<I>Cittarium Pica</I>

BULLETIN OF MARINE SCIENCE, 51(2): 250-266, 1992 CORAL REEF PAPER

REPRODUCTION AND LARVAL DEVELOPMENT OF THE WEST INDIAN TOPSHELL, CITTARIUM PICA (TROCHIDAE), IN THE BAHAMAS

Lori J. Bell

ABSTRACT The reproduction and larval development of the West Indian topshell, Cittarium pica (Linnaeus), was studied in the southern Exuma Cays, Bahamas, between June 1990 and June 1991. Topshells held in outdoor water tables spawned unpredictably, possibly in response to simulated low and high tides and the moon phase. Eight of nine spontaneous spawnings occurred within 4 days of either the full or new moon between 20 June and 4 November. Larvae of C. pica were lecithotrophic with a relatively short larval life of 3.5-4.5 d, when reared at 26.5-27.5·C. Emergence from the egg membrane occurred at the trochophore stage. At this stage the larval shell covered about two-thirds to three-fourths of the posterior end of the larvae, with the velum not yet developed. Settlement could be induced by providing a bacterial/algal film substratum. Oocyte diameters from histological slides of ovaries were used to determine reproductive seasonality. Females began to mature in July. A gradual increase in oocyte diameters occurred over the summer until they peaked in late September and early October, followed by a significant decline. Oocyte size was variable between October and January, after which the mean size was consistently small with little variability. The data suggest a natural spawning period in early October. Mean oocyte diameter was highly correlated with seawater temperature. An influx of juvenile C. pica (1-2 mm shell width) was observed in January. Using growth rates of laboratory-reared juvenile C. pica, a spawning date corresponding to the significant decline in oocyte diameter in early October was extrapolated.

The West Indian topshell, Cittarium pica (Linnaeus, 1758) (Trochidae), is an intertidal gastropod found along rocky shores in the Bahamas and Caribbean, south to Trinidad (Abbott, 1974). It is very rare in south Florida (Abbott, 1976) and became extinct in Bermuda in comparatively recent times (probably early 1800's, Verrill, 1901). It is an important food item, second only to the queen conch, Strombus gigas, in value among Caribbean gastropods (Randall, 1964; Flores and Talarico, 1981), and it has been overexploited in many of the dense1y- populated areas of the West Indies (e.g., Cayman Islands, P. Bush, pers. comm.; Virgin Islands, R. Boulon, pers. comm.; Venezuela, Flores and Talarico, 1981). The biology of C. pica was first studied in the Virgin Islands by Randall (1964). More recently Castell (1987) and Debrot (1990a; 1990b) studied various aspects of its biology. Aside from seasonal trends in gonad indices (Castell, 1987; Debrot, 1990a), little is known of the reproductive activity of C. pica. Trochids are found in tropical and temperate waters and the reproductive cycles of temperate intertidal trochids have been fairly well documented (Fretter and Graham, 1977; Garwood and Kendall, 1985; Lasiak, 1987). Colman and Tyler (1988) have provided the first insight into the reproduction of a deep-sea (990- 2,450 m) trochid. Spawning in the laboratory, as well as larval development, has been described for several temperate species (Desai, 1966; Underwood, 1972; Holyoak, 1988a; 1988b). Much less is known about reproduction of tropical trochid gastropods. Spawning and larval development offive tropical species have been described from the Pacific and Red Sea (Gohar and Eisawy, 1963; Duch, 1969; Eisawy, 1970; Heslinga, 1981; Hulings, 1986), although little has been reported of their reproductive periodicity (Randall, 1964; Heslinga and Hillmann, 1981).

250 BELL: TOPSHELL REPRODUCTION AND LARVAL DEVELOPMENT 251

The objectives of the present study were to describe and illustrate the larval development of C. pica and to examine aspects of its reproductive periodicity. This information could be applied to management and possible mariculture of the species.

