SEX BEHAVIOUR AND SEX DETERMINATION IN CREPIDVLA FORNICATA L.

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1 [34] SEX BEHAVIOUR AND SEX DETERMINATION IN CREPIDVLA FORNICATA L. BY JAN Z. WILCZYNSKI Lebanese State University, Beirut, Lebanon (Received 21 November 1957) INTRODUCTION The problem of sex determination in relation to the formation of chains in Crepidula fornicata L. was first raised by Orton (1909) and was further dealt with by him in a series of papers of which the last is Orton (1952). The same problem has also been studied on other species of the same genus, e.g. on C. plana by Gould (1952 and earlier papers) and by Coe (1953 and earlier papers); and on C. aculeata and C. icalshi by Hitoshi Ishiki (1936, 1938). The present investigations were directed towards (i) the determination of the normal sex ratio in chains, (ii) the possibility of inducing changes of sex by artificial means and (iii) the cytology of the gonads. THE NORMAL SEX RATIO Statistical analysis was based upon the examination of five separate groups, containing 15, 47, 24, 19 and 18 chains. These included single, double, triple and quadruple chains, ranging from 7 to 19 individuals per chain. Taking all groups together there were: 122 basal empty shells, 404 mature females without trace of penis, 81 'transient' individuals in the sexually inactive condition, 739 mature and immature males with normal penis. 'Visiting' males, not definitely attached to chains, were not taken into account. On a percentage basis there were 39 % mature females + empty shells, 6% 'transients' and 55% normal males. On further examination it was found that in 42 chains, in which the ' transient' condition of individuals in the middle was less obvious than usual, males were directly followed by females. Although the gonads of the first female and the last male were not examined histologically it is reasonable to suppose that they were not in full development and it is certain that copulation between them could not have taken place, the distance being too great. The majority of chains show one or two individuals in the ' transient' state, and in view of the range of variation encountered it seems likely that the complete absence of ' transient' individuals from a chain is not of any significance. Double, triple and quadruple chains were encountered more frequently in these experiments than in Nature. This may be due to originally single chains having been broken during manipulation and having joined up with other chains in an irregular manner. The side chains, no matter of how small a number of individuals they may be composed, provided only that the individuals are of different sizes,

2 Sex determination in Crepidula fornicata L. 35 invariably include both sexes. This suggests that sex determination is related to size, all small animals being males and all large animals being females, with representatives of both sexes at intermediate size. In the intermediate size-range it often happens that the normal male/female sequence along the chain is locally reversed, and from this it follows that the sex-differentiating factors cannot be dependent upon such external influences as that of position. In addition to the factor of size there must be some internal factor, possibly genetic or hormonal, as yet undisclosed. ATTEMPTS TO ALTER THE CHANGE OF SEX (i) By re-association of separated males. In September 1954, 456 males (131 large, 87 medium, 238 small) were separated and placed in a wire basket attached to a floating car and submerged in the sea off Woods Hole. The cage was taken up in July 1955 and the animals were accounted for as follows: 153 males, 83 females, 210 dead, 10 lost. Of those remaining alive there were 35% females, 5% 'transients' 60% males, figures which are remarkably close to those obtained from natural populations. It is therefore concluded that a population of males isolated in Nature will tend to revert to the normal sex ratio. (ii) By temperature changes. Evidence of the effects of temperature upon sex change in other animals suggests that the effects are noticeable within a few days. Three experiments were carried out on separated males which were kept in running sea water at C. and at 10 C.: (a) 157 males; 29 days at C.; no sign of sex changes. (b) 274 males; 6 weeks at C.; 3 small males became females; 1 large male showed reduction of penis. (c) 50 small males; 24 days at io C.*; 10 survived; no sign of sex change. 55 large males; 24 days at 10 C.*; 20 survived; no sign of sex change. There is thus no evidence for an effect of temperature upon sex change in Crepidula. (iii) By injection of extracts of the other sex. This method was suggested to me by Prof. Moenkin. An extract was prepared in the following way. Twenty-five large females were taken and their pallial organs were ground up with fine sand in 20 ml. sea water and centrifuged. The supernatant was boiled for a few minutes (for purposes of sterilization) then cooled to 20 C. Two series of males were chosen for injection: (a) 25 of the largest size and (b) 22 of smaller size but with distinct penis. Of (a) 6 survived for 23 days without sign of sex change. Of (b) 9 survived for 23 days and one specimen underwent reduction of the penis; it should be remembered, however, that the experiment was carried out in September when reduction and discoloration of the penis is seen in natural populations. There is therefore no evidence that female sex hormones have any effect in changing the sex of the male. I take this opportunity of drawing attention to a misprint in an earlier paper (Wilczynski, 1935, Biol. Bull., Woods Hole, 109, 353): for 'o C read ' io C.\ 3-2

