua de mar. El molusco gaster6podo Crepidula plana pasa durante su vida por una fase de macho, una fase de transicih y una fase de hembra. La fase de macho es inestable y se presenta solamente como resultado de un estimulo susministrado por un individuo de la misma especie m&s grande que el individuo estimulado. El aislamiento completo de 10s individuos pequeiios no desarrollados sexualmente, durante largos periodos, demuestra que baj o tales condiciones no tiene lugar m&s desarrollo de 10s caracteres machos que la formaci6n de unas pocas espermatogonias. En su debido tiempo aparecen 10s caracteres de la hembra. Los individuos pequefios y no desarrollados sexualmente confinados a distancias fijas de 4 a 7 mm. de hembras grandes, impidihdose de este mod0 todo contacto, desarrollan en la mayor parte de 10s casos caracteres del macho en varios estados de madurez sexual. Bajo tales condiciones se producen menos machos y peor desarrollados que cuando 10s animales pequeiios esth m&s cerca del origen del estimulo. Los individuos grandes de Crepidula fornicata, una especie pr6xima a Crepidula plana, no inducen desarrollo alguno sobre 10s individuos pequefios de esta liltima especie, except0 en unos cuantos casos dudosos. El estimulo que provoca el desarrollo de machos actda de tal mod0 que indica que es una substancia que sale de 10s cuerpos de 10s individuos grandes de Crepidula plana, la cual substancia es difusible en el agua de mar, per0 es muy inestable.

Translation by Jose F. Nonidez Carnegir Institiition of Washington ACTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, AUGUST 4

STUDIES ON SEX IN THE HERMAPHRODITE MOLLUSC CREPIDULA PLANA

111. TRANSFERENCE OF THE MALE-PRODUCING STIMULUS THROUGH SEA-WATER HARLEY N. GOULD Marine Biological Laboratory, Woods Hole, and School of Medicine, University of Pittsburgh

ONE TEXT FIGURE The second paper of this series' described a number of experiments showing the instability of the male phase in the marine gastropod Crepidula plana. In common with other members of the family Calyptraeidae, C. plana passes through a sperm-producing phase during the early part of its life while it is small (up to about 15 mm. in length) followed by a transitional phase (15 to 20 mm.) and later by an egg-producing phase (20 to 40 mm.). Growth goes on with varying degrees of rapidity during life. The functional females are the largest and oldest. The species has a peculiarity in that the development and mainte- nance of the male phase requires a stimulus from the outside, which is furnished by the presence of a larger individual, usually transitional or female, in the immediate vicinity of the potential male. The animals are most commonly found in colonies adhering to the inner surface of shells inhabited by hermit crabs. The younger, smaller Crepidulas have various degrees of male development, those directly attached upon the shells of the large females as a substratum, or close beside them, being nearly all fully developed males, while those at a distance of 5 mm. or over are more likely to have only partially developed male organs; the degree of development being less in the specimens farther from the source of the stimulus, i.e., the large individuals of the colony.

1 Gould, 1917, 11. 113 114 HARLEY N. GOULD

In a group in which there are no females and all the members are less than 10 or 12 mm. in length, there are seldom any adult males; the majority being, instead, sexually undeveloped (neuter) ; but often the smaller members of such a group have a rudimentary male development, evidenced by the presence of many spermatogonia in the sex gland, even some spermatogenesis and a rudimentary penis. In fact, wherever two members of the species are attached close together, however insignificant the difference in size between them, the smaller tends to begin male development.

ISOLATION OF NEUTERS The adult male stage is never developed in isolated animals, nor can it be maintained after removal of a male from the colony. Wishing, however, to determine whether any partial development of male characters would take place in completely isolated specimens, the writer allowed young neuter animals to attach themselves to the inner surface of glass vials, one to each vial. These were all kept in salt-water aquaria. Selection of the specimens for the experiment was made with care from hermit shells containing only a new small C. plana. Each was examined with a lens, and only those quite devoid of rudimentary male characters were used. After isolation a few specimens were taken from time to time, examined, then fixed and sectioned for study of the gonad. At the beginning all were from 5 to 12 mm. in length, and were thus at the size when male development can easily be induced. They grew during the period cf isolation, and the last lot, taken at fifty days, were much larger. Slides were made from twenty-four specimens; two at twenty-two days’ isolation, four at twenty-four days, three at twenty-six days, five at thirty- three days, five at forty-three days, and five at fifty days. The results may be summarized as follows: External male characters: In three animals only, two twenty-four days and one at thirty-three days, there was a very small stump at the spot where the penis forms. No other external signs of the male condition appeared. STUDIES ON SEX IN CREPIDULA 115