MATERIALS AND METHODS

This study was conducted from June 1990 through June 1991 at Lee Stocking Island (23°46'N, 76006'W) in the southern Exuma Cays, Bahamas. The islands in this area are uninhabited, but topshell harvesting does occur. The intertidal habitat in the Exumas is described by Debrot (1990b). The tides in the Exuma Cays are semi-diurnal with a tidal range of approximately 1.5 m. Daylength for the study area was calculated (The Astronomical Almanac, 1990). Seawater temperature was monitored from May 1989 to February 1991 using a recording thermograph (Ryan Tempmentor, Ryan Instru- ments), located on the bank at a depth of 2.8 m. From June to November 1990, weekly collections of approximately 75 adult C. pica were made from islands within 3 km of Lee Stocking Island. Spontaneous spawnings could be observed when holding animals for I week in outdoor water tables equipped with flow-through running seawater. Alternating low-tide and flood-tide environments were simulated in these tanks as follows: animals were maintained dry for periods typically ranging from 3-36 h then flooded with fresh seawater, and maintained as such for 1-2 days. These dry/wet cycles were continuously repeated for all collections, and included all moon phases of each month during the 5-month period. All water tables were continuously drained through 125 !tm mesh nets that were checked twice daily for egg release. Animals were returned to their collection site after I week. Larval development of C. pica was described from eggs spontaneously spawned or from newly- released eggs that were fertilized in small glass dishes. Fertilized eggs were maintained indoors in fresh seawater in glass or plastic containers, without aeration, at an ambient temperature of 26.5-27 .5°C. Specimens were preserved in 5% buffered formalin in seawater, and drawings oflive specimens, noting live coloration, were made. Hatching, used herein, will refer to the emergence of larvae from the egg membrane. Once the larvae began to crawl they were placed in biofouled petri dishes, containing a bacterial/algal film, to induce metamorphosis. The dishes had been hung outdoors in seawater for 1- 2 weeks. A growth equation was obtained from regular measurements of reared juveniles maintained in heavily fouled dishes in the laboratory through mid-November, when summer field work was completed. Drawings of preserved embryos and larvae were made using a camera lucida attachment on a Wild compound microscope. Measurements of specimens were made using a calibrated ocular micrometer. Developmental times are described as min or h post-fertilization. A settlement experiment was conducted to determine the effect of unfouled and biofouled substra- turns on the occurrence and timing of metamorphosis. Five sterile and five biofouled dishes were used for the two treatments. Each dish was filled with I !tm-filtered natural seawater, stocked with 20 2-day-old post-torsional veliger larvae and maintained at an ambient temperature of 26.5-27.5°C. The water in each dish was changed daily and the number of metamorphosed and non metamorphosed larvae, as well as survival, were scored for 5 days, until mean survival was less than 50% for each treatment. Results were analyzed comparing the number of metamorphosed individuals in each treatment on day 7 using the Mann-Whitney U test (Sokal and Rohlf, 1981). Beginning July 1990, weekly collections of 10 or more C. pica were made on or near days of the moon phases for histological examination of the ovaries. These weekly collections continued through the new moon in November. Samples of approximately 30 animals were then collected monthly on or near the new moon from November 1990 through June 1991. A total of 27 collections were made during the 12-month period. Specimens were processed in the laboratory on the day of collection. Shell length and width measurements (Debrot, 1990a) were taken for each animal and shells were cracked and animals sexed, when possible, upon the basis of gonad color. Ovaries were green and testes a cream color. Gonads were preserved in 10% buffered formalin in seawater for a minimum of one month, transferred to 35% ethanol for one week, and then placed in 70% ethanol for storage. Collections were made at numerous locations in an attempt to avoid greatly reducing the population of C. pica at anyone island. Five females between 40-55 mm shell width, from each of the 27 collections, were randomly selected for histological analysis. A cross-section of the visceral coil (gonad and digestive gland) was taken from each specimen just behind the gastric cecum, embedded in paraffin, sectioned at 7 !tm and stained with hematoxylin and alcoholic eosin. A minimum of 50 oocytes per animal was measured at 100 x magnification using a computer video image analysis system (Java, Jandel Scientific). Only pre-vitellogenic and vitellogenic oocytes in which the nucleolus was visible were measured. The area of each cell was measured and then converted to 252 BULLETIN OF MARINE SCIENCE, VOL. 51, NO.2, 1992 a theoretical diameter, as if the cell was perfectly round, Relative size-frequency histograms of oocyte diameters were averaged for the five animals of each collection. Data on oocyte sizes were analyzed following Grant and Tyler (1983), A Kruskal-Wallis test was performed to compare mean oocyte sizes while a Kolmogorov-Smirnov two-sample test for goodness of fit was used to compare sequential distributions of oocyte sizes among samples. Recruitment was studied by monitoring the appearance of juveniles, 1-2 mm width, into the population at four sites of moderate wave exposure from July 1990 to May 1991. This monitoring was done monthly from July to November 1990, and bimonthly thereafter. Each site was marked at 4-m intervals along a 20-m transect using underwater epoxy. All ::520-mm-wide C. pica found within each transect between the high-water and low-water spring tide marks were collected. Animals were measured on site and returned to the same 4-m quadrant from which they were collected,

