THE BREEDING CYCLE OF THE KEYHOLE FlSSURELLA BARBADENSIS GMELIN

JONET WARD Bellairs Research Institute of McGill University, St. James, Barbados, West Indies

ABSTRACT The breeding cycle of barbadensis is defined from the gonad stages of bimonthly samples from June 1964 to November 1965. The results are compared with the breeding cycles of temperate climate .

INTRODUCTION Reproductive cycles of the Zeugobranchia and Patellacea have been described for Haliotis spp. (Crofts, 1929; Bonnot, 1940; Leighton & Boolootian, 1963; Boolootian et al., 1962), Acmaea spp. (Fritchman, 1961a, b, c, 1962), Patella vulgata (Orton et al., 1956) and P. depressa (Orton & Southward, 1961). With a few exceptions, spawning in these is seasonal. In Diodora apertura (=Fissurella reticulata) the eggs are shed from the anterior end of the mantle cavity of the female and are spread by the foot in gelatinous masses on a suitable surface. Boutan (1886) records that an adhesive substance comes from an accessory gland in the urinogenital duct. Fretter & Graham (1962) suggest that the shell, which surrounds the gelatinous coat of the ovum in the ovary and swells when the egg is released, may be the only cause of adhesion. The egg-laying process takes 2 to 3 hours and fertilization occurs after the eggs are laid. The spermatozoa are liberated through the apical hole of the male (Boutan, 1886) . The trochophore and veliger stages are passed through in the gelatinous material and the young emerge as miniature adults. F. barbadensis has a free swimming trochophore and the pelagic life lasts for 2 to 3 days in Barbados (Lewis, 1960). The eggs of Fissurella (=Diodora) nubecula are fertilized within the ovary and the spermatozoa are grouped into spermatophores according to Medem (1945). Only one type of spermatozoon is found in Fissurella, Haliotis, and Patella (Tuzet, 1930) and four gamones facilitating external fertil- ization have been located in these genera (Medem, 1945). Sex reversal occurs in 12 per cent of a population of F. nubecula (Bacci, 1947) and in at least 90 per cent of a Patella vulgata population (Orton, 1920, 1928, 1946). Nearly all P. vulgata specimens below 10 mm in shell length are neuter. From 16 to 25 mm, 90 per cent of the specimens are male. At 40 mm the numbers of the two sexes are equal 686 Bulletin of Marine Science [16 (4) and at 60 mm between 60 and 70 per cent are female (Orton et al., 1956). P. coerulea shows a 92 per cent sex reversal (Bacci, 1947) whereas it is rare or absent in P. aspera and P. depressa (Dodd, 1956). Hermaphrodites occur occasionally in P. vulgata, P. aspera, and P. depressa. This study on F. barbadensis Gmelin was supported by a grant from the National Research Council of Canada. I thank Dr. J. B. Lewis of the Bellairs Research Institute, McGill University, for his helpful suggestions and Mr. J. W. Pollock of the Zoology Department of McGill University for photographing the gonad sections.

METHODS Random samples of 35 specimens of F. barbadensis over 11.5 mm in shell length were collected on the southwest coast of Barbados in an area of large boulders. Collections were made at bimonthly intervals from June 1964 to November 1965. The length, width, and height of each shell was measured to 0.5 mm with vernier calipers and the gonad was examined by cutting away the posterior part of the foot from the shell and reflecting the foot anteriorly. The sex, size, position, and maturity of the gonad were noted by observation under a dissection microscope. High power magnification was necessary to determine the sex of the gonads in the very early phase of development. The virgin gonads, whose sex could not be defined by this method, were termed neuter. The non-virgin gonads were divided into developing, fully developed, and spawning according to the criteria given in the following section. Samples of all stages of gonad development and spawning were preserved in Bouin's solution, dehydrated in alcohol, cleared in benzene, and sectioned at 8 1-'-. The sections were stained with Ehrlich's haema- toxylin and eosin Y (HE) and the microscopic structure of the gonad was examined. RESULTS Both the male and female gonads are enveloped in connective tissue and a layer of muscle surrounded by the pallial epithelium. The repro- ductive cells were identified as follows: Spermatogonia.-These are pale-staining cells, 5 I-'- in diameter, with a little cytoplasm. The small centrally placed nucleus, about 2 I-'- in diameter, has granular chromatin and a prominent nucleolus. The cells are supported by the connective tissue base of the seminiferous tubules and may be found in the gonads of all mature limpets throughout development and spawning. Spermatocytes.-The cells are 3 to 5 I-'- in diameter with a dark-staining spherical nucleus, 2 I-'- in diameter, containing scattered chromatin. The cytoplasm is granular. 1966) Ward: Breeding Cycle of a Keyhole Limpet 687