Gonad: In three cases there were a few spermatogonia in th,e sex gland; one at twenty-two days, one at twenty-four days, and one at forty-three days. None of these corresponded with any one of the three having a rudimentary penis. In sixteen cases the gonad was inactive (containing only primordial male and female cells). The remaining five were the animals sectioned after fifty days’ isolation. They had passed from the neuter to the incipient female condition, having various stages in early growth periods of oocytes, and had grown considerably in size, being now from 14 to 23 mm. in length. A similar record was made of males removed fro% colonies and kept isolated in vials. As was shown in a former paper, all males lose their male characters after removal from the colonies. Four samples were taken from the vials after thirty-six days, four after forty-six days, four after fifty-three days, and four after sixty days. There was no resumption of spermatogenesis or redevelopment of external male organs after the degenerati0.n in any case. The only hint of any such activity was the presence of a few dividing spermatogonia in the gonad of one isolated forty-six days. It should be recalled that previous experiments demonstrated the ability of degenerate males to reassume the functional male state under stimulus from larger individuals. It is thus indicated that the gonads of isolated small specimens may produce a few spermatogonia, but proceed no further toward spermatogenesis; and the spermatogonia so formed later degenerate, as sections-show. The isolation experiment is meant to clear the way for others, i.e., to show, in cases where partial male development is induced under weak stimulus, how much of this is due to internal causes. The writer concludes that rapid sperma- togonial multiplication, formation of spermatocytes, or any later stage of spermatogenesis is an indication of an external stimulus. In previous experiments where they developed male characters under observation, the neuters were placed as closely as possible to the larger animals. Only in this way could the stimulus be clearly shown. The writer failed to find positive evidence 116 HARLEY N. GOULD of a stimulating secretion thrown into the sea-water. The ques- tion arose whether physical contact is necessary for the transference of the stimulus.

STIMULUS WITHOUT CONTACT A simple apparatus (fig. 1) was devised to hold a large female Crepidula at a definite distance from a small neuter without al- lowing them to touch or to move farther apart. The female was removed from the inner surface of a hermit crab’s shell and allowed to attach herself to the concave surface of a watch crystal. The small neuter was placed on the floor of the flat-bottomed depression in a hollow-ground slide. Mosquito netting was fastened over the depression to prevent the neuter from escaping. ab d I! y I I 8 I

Fig. 1 Diagram showing method of preventing contact between specimens a, large female; b, small neuter; c, depression slide; d, watch crystal.

The depression slide was inverted and fastened over the watch crystal containing the large female; leaving the neuter animal, imprisoned in its cell, at a distance of from 4 to 7 mm. from the top of the female’s shell. There was no possibility of contact, yet there was little hindrance to diffusion currents in the sea- water between the two. The variation in the distance between female and neuter was due to irregularities in the curvature of the watch crystal and in depth of depression of the slide. The average distance was 6 mm. ,After various periods, samples of the originally neuter C. plana were fixed and sectioned. The results are tabulated below (table 1). Those specimens the gonads of which showed any male development beyond the mere presence of spermatogonia are marked ‘male.’ ‘Inactives’ are specimens with primordial germ cells only, or with these plus spermatogonia. ‘Females’ are animals where some development of oocytes could be detected. STUDIES ON SEX IN CREPIDULA 11.7

TABLE 1

NUMBER OF NUMBER OF DURATION SPECIMENS MALES

days 14 7 4 3 0 15 20 11 3 6 17 21 14 3 4 21 19 14 5 0 - Total...... 67 43 14 .lo

Thus, forty-three out of sixty-seven, or about 64 per cent, showed spermatogenetic activity of some sort more than isolated neuters show. Classifying these forty-three with regard to degree of male development, we have: fully developed testis, twenty- five; testis containing sperm, but with some missing stages of spermatogenesis, four; testis developed as far as spermatids, eight; spermatogonia and spermatocytes, two ; spermatogonia in multiplication period, four. The occurrence of incipient female development in some of thLe specimens will be understood if we assort them all in the order of their size, indicated by the length of the shell in millimeters. This is done in table 2. All those having early stages of developing oocytes (‘female’) are seen to be among the larger animals used for the experiment.