RESULTS Spawning. -c. pica released eggs and sperm into the water where external fertilization occurred. In all instances where spawning was observed, sperm release was seen before release of eggs. Not all animals were found to release gametes, but in all cases at least several animals of each sex spawned. Spawning topshells were found in all areas of the water tables, from just below the surface to crawling on the bottom. Male C. pica releasing sperm were observed both individually and aggregated together with other C. pica. Males released sperm either gradually, in a slow, steady stream, or in dense clouds. Spawning females were detected when they were removed from the water and eggs gushed out from the aperture when they retracted into their shell, or when a stream of eggs could be seen in the water. Often animals were clumped together and it was difficult to determine which one was releasing the eggs. Females placed in glass bowls usually continued to release eggs in a slow, steady stream; the eggs were either suspended in the water or accumulated at the base of the female. Eggs were slightly negatively buoyant and most sank to the bottom in still water, but were easily suspended by only slight water movement. There was no evidence of intentional pairing or aggregating of spawning animals within the tank, and no obvious behavioral patterns associated with spawning were observed. While collective spawning lasted from 3-5 h on the two occasions it was observed in its entirety, the duration of spawning of individual topshells was not determined. C. pica spawned unpredictably in outdoor water tables during most hours of the day and night. Out of72 simulated low tides between 20 June and 4 November 1990, nine spawnings occurred with spawning in each month except November. Five of the spawnings occurred within 4 days of the new moon, three within 4 days of the full moon, and one within I day of the last quarter moon, in August. There was no apparent relationship between the duration of the simulated low tide and occurrence of spawning. Spawning never occurred when animals were maintained in continuous running seawater for weeks at a time, nor could it be induced with any predictability by simulating low-tide environments. Mean daily seawater temperatures on days which spawning occurred ranged from 28.7-30.9°C. Temperature of running seawater in the water tables was the same as in the field. Spawning could not be induced using serotonin, which stimulates bivalves to spawn (Matsutani and Nomura, 1982; Braley, 1985) or hydrogen peroxide, which induces gamete release in abalone (Morse et al., 1978). In several trials in June and July, 1 ml of 1 mM serotonin in seawater was injected directly into the gonad of male and female topshells, through a small hole drilled in the shell without disturbing the tissue inside, which resulted in increased movement of the animal but no gamete release. In addition, in a trial run in August, C. pica showed no reaction when held in buffered seawater containing a final hydrogen peroxide concentration ranging from 0.001 M to 0.01 M. BELL: TOPSHELL REPRODUCTION AND LARVAL DEVELOPMENT 253

Table 1. Stages of embryonic and larval development of Cittarium pica reared at 26.5-27.5°Cin the laboratory (sizes given are the widest diameter or width)

Developmental stnge Size (pm) Time after fertilization Unfertilized egg 170-175 o min Inner jelly layer 195-210 o min Outer jelly layer 340 o min Release of polar bodies 10 min* 2 cells 195 30 min 4 cells 195 60 min 8 cells 90 min 16-32 cells 2-3 h Gastrula 6-8 h Trochophore 200 8 h Shell cap 9 h Hatching (see text) 9-11 h Pre-torsional veliger 190t 17-22 h Post-torsional veliger 270-280t 23-26 h Crawling veligers 60 h Metamorphosis 270-280t 3'/:z-4I/2 d • After addition of spenn. t Shell width al widesl point.

Spawning of C. pica in the field was not observed, nor was there any indirect evidence of its occurrence. Also, no C. pica embryos or larvae were found in 32 biweekly plankton tows made between August and October. However, larvae of at least three other topshell species were collected in these tows, and reared to at least 1 mm shell width to confirm the species identification. Larvae of Tegula fasciata were often collected, as unhatched trochophores, hatched trochophores or veligers. They differed from C. pica larvae in that T. fasciata trochophores lacked the green dots above the prototroch, veligers had a clear to very pale green velum and the fully-formed larval shell was slightly smaller, measuring 240 J,Lm (see below for comparisons). Larval Development. -A summary of the embryonic and larval development of C. pica is given in Table 1. In the undisturbed ovary, oocytes were misshapen and surrounded by a single jelly layer of albumen, a characteristic of archaeogastropod oocytes (Fretter and Graham, 1962). When spawned, eggs were nearly spherical, measuring 170-175 J,Lm in diameter, and were surrounded by this inner jelly layer and, additionally, an outer jelly layer (Fig. lA). In artificial fertilization trials, the elevation of the outer jelly layer occurred immediately upon contact with seawater, before fertilization. The inner jelly layer measured between 195 and 210 J,Lm in diameter, and the outer approximately 340 J,Lm in diameter. The outer jelly layer was not as distinct as the inner one and disappeared about 30 min after the egg was released into seawater. Individual eggs were a melon-green color with a dark green splotch at the animal pole. Fertilization was rapid, with the release of polar bodies occurring as early as 10 min after the addition of sperm. The first cleavage occurred about 20 min later (Fig. 1B). The first two cleavages were typically holoblastic, whereas subsequent ones were unequal. Dextral spiral cleavage was observed, and cell divisions occurred about every 30 min. The dark green pigment at the animal pole was divided at the first two cell divisions, with the result that new cells also had the pigment (Fig. IB, C). At the 8-cell stage, the four micromeres situated at the animal pole contained the dark green pigment. Embryos contained 16-32 cells 2-3 h after fertilization. At the 16-cell stage, the macromeres at the vegetal pole of the embryo 254 BULLETIN OF MARINE SCIENCE, VOL. 51, NO.2, 1992