3 36 JAN Z. WILCZYNSKI CYTOLOGY OF THE GONAD Cytological observations were made on fresh material of American (Woods Hole) and English origin. The gonad is an organ with many branches lying mainly on the left of the pallial complex when seen in dorsal view. There is no difference between the sexes in the anatomy of the gonad (Fig. 1). Fig. I. a, Young male in ventral view; b, upper part of male gonad showing the beginning of the vas deferena; c, female in ventral view. The branched gonad is shown in black, the hepatopancreas is stippled. Histologically the gonad has the same appearance (as seen in 'squashed' preparations) throughout all the stages of sex change, male and female sex cells being present together all the time as described by Coe (1936, 1940) for C. plana. Spermatozoa are to be found as free aggregates (Fig. 2 a, b). A few sperm tails are to be seen attached to the germinal epithelium. These were described by Gould (1917a, b) in C. plana and he suggested that they were in process of being eliminated by histolysis; but as they are also to be seen in very young males this is unlikely. Spermatozoa of at least five different types have been recognized: (i) a spermatozoon with one or sometimes two tails (Fig. 2 c) probably representing two

4 Sex determination in Crepidula fornicata L. 37 spermatozoa with their heads stuck together; (ii) a long spermatozoon with a small head extending into a long perforatorium (Fig. zd, e)\ (iii) a spermatozoon with a large thick head (30-50 x normal) and a comparatively short thick tail, the perforatorium being shorter than in other forms (Fig. 2/); (iv) a rare type, probably a developmental stage of (iii); (v) a spermatozoon which is possibly a modification of an apyren spermatozoon (Fig. 2/1); it is possible that the structure illustrated in Fig. zg is the mother cell of several apyren spermatozoa as suggested by Gould for C. plana. Fig. 2. a-h, Spermatozoa and stages in their development; i k, eggs and stages in their development; /, yolk cells. In the same preparations as those in which spermatozoa appear there are to be seen primordial cells, oogonia and oocytes in all stages (Fig. 21, j, k). There are three types of female cell: (i) round, with pale cytoplasm and with a nucleus showing granules and fine chromatin network (Fig. 21); (ii) elongated, with elongated nucleus (Fig. 2 k); (iii) small with compact nucleus and little cytoplasm (Fig. 2/). Besides the oogonia and oocytes mentioned above there are scattered cells of irregular outline containing yellow droplets suggestive of yolk (Fig. 2 0-

5 38 JAN Z. WILCZYNSKI The chromatin in the nucleus is everywhere in the form of granules and of fine interwoven filaments, and it has therefore not been possible to establish the chromosome number. CONCLUSION Earlier studies on the biology of Crepidula, already referred to in the Introduction, together with the results reported in this paper indicate that the chains of Crepidula are not breeding associations as was at one time supposed. The distance between males and females in the chain is too great to allow of copulation. Insemination of females must be brought about by small 'visiting' males. It is observed that the seminal vesicles of spawning females are always full of sperm and this suggests that repeated insemination at frequent intervals is unnecessary. The tendency for the normal sex ratio to be re-established among groups of separated males and the regular arrangement in order of size of the individuals forming a chain, together argue that sex determination is a matter of age and/or size. It has yet to be demonstrated that sex determination is susceptible to any other influences. SUMMARY 1. The percentages of males and females in naturally occurring chains of Crepidula fornicata L. were found to be 39% females, 6% 'transients' and 55% males. 2. Disconnected males kept in cages in sea water for 9 months re-established the normal sex ratio. 3. Disconnected males were kept for periods of 4-6 weeks at temperatures of 30 0 and io C. No significant tendency to change sex was observed. 4. Males were injected with extracts of females. No tendency to change sex was observed. 5. In animals of all sizes from small males to large females both male and female gametes are invariably present together. The gametes of both sexes are polymorphic. My thanks are due to Prof. Armstrong, Director of the Marine Biological Laboratory at Woods Hole, Massachusetts, where much of the work was done, and to the American Philosophical Society for a grant during my stay there. I also wish to thank Mr H. A. Cole for sending living specimens by airmail from the Fisheries Laboratory, Burnham-on-Crouch, Essex, to Beirut. REFERENCES COB, W. R. (1936). Sexual phases in Crepidula. J. Exp. Zool. 72, COE, W. R. (1940). Divergent pathways in sexual development. Science, 91, COB, W. R. (1953). Influences of association, isolation and nutrition on the sexuality of snails of genus Crepidula. J. Exp. Zool. 133, GOULD, H. N. (1917a). Studies on sex in the hermaphrodite mollusc Crepidula plana, I. J. Exp. Zool. 33, 2*5-50. GOULD, H. N. (19176). Studies on sex in the hermaphrodite mollusc Crepidula plana, II. J. Exp. Zool. 33, GOULD, H. N. (1952). Internal and external factors influencing growth and sex development in Crepidula plana. J. Exp. Zool. 119,

6 Sex determination in Crepidula fornicata L. 39 HITOSHI ISHIKI (1936). Sex changes in the Japanese slipper-limpets Crepidula aculeata and C. wahhi. J. Sci. Hiroshima Univ. Ser. B, Div. I, 4, HITOSHI ISHIKI (1938). Histological studies on the sexual organs during sex changes of Crepidula aculeata and Crepidula tvalshi. J. Set. Hiroshima Univ. Ser. B, Div. I, 6, ORTON, J. H. (1909). On the occurrence of protandric hermaphroditism in the mollusc Crepidula fornicata. Proc. Roy. Soc. B, 8i, ORTON, J. H. (195a). Protandry and self-fertilization in Crepidula. Nature, Lond., 169, 279. WILCZYNSKI, J. Z. (1955). On sex behaviour and sex determination in Crepidula fornicata. (Abstract.) Biol. Bull., Woods Hole, 109,