LENQTH NUMBER OF MALES NUMBER OF INACTIVES NUMBER OF FEMALELl

mm. 8 6 0 0 9 10 2 0 10 12 2 0 11 6 1 0 12 5 3 1 13 3 1 2 14 1 3 1 15 0 2 3 17 0 0 1 18 0 0 1 20 0 0 1 118 HARLEY N. GOULD

Female development is much slower than male, and it is likely that the most of these animals were already in the course of female differentiation when selected as neuters. The percentage of ‘inactives’ is also greater among the larger specimens. It has been evident to the writer from many observations that the tend- ency to male development under stimulus gradually wanes as the period approaches when female development may set in. It is, however, sometimes possible to superimpose male on early female development, as shown in the former paper. In the watch-crystal experiment forty-three animals out of a possible sixty-seven showed some degree of male activity in the sex gland, twenty-five of them being fully developed males. Compare this with the result obtained when neuters were placed on and closely around females. In the latter case (from records in previous paper) fifty-one out of a possible fifty-three showed some degree of male development, and thirty-four of them were adult males. It is clear that more males develop when the neuters are close to the source of the stimulus than when sep- arated by seSeral millimeters; and furthermore, the difference in the results of these two experiments cannot be adequately set forth in tabular form. Examination of the gonad under the mi- croscope shows it more strikingly. Many marked ‘adult testis’ in specimens from the watch-crystal experiment are only a fraction of the size of the gonads developed in those placed close to or on the large females. There are often signs of arrested development in the former, shown by the paucity or absence of some stages of spermatogenesis. An examination of the small individuals in a large number of normal colonies shows about 62 per cent adult males (determined from external characters). By placing neuters on and close to females, about the same percentage of adult males was obtained, and this could have been raised considerably by rejecting all those specimens which had moved several millimeters from the females during the course of the experiment. In the watch- crystal experiment only about 38 per cent became adult males. The development of the male phase by neuters imprisoned in depression slides thus shows that the male-producing stimulus is STUDIES ON SEX IN CREPIDULA 119 able to act in the absence of physical contact and through several millimeters distance in sea-water. A comparison with other experiments indicates that fewer and less fully developed males are produced under such conditions than when the stimulus acts more directly. The writer has tried several times to determine whether a large female of Crepidula fornicata, another species of the same genus, could furnish the stimulus for male development in a small neuter Crepidula plana. The experiment has been difficult to carry out, as the little C. plana neuters were generally crushed by the twisting and turning movements of the great C. fornicata before sufficient time elapsed to make the experiment valuable. The writer has, however, slides made froa thirty-two C. plana selected as neuters and kept near the C. fornicata for various periods. Of these, twenty-one remained entirely neuter and six became incipient females. The remaining five show traces of male development. Two of these must be counted out because the microscopic appearance of the gonad shows that the few products of spermatogenesis there must have been formed and further activity must have ceased before the experiment began. This leaves only three which seem to have developed any male characters during association with C. fornicata, and they are as follows : a. Penis partly developed and small testis as far as spermatids, not very active. Time, seventeen days. b. No penis. A few spermatogonia and spermatocytes. Time, eleven days. c. No penis. Spermatogonia and a few spermatocytes. Time, eight days. Thus, there are no adult males developed out of twenty-six neuter specimens (leaving out of consideration those which had begun female differentiation), but there are three with partial male development during the experiment. This result is rather perplexing. One would naturally expect either an appreciable proportion of males, if the C. fornicata exerted any influence, or none at all, if they did not. However, we may draw the conclu- sion that the male-producing stimuhs is not due to any general

THE JOURNAL OF EXPERIMENTAL ZO~LOQY,VOL. 29, NO. 1 120 HARLEY N. GOULD change in the medium (sea-water) caused by C. plana which would be similarly caused by other species. The C. fornicata females used for the experiment were larger than the largest C. plana females, and would be expected to throw into the sea- water at least as much of the general katabolic products, for in- stance, as the latter, yet they had almost no effect in stimulating development of the testis. It should be emphasized that the power of large animals of the species C. plana to stimulate spermatogenesis in the smaller is not limited to females. A number of unusually large males were removed from a colony and imprisoned in a watch crystal with nine small neuters. In eighteen days five of the nine showed some degree of male development, mostly immature. The large males were in the meantime losing their male characters. They were kept forty days after this losing all signs of maleness and growing larger. A second lot of nine small neuters was placed with them. In sixteen days eight of the nine had some degree of male development, averaging nearer the mature male phase than the first nine. The numbers are too small to speak for the relative effectiveness of large males and large transitionals, but show the ability of both to produce the stimulus.

SUMMARY The stimulus passing from larger to smaller Crepidula plana, causing the latter to assume and retain the male phase, can be transmitted for several millimeters through sea-water, though its effectiveness is reduced at this distance. Indication that the stimulus may be given faintly by Crepidula fornicata, a related species, was given in only three out of twenty-six cases. The stimulus acts in such a manner as to suggest that it is a specific substance given off from the bodies of the animals, dif- fusible in sea-water, but very unstable.

BIBLIOGRAPHY

GOULD,H. N. 1917 Studies on sex in the hermaphrodite mollusc Crepidula plana. I. History of the sexual cycle. Jour. Exp. Zool., v. 23, no. 1. 11. Influence of environment on sex. Jour. Exp. Zool., v. 23, no. 2.