vitelline membrane

inner jelly layer B polar bodies

c D

apical tuft '\

E F 100 pm Figure 1. Embryonic and larval stages of laboratory-reared Ciuarium pica at 26.5-27.5°C in the Bahamas, A: Newly-spawned egg showing two jelly coats surrounding the egg. B: Two-cell stage, 30 min post-fertilization. C: Four-cell stage, 60 min post-fertilization. D: Early trochophore, prehatching, 7-8 h post-fertilization. E: Trochophore with shell cap, 9 h post-fertilization. F: Advanced trochophore, 10 h post-fertilization, most common hatching stage. began to acquire a light turquoise color. By 4.5 h, past the 32-cell stage, the green pigment had the appearance of small dots aggregated on the animal pole side of the embryo, while the other side was turquoise. A small depression at the embryonic animal pole, or an apical invagination (Robert, 1902, cited in Underwood, 1972), was evident at 5-6 h but disappeared within the subsequent hour. At 6- 8 h, gastrulation by epiboly occurred, and cilia developed. By 8 h, the prototroch was well-developed, an apical tuft present and the cilia were beating (Fig. 1D). Green-pigmented dots were restricted to the area above the prototroch, while the turquoise area was below. By 8-9 h, the trochophore was spinning within the egg BELL: TOPSHELL REPRODUCTION AND LARVAL DEVELOPMENT 255 membrane in an irregular manner. Polar bodies were visible within the egg membrane until hatching occurred (Fig. 1D). Hatching occurred between 9 and 11 h at the trochophore stage. The earliest occurred before the shell could be discerned, while some larvae hatched with a small shell cap already formed (Fig. IE). Most often, however, the trochophore had a more developed larval shell at hatching, covering about two-thirds to three- fourths ofthe posterior end ofthe larvae, with a slight bulging of prop odium (Fig. 1F). The larval shell at all stages was transparent white, marked with irregular, broken spiral striations. Larvae were active swimmers, with much vertical movement. The pre-torsional veliger, with a well-developed velum, was noted between 17 and 22 h (Fig. 2A). The propodium was large, and the larval shell had a distinct whorl. The velum was round and single-lobed, resembling that of a bivalve veliger. As the velum developed, the green pigment aggregated around the outside of the velum in large, elliptical spots with the smaller dots still present in the prevelar area (Fig. 2B). The turquoise pigment was concentrated in the digestive gland area and indicated the larva's yolk supply. At 23-26 h the larval shell was complete, having one complete whorl, and measured 270-280 /.tmat its greatest width (Fig. 2B, E). The operculum was well-formed, the mantle cavity evident and the larva could withdraw completely into the shell. Two tiny black eye spots were apparent on the prevelar area, and torsion was complete (Fig. 2B). Coloration had not changed appreciably. Veligers provided with unicellular brown algae, Isochrysis galbana, showed no uptake of this food, suggesting lecithotrophy. The velum of the veliger gradually became bilobed between 24-48 h and was accentuated by two rings of green chromatophores, but it remained small. The eye spots became larger and more distinct, and the foot became noticeably larger. Between 48 and 72 h, the velum had distinct, dark green "rims" on the outer side of each lobe, and small protuberances of the tentacles were evident near the eye spots. Larvae on the bottom of the dish were often seen with their foot, now ciliated, outstretched and "searching" for the substratum. By 60 h many larvae were crawling, with the twirling velum protruding from beneath the larval shell opening (Fig. 2C). Larvae altemated between swimming and crawling during this period. At about 3 days, the foot became very sticky, and the larvae could attach quickly to a substratum.

Metamorphosis. -Metamorphosis in biofouled dishes occurred as early as 3.5- 4.5 days post-spawning. It was characterized by shedding of the velum and elon- gation of the tentacles. Metamorphosed individuals were easy to detect if crawling; the tentacles protruded from the shell opening (Fig. 2D, F), in contrast to the non- metamorphosed, crawling individuals. Shell measurements of newly-metamorphosed juveniles were not significantly different from those of the post-torsional veliger (N = 14 and N = 30, respectively), indicating that further growth of the protoconch had not occurred. The digestive gland area still retained turquoise pigment after metamorphosis. Larvae rarely metamorphosed in unfouled dishes, viz., once each at 4 d and 10 d post-spawning. Typically, larvae in unfouled dishes gradually assumed a more benthic mode, and the velum became reduced. Evidence of ingestion of algae prior to metamorphosis in these animals was never observed, and the turquoise-pigmented yolk/digestive gland gradually disappeared. Larvae maintained in unfouled dishes typically lived up to 10-14 d. In a settlement experiment, C. pica metamorphosed faster when on a biofouled substratum than on an unfouled substratum (Fig. 3). Survival did not differ be- 256 BULLETIN OF MARINE SCIENCE, VOL. 51, NO.2, 1992

eye spots ~velum~

propodium /

shell A B shell

eye spots

operculum c E

eye spot

\ operculum G 100 pm Figure 2. Larval and post-metamorphic stages of laboratory-reared Ciltarium pica at 26.5-27.5°C in the Bahamas. A: Pre-torsional veliger, 17-22 h post-fertilization. B: Post-torsional veliger, 23-26 h post-fertilization. C: Crawling veliger, 60 h post-fertilization. D: Crawling post-metamorphic juvenile, 3.5-4.5 d post-spawning. E: Larval shell. F: Post-metamorphic juvenile, 3.5-4.5 d post-spawning. G: Juvenile topshell, 10 d post-spawning. tween treatments and was low with less than 50% of the larvae surviving at 7 days of age, after which the experiment was terminated. Even so, the effect of substratum type on timing of metamorphosis at day 7 was significant (Mann-