FIGURE 1. Section through neuter gonad. HE, X 570.

EXPLANATION OF LETTERING ct = connective tissue 10 = large oocyte mo = medium oocyte og = oogonium sc = spermatocyte sg = spermatogonium so = small oocyte st = spermatid sz = spermatozoon

Spermatids.-The young cells are 2 fL in diameter and have scattered chromatin and a granular cytoplasm. Older spermatids possess a dark-staining nucleus with densely packed chromatin. Spermatozoa.-Only one type of spermatozoon is found with a head about 4 fL long.

Oogonia.-These are 6 fL cells, eac·h with a pale-staining, oval-shaped nucleus containing scattered chromatin and a nucleolus. There is very little cytoplasm and the cell boundaries are often hard to define. The cells are embedded in the connective tissue trabeculae, usually in groups, and they may be found in the gonads of all mature specimens throughout development and spawning.

Small Oocytes.-The cells have a diameter ranging from 7 to 40 fL. They are dark-staining with a large nucleus, 5 to 17 fL in diameter, scattered chromatin and a prominent nucleolus.

Medium Oocytes.-The diameter of these cells reaches 80 fL, including the gelatinous coat which surrounds the cell. The nucleus has a diameter of up to 27 fL with reticular chromatin and a prominent nucleolus. 688 Bulletin of Marine Science [16 (4)

FIGURES 2-5. 2, Section through developing male gonad. HE, X 208. 3, Section through fully developed male gonad. HE, X 208. 4, Section through early spawning male gonad. HE, X 208. 5, Section through late spawning male gonad. HE, X 380.

Large Oocytes.-These cells range from 80 to 180 JL in diameter, including the gelatinous coat which increases in thickness as the cell becomes larger and swells on contact with sea water. The gonad stages were grouped according to the following criteria: Neuter.-At this stage, when the gonad is just beginning to show develop- ment (Fig. 1), the sex can only be determined after the gonad has been sectioned. The gonad is a thin elongate structure lying between the visceral mass and the foot. It is situated just posterior to the large loop of the intestine and is invisible to the naked eye. The covering membrane is mottled green or brown and the gonadal tissue varies from translucent to beige. Oogonia (Fig. 1, og) and spermatogonia are embedded in the connective tissue of neuter gonads just prior to the onset of development. Developing.- The sex can be determined by the colour of the gonad under high magnification in the early stages of development and with the naked Ward: Breeding Cycle of a Keyhole Limpet 689

...• to

6 ".

FIGURES 6-9. 6, Section through developing female gonad. HE, X 80. 7, Section through fully developed female gonad. HE, X 80. 8, Section through early spawning female gonad. HE, X 80. 9, Section through late spawning female gonad. HE, X 80. eye in the later stages. In early development the testis is brown with white mottling due to the ripe spermatozoa in the seminiferous tubules. Later, as the number of spermatozoa increase, the gonad appears beige and the active sperm may be seen under low power with their tails extending into the cavities of the tubules. The ovary in early stages of development is translucent to beige and is tinged with green from the developing eggs. As development continues the olive green or bright green eggs gradually mask the yellow-beige connective tissue trabeculae that extend through the gonad. Growth of the gonad in both sexes occurs posteriorly and laterally and is accompanied by an increase in thickness. At the end of the developing stage it forms a thick extension to the posterior end of the visceral mass and extends dorsally. During early development in the testis, several layers of spermatogonia are supported by the connective tissue base of the tubules and the tubule walls are also lined with spermatocytes, spermatids, and a few spermatozoa. 690 Bulletin of Marine Science [16(4 )

(/) 6 z ,.... , \ w \ Neuter , i\ ~ , \ / \ Male ...J , " \

FIGURE 10. The shell length distribution curves for the neuter, male, and female specimens examined.