Whitney U: U = 0, nl = 5, n2 = 5, P = 0.004). BELL: TOPSHELL REPRODUCTION AND LARVAL DEVELOPMENT 257

100

-0 7 --- Q) .. til ----e-, 0 :s:: 6 CD .!: 75 0 Q. •. -- .• Survival :J •.... 5 0 -0 6. - 6. Metamorphosed CD E .I: -0 III ..., 0 4 biofauled CD () +- ..., Ci (]) Q) A .-- A Metamorphosed 50 I... 0 :J ::E Q) 3 -+- a. un fouled III 0 "3 <.n +- C 0 2 ..., l- < 25 <' e 0 0 Q) ::E o a 3 456 7 Age Post-spawning (Days) Figure 3. Mean number of metamorphosed individuals of laboratory-reared Cittarium pica larvae (26.5-27.5"q in biofouled (open triangle) and un fouled (shaded triangle) dishes from 3-7 days post- spawning. Mean survival oflarvae or metamorphosed juveniles in all dishes is indicated by the dashed line (closed circle).

Juvenile Growth and Survival. - The first growth of the juvenile shell was evident at 6.5 d. The new juvenile shell was white, not as transparent as the protoconch, and was marked distinctly with microscopic pits and five thin spiral ridges (Fig. 2G). The turquoise digestive gland area became paler every day following metamorphosis, and on 7.5 d the first ingested food was noted, detected by a gold color in the gut. This gold color expanded and darkened daily, and the juvenile shell became less transparent. The shells ofthese small C. pica remained smooth and opaque white until about 1.2 mm in width, when they often began to get the first black spot on the shell. By 1.8-2.0 mm the spiral ridges typical of the juvenile shell began to form. At 2-3 mm the juvenile shells were white with several black spots and had three distinct spiral ridges. A mean growth rate of 10.8 J.Lm·day-1between 6-90 days age (post-spawning) was estimated using the following growth equation for laboratory-spawned and reared C. pica: Log size (J.Lm)= 2.48 + 0.00682 Age (days)

(R2 = 0.881, N = 492) (Bell, submitted). I Survival post-metamorphosis to 30 days age was approximately 57.3%, and 16.6% at 90 days. The low survival of post-metamorphic C. pica was in part due to desiccation of animals apparently caught in the miniscus in the rearing dish. Reproductive Periodicity.-Small oocytes, 10-20 J.Lmin diameter, were typically present in the gonad of C. pica year round, while larger oocytes, 140-150 J.Lm diameter, were present only during the summer and fall months, peaking in July-

I Bell. L J. Maricullure prospects for the West Indian topshell, Curarium pica. Prac. Gulf and Carib. Fish Inst. Submilled. 258 BULLETIN OF MARINE SCIENCE, VOL. 51, NO.2, 1992

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Jan 1991

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Oocyte Diameter (J.lm) Figure 4. Relative frequency of size classes of oocyte diameters (/Lm) of Ciuarium pica collected on or near the new moon between July 1990 and June 1991 in the southern Exuma Cays, Bahamas. N = 5 animals per month; each frequency distribution is an average of 5 distributions; N = 50-60 oocytes per animal.

September, with a noticeable decline in frequency in October (Fig. 4). Sequential relative frequency distributions were significantly different between September and October (P = 0.004), October and November (P < 0.001) and January and February (P = 0.041). Mean oocyte size peaked in August and September, declining thereafter (Fig. 5). Mean oocyte diameters among the 12 months were significantly different (Kruskal-Wallis: H = 37.15, df= 11, P < 0.001). Variation BELL: TOPSHELL REPRODUCTION AND LARVAL DEVELOPMENT 259

180 "...... E 160 ::t '--' l..- 140 v .-v 120 E T 0 , 100 1 0 , • 1 v 80 r .->- • u 1 0 GO • 1 ! 0 1 f • c • T I 0 40 • v T ~ •1 • 20 1 I •1 • • 1 0 Jul A S 0 N 0 J F M A M Jun 1990 1991 Month of Year Figure 5. Mean monthly oocyte diameters (J!m) of Cittarium pica collected on or near the new moon between July 1990 and June 1991 in the southern Exuma Cays, Bahamas. N = 5 animals per month; N = 50-60 oocytes per animal; error bars indicate ± 1 standard deviation. in oocyte size between individuals within each monthly sample was greatest during the declining months of October-January and smallest during the ripe summer and nonripe winter and spring months. Weekly gonad samples indicated a gradual ripening of oocytes from July to October with a significant decline in mean oocyte diameter specifically between 6-13 October (Fig. 6). Yisually, gonads were full of ripe oocytes during July-September. Typically during February-May, oocytes were small and attached to the lumen wall with much empty space in the gonad. Mean monthly and weekly oocyte diameter was significantly correlated with mean seawater temperature (Spearman Rank Correlation: rs = 0.929, df = 6, P < 0.05 and rs = 0.663, df= 18, P < 0.005, respectively). Daylength was inversely correlated with mean monthly oocyte diameter (Spearman Rank Correlation: rs = -0.731, df= 10, P < 0.01). Recruitment. -A large influx of juvenile C. pica, between 1 and 2 mm shell width, was seen in January 1991 (Fig. 7), with all four sites exhibiting similar patterns. All sites indicated a low occurrence of 1-2 mmjuveniles until the January sample. At low tide the small 1-2 mm juveniles were found just above the low-tide bench (where the sea urchin Echinometra lucunter was common) up to the high tide level, the same zone where small «20 mm) C. pica were typically found. Most were cryptic, occurring in holes or crevices, or under a tuft of macroalgae. The smallest ones (1.0-1.5 mm) were all white, sometimes partly covered by encrusting coralline algae. Juveniles larger than about 1.5 mm typically had at least one black spot on the shell. The mean sizes of the new January cohorts were not significantly different among Sites 1-3 (ANOYA: F = 0.55, df = 2,146, P = 0.576) (Site 4 was not used due to an irregular cohort distribution). Recruitment cohorts from the 3 sites were therefore combined and a mean size of 1.83 ± 0.65 mm (N = 149) was calculated. 260 BULLETIN OF MARINE SCIENCE, VOL. 51, NO.2, 1992

180 ,...... 160 E 0 0 0 0 0 ::l • • • • • '--'" 140 ~ (!) 1 120 .-(!) T T T E T • T • 1 0 100 ~ I • •1 •1 0 • 1 r •1 (!) 80 •1 1 .->- r 1 • u d 1 0 60 • 1 1 • 0 j lId c 40 r T 0 1 1 (!) • • ~ 20 1 1 T • T 1 0 1 7 142228 4 11 1825 1 8 152229 6 13 2027 3 10 17 July Aug Sept Oct Nov Figure 6. Mean weekly oocyte diameters (JLm) of Cittarium pica collected on or near each moon phase between 7 July and 17 November 1990 in the southern Exuma Cays, Bahamas. N = 5 animals per month; N = 50-60 oocytes per animal; error bars indicate ± I standard deviation; open circles above: full moon; closed circles above: new moon.

The age of this size juvenile was estimated, and the spawning date then back- calculated, using the above growth equation from laboratory-spawned and reared C. pica. An age of 114.7 days was calculated and the estimated spawning date, back-calculated from 17 January 1991, was 24 September 1990.

DISCUSSION Spawning. -Fertilization of archaeogastropod eggs is external (Fretter, 1984). In trochids, eggs are laid in ribbons or jelly masses on the substratum (Gohar and Eisawy, 1963; Holyoak, 1988b) or broadcast into the water (Eisawy, 1970; Un- derwood, 1972; Heslinga, 1981). The spawning of C. pica belongs to the latter group; eggs and sperm are broadcast into the water and fertilization follows. In C. pica, the sex which initiates spawning was not confirmed. Sperm release was the indication of spawning of C. pica in all cases where spawning was observed in the present study. In many broadcast spawners, the males initiate spawning and the sperm induces the females to spawn (Thorson, 1950). In contrast, Un- derwood (1972) found that for the trochid Gibbula cineraia, females sometimes spawned first in tanks. Because spawning occurred in water tables during most hours of the day and night, there was no suggestion of any die 1 periodicity to spawning. On a larger time scale, the present study might suggest a lunar periodicity to spawning, with eight of the nine spawning incidences in water tables occurring around the new or full moon. Total spawning occurrence in water tables was low, however, and conclusions must be viewed cautiously. The spawning event observed by Castell (1987) occurred 3 days after the new moon, in late June. Larval Development. - Trochid larvae of eggs that are broadcast-spawned (Eisawy, 1970; Heslinga, 1981; Holyoak, 1988a), and some which are laid in egg ribbons BELL: TOPSHELL REPRODUCTION AND LARVAL DEVELOPMENT 261

80 60 23 Jul 1990 n = 19.3 '0 20 80 0 30 Aug 1990 80 60 n = 409 60 18Au91990 .0 n = 206 '0 20 20 0 0 80 80 30 Sep 1990 60 n - 29.3 60 18 Sep 1990 40 n = 239 '0 20 20 !!l 0 0 0 80 :J 80 4 Nov 1990 "0 60 n ~ 265 60 19 Oct 1990 :~ n :; 272 .0 "0 '0 E 20 '0 0 ~ 80 80 10 No .•• 1990 .D'" n ~ 267 E '0 :J 20 Z 0 80 19 MOr" 1991 18 Jon 1991 60 n;:;; 284 n .0 = 190 20 0 80 80 9 Moy 1991 '0 120 n '"" 473 20 100 0 80 80 60 60 '0 ADO20 012 J 4 5 6 7 B '3 IOI1121Jl.1~161718192021 012 J 4 5 5 7 B 910'1'21)14'5161718192021 Shell Width (mm) Shell Width (mm) Figure 7. Size frequency histograms of Cillarium pica between 1-20 mm shell width at two recruitment sites in the southern Exumas, Bahamas, monitored from July 1990 to May 1991. A) Site 1. B) Site 2. or gelatinous masses (Gohar and Eisawy, 1963), go through a brief planktonic stage, whereas some other benthic trochid eggs are direct developers (Fretter and Graham, 1962; Holyoak, 1988b) (Table 2). The embryology and larval development of C. pica are similar to those of other broadcast-spawning trochids as described by Desai (1966), Eisawy (1970) and Underwood (1972). Larval feeding was not observed in C. pica. Archaeogastropod larvae are not planktotrophic because the veligers lack the physical mechanisms for planktonic feeding (Strathmann, 1978). Some archaeogastropod veligers, however, do feed as benthic larvae (e.g., Haliotidae: Ha/iotis tuberculata, Strathmann, 1978), as evidenced by the color of the digestive gland, which is affected by the larval food (Strathmann, 1987). C. pica has a dense, yolky egg, and the presence ofthe yolk- coloration in the gut area from the veliger stage through metamorphosis, even though algae was provided as food (planktonic and benthic), suggests that the larvae live off yolk stores throughout this period. Additional larval nutrition may also be derived from dissolved organic matter in seawater, as demonstrated for the lecithotrophic larvae of Ha/iotis rufescens (Jaeckle and Manahan, 1989). The melon-green coloration of C. pica eggs is strikingly similar to that of all other broadcast-spawned trochid eggs reviewed, which are green (Table 2). In contrast, none of the eggs spawned in benthic ribbons or egg masses are green. The advantage of green eggs is unclear; for C. pica, a light colored melon-green egg might be more cryptic in the water column than a white egg. The advantage of cryptic eggs is more obvious; an egg must be large and yolky, and thus opaque, 262 BULLETIN OF MARINE SCIENCE, VOL. 51, NO.2, 1992