As development continues, the numbers of spermatocytes (Fig. 2, sc) and spermatids (st) increase, followed by an increase in the proportion of active spermatozoa (sz). Later, the walls are lined mainly with spermatids and spermatozoa but there are still spermatocytes and spermatogonia (sg) at the periphery of the tubules. The amount of connective tissue decreases as development advances. In the ovary, groups of oogonia, small oocytes, and a few medium oocytes are attached to the connective tissue trabeculae during early development. Gradually the numbers of small (Fig. 6, so), medium (mo), and large (10) oocytes increase and in late development the eggs are more uniform in size than in the early stages. However, considerable numbers of small oocytes are attached to the connective tissue with a few oogonia. The trabeculae become thinner in later development as the amount of connective tissue decreases. Fully Developed.-The gonad in this stage is ripe and ready to spawn. It is full and loose with maximum extension posteriorly and dorsally. The testis is pale beige to white and there is only a small amount of connective tissue. Spermatozoa are abundant but considerable numbers of develop- mental stages arc still present (Fig. 3). In the female gonad the trabeculae 1966] Ward: Breeding Cycle of a Keyhole Limpet 691 TABLE 1 THE PERCENTAGE OF DEVELOPING, RIPE, AND SPAWNING SPECIMENS OF F. harbadensis FROM JUNE 1964 TO NOVEMBER 1965

Mean of both Male Female sexes % % % Developing 68.6 63.9 66.25 Ripe 14.7 15.4 15.05 Spawning 16.7 20.7 18.70 Total 100.0 100.0 100.00 No. of specimens 618 527 Total = 1145 ------

(Fig. 7, ct) are very thin and most eggs are mature, fully grown, and loosely packed. Numbers of small oocytes and a few medium oocytes are still attached to the trabeculae. Spawning.-The gonad decreases in size as spawning advances and reverts to the same position that it occupied in early development. The seminif- erous tubules begin to break down as spawning commences (Fig. 4) and this process is completed by the time the testis is about one-third of the full size (Fig. 5). The numbers of ripe spermatozoa decrease until spawning is completed when a few spermatozoa, numbers of developmental stages, and a large amount of connective tissue are found in the gonad. In the ovary, the numbers of large and medium oocytes decrease as spawning advances (Figs. 8 and 9). At the completion of spawning the gonad contains a large amount of connective tissue (Fig. 9, ct), numbers of small oocytes (so), and a few oogonia (og), medium (mo), and large oocytes. Approximately equal numbers of neuter, male, and female gonads were found in the 12 to 12.5 mm limpets collected (Fig. 10). Most of the smaller , examined at various times during the period of study, had neuter gonads. Tn 1,190 limpets examined, all specimens over 18 mm long (comprising about 60 per cent of the total number) had developing, ripe, or spawning gonads. These results indicate that there is no resting phase between spawning and redevelopment of mature limpets. Since a random sample of 35 specimens over 11.5 rom was collected at bimontWy intervals, the number of neuter specimens in each sample varied according to the size of the limpets. During the 17 months, 45 neuter animals were coHected. In Figure 11 the neuter specimens are therefore omitted and the two sexes are considered together as there is no significant difference between their gonad development (Table 1). 692 Bulletin of Marine Science [16 (4)

100

80

60

40 \ \ \

20 ,\ \\~\\ :. : \ ...... ", . o :' ::::: . :::::: J J A SON 0 J F M A M J J A SON FIGURE 11. Variation in breeding activity from June 1964 to November 1965 showing the percentages of developing, ripe, and spawning specimens.