Table 2. Stage at hatching, color of eggs and time to metamorphosis in days (ambient water temperature in parentheses) for species of temperate and tropical gastropods of the family Trochidae with planktonic and benthic eggs. direct dev: direct development

Time to meta- Hatching Egg color morphosis ("C) Reference Planktonic Eggs Calliostoma annulatum* veliger green ? Strathmann, 1987 C. ligatum* veliger green 12 d (7-9) Strathmann, 1987 Ciltarium pica trochophore green 31/2-4'12d Present study (26.5-27,5) Gibbula cineraia trochophore green 9 d (12) Underwood, 1972 G, umbilicalis ? green ? Underwood, 1972; Fretter and Graham, 1977 Monodonta dama ? green ? Hulings, 1986 M.lineata veliger green 7 d (15-20) Desai, 1966; Fretter and Graham, 1977 Tegula brunnea ? green ? Belcik, 1965 T.Jasciata trochophore green ? Present Study T. Junebralis* ? green ? Strathmann, 1987 Trochus dentatus trochophore blue-green 3-4 d (27,5) Eisawy, 1970 T. niloticus trochophore green 3 d (27-30) Heslinga, 1981 Benthic Egg Ribbon or Mass Calliostoma zizyphinum direct dey yellowish 7-8 d (?) Fretter and Graham, 1977 Cantharidus exasperatus direct dey white 5-6 d (?) Fretter and Graham, 1977 C. striatus direct dey white ? Fretter and Graham, 1977 Euchelus gemmatus direct dey brown ? Ouch, 1969 Margarites helicinus = direct dey orange-red 12 d (7-9) Holyoak, 1988b M. marginatus Trochus erythraeus veliger white ? Gohar and Eisawy, 1963 • Eggs are in soft, gelatinous coat; eggs disperse with water movement (Strathmann, 1987; Holyoak, 1988a). to provide enough energy for the development of a lecithotrophic larva, and any camouflage would appear to increase the survival of the egg or larva through reduced predation. Metamorphosis. - Cittarium pica were capable of metamorphosing 3]12days after spawning, at 26.5-27.5°C. This observation is similar to that oftwo other tropical broadcast-spawning trochids, Trochus niloticus (Heslinga, 1981) and T. dentatus (Eisawy, 1970), and considerably faster than times for temperate species (Table 2). The life span of the planktonic archaeogastropod larvae is typically short (Webber, 1977; Fretter, 1984). The duration of the planktonic larval stage is partly limited by the larvae's yolk supply and by temperature wherein shorter larval stages are correlated with higher seawater temperature (Kinne, 1970; Fretter, 1984; Strathmann, 1987). The settlement experiment conducted indicated that larvae metamorphosed faster when provided with a suitable substratum (biofouled dish) than an un- suitable substratum (unfouled dish). As with other herbivorous intertidal gastropods, C. pica probably shows little substrate specificity (Heslinga, 1981). This finding is in contrast to other gastropods for which specific substrates are required to induce settlement (Scheltema, 1961; Hadfield, 1976). BELL: TOPSHELL REPRODUcrION AND LARVAL DEVELOPMENT 263