The principal spawning periods were recorded from September to November in 1964 and from March to June in 1965. Spawning specimens occurred in all except two samples throughout the collecting period (Fig. 11). The small size of the samples and the sampling frequency may account for the absence of spawning and fully developed limpets in early January and early April and for the pronounced fluctuations in the proportion of spawning specimens between March and May. The population of F. barbadensis in Barbados was not large enough to allow collection of larger samples from anyone habitat. The total proportion of spawning specimens collected throughout the study is slightly more than that of the fully developed limpets but considerably less than that of the animals with developing gonads. The results (Table 1) indicate that, during the reproductive life of the limpet, the total time taken by a gonad to develop is about 4% times the period spent in the fully developed state and about 3% times the spawning period. About 7% per cent more males than females were collected throughout the year. A chi-square test (chi-square = 7.232) shows that, for 1 degree of freedom, there is a probability of less than 1 per cent that the differences between the observed proportions and equal numbers of both sexes could have arisen by chance. Therefore, significantly more males than females were collected. There was no indication of hermaphroditism or change of 1966J Ward: Breeding Cycle of a Keyhole Limpet 693 sex at any shell length. The proportions of the two sexes in limpets of all lengths over 11.5 mm are relatively constant (Fig. 10).

DISCUSSION A reticulate shell, such as was found surrounding the eggs of Diodora apertura (Fretter & Graham, 1962), was not identifiable in the eggs of F. barbadensis with the histological methods used. However, an increase in thickness of the gelatinous coat was noticed when the egg was in contact with sea water, possibly indicating a similar method of adhesion of the eggs in these two species. An accessory gland in the urinogenital duct mentioned by Boutan (1886) in D. apertura was not found in F. barba- densis. The yellow eggs of D. apertura are slightly smaller, 140 !.t in diameter, than the green oocytes of F. barbadensis when they are shed. The mature eggs of Patella vulgata are also green and Goodwin & Taha (1950) suggest that the colour may be due to "chromoprotein Y" extracted from the female gonads of this species. Spermatogenesis in F. barbadensis appears similar to that described for Trochus spp. (Tuzet, 1930). The position of the gonad in F. barbadensis resembles that in Acmaea spp. (Fritchman, 1961a) and Patella spp. (Orton et al., 1956; Orton & Southward, 1961) but a fully ripe gonad appears to be relatively larger in P. vulgata than in F. barbadensis. Histologically, the gonads of these two species are similar in development and spawning. In P. vulgata spawning is interrupted by periods of development (Orton et al., 1956) and a similar phenomenon may occur in F. barbadensis since a large number of immature stages are found in discharging gonads of both sexes (Figs. 5 & 9). However, in the absence of a resting period, the presence of young developmental stages in spawning gonads probably reflects the continuous reproductive cycle of this species. Specimens of both P. vulgata and F. barbadensis less than 10 mm long are mostly neuter. Although signifi- cantly more Fissurella males than females were collected, no predominance of one sex over a certain size range was obvious as in P. vulgata (Orton et al., 1956). Clustering, or pairing of the two sexes, during spawning was not observed in F. barbadensis as it has been in P. coerulea, P. lusitanica, and Patina pellucida (Fretter & Graham, 1962). Like F. barbadensis, Acmaea jamaicensis appears to have a prolonged breeding season in Barbados (Lewis, 1960). In the more northern forms, A. scabra spawns throughout the year whereas many other Acmaea species are seasonal spawners (Fritchman, 1961a, b, c, 1962). At the northern limits of the range, in southeast Florida and Bermuda, F. barbadensis apparently reaches a larger size (Farfante, 1943) than in Barbados where no specimens above 35 mm long have been found and few exceed 30 mm.. Perhaps the smaller size in Barbados is due to more intensive repro- ductive activity. 694 Bulletin of Marine Science [16(4)

SUMARIO

CICLO REPRODUCTOR DE LA LEPA Fissurella barbadensis G1\1ELIN Se definen los estados neutro, de desarrollo, madurez y desove en el cicIo reproductor de F. barbadensis sobre la base de las diferencias macrosc6picas y microsc6picas de las gonadas. En la mayoria de las lepas la actividad reproductora ha comenzado para la epoca en que miden 12.5 mm de longitud. La reproducci6n es continua durante el ano y se obtuvieron ejemplares desovando en todas las muestras, excepto dos bimensuales, durante un periodo de 17 meses. Despues que se inicia la actividad reproductora, no hay periodo de descanso entre reproducci6n y reconstrucci6n. No hay indicaci6n de hermafroditismo 0 inversi6n de sexo.

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