Juvenile Growth Rates. - Growth rates of C. pica in the field have been documented for juveniles (> 1.5 mm) and adults (Randall, 1964; Debrot, 1990b). The present data are the first estimates of growth rates for post-metamorphic juveniles. Es- timated growth rates for older, 1.5-8.2 mm, juveniles and 5.8-35.7 mm C. pica are 0.0372 mm/day and 0.0597 mm/day, respectively (calculated from Randall, 1964), which are higher than those estimated presently for 6-90 day old juveniles (0.0108 mm/day). Reproductive Periodicity. - Data presented herein suggest an annual reproductive season of July to October with an early October spawning period for C. pica in the Bahamas. Weekly gonad samples did not reveal any intra-seasonal periodicity to spawning prior to October. The greater variation in oocyte size in October- January compared to the summer months, in conjunction with the gradual decline in frequency of large oocytes, suggests that some females retained large oocytes throughout these months and that spawning might not have been completely synchronous. Although animals did not spawn in water tables after 20 October, occasional spawning could well have occurred into January. The significant change in oocyte-size distribution between January and February, due to a decrease in large oocytes, suggests the start of a non-reproductive period between February and July, when oocytes once again began to mature. Debrot (1990a) described a reproductive periodicity for C. pica in the Exumas, Bahamas, similar to that found in the present study, using gonad indices of both sexes. A different pattern of reproduction for C.pica was observed at the Caribbean islands off Venezuela. Using gonad indices, Castell (1987) suggested two spawning periods each year, between the winter and spring months, and between May and June. Her data of gonad indices and oocyte diameter frequencies appear to be more consistent with those of a continuously breeding population. Recruitment. - The fall recruitment peak of 1-2 mmjuvenile C. pica first detected in January 1991 corresponded closely with the suggested spawning period in early October 1990. Differences in food type and abundance, and ambient temperature available to laboratory-reared and field C. pica, would influence growth rates and could account for any variation between the inferred and extrapolated spawning period. C. pica were reared at a temperature of 26.5-27.5°C, while temperatures in the field ranged from 29.0°C in October 1990 down to 23.9°C in January 1991. The single annual influx of C. pica juveniles further supports the occurrence of a single period of spawning in early October, as opposed to multiple peaks of both. The chance that the January juveniles of 1-2 mm were the result of the October spawning period is high. Because the planktonic phase of C. pica larvae is short (about 2.5 d), the planktonic dispersal abilities must be limited. Debrot (1990a) stated that recruitment of juveniles in the Bahamas peaked in the summer and coincided with the peak in reproduction. His "recruitment peaks," measured in October of two consecutive years, had mean shell widths of 7 and 13 mm (Debrot, 1990a). Small C. pica, less than 5 mm width, were not apparent on his size-frequency distributions, and I suggest that his summer recruitment peaks were comprised of one-year olds. Randall (1964) inferred a similar spawning season for C. pica in the Virgin Islands from the presence of small «4 mm width) juveniles which were found during all months of the year at a low frequency. She found a large influx ofthem, with a mean size in the 1-1.9 mm size class, in mid-January and concluded that a low level of spawning probably occurred for most of the year, with a peak "some weeks" before the January peak in recruitment. 264 BULLETIN OF MARINE SCIENCE, VOL. 51, NO.2, 1992

Reproduction and its Relation to Environmental Factors. - The annual reproductive cycle of C. pica in the Bahamas showed a significant correlation with the annual seawater temperature cycle and daylength. Gametogenesis in marine invertebrates has been related to a change in seawater temperature (Kinne, 1970; Himmelman, 1978) and photoperiod (Himmelman, 1979; Pearse and Eernisse, 1982; Pearse et al., 1986). A review of temperature and daylength at different latitudes within the range of C. pica suggests that both might influence gametogenesis in this species. The southern Bahamas (Lee Stocking Island) has an annual mean seawater temperature range of approximately 6°C and an annual daylength range of 3 h 4 min, while the Virgin Islands (St. John) has an annual mean temperature range of approximately 5°C (Caribbean Marine Research Center, unpubl. data) and a daylength range of 2 h 22 min. In these two areas the reproductive cycles of C. pica appear similar. In contrast, the southern Caribbean, such as the Los Roques Archipelago, Venezuela, has an annual mean seawater temperature range of 2-3°C (Oceanographic Monthly Summary, 1990-1991) and a daylength range of 1 h 32 min. Castell's (1987) evidence of a continuous breeding season for C. pica in Venezuela may be related to the more consistent temperature and daylength, while more variable temperature and photoperiod further north might provide a cue for seasonal gametogenesis in the Bahamas and Virgin Islands. It is well-known that many marine organisms spawn on lunar cycles. Korringa (1947), Webber (1977) and Fretter (1984) specifically report lunar periodicity as a spawning cue in many prosobranch gastropods. Monitoring spawning of C. pica in the Bahamas during a different year with a similar temperature and daylength regime, but shifted moon phase, would be necessary to distinguish between the effect of these factors and moon phase on its spawning. Further speculation of the effect of environmental factors on spawning of C. pica are presented by Colin (1991). The short planktonic life of C. pica is typical oftrochid larvae (Webber, 1977), and consequently their occurrence in the plankton is not common (Fretter, 1984). Heslinga (1981) attributed the limited indigenous range (locations where it has not been introduced) of Trochus ni/oticus in the Pacific to its limited larval dispersal ability. It has, however, been successfully introduced to many Pacific islands (Heslinga, 1981). C. pica is known from all the islands of the Caribbean and Bahamas. Interestingly it is known only from fossil shells in Bermuda and in the Florida Keys it is very rare (the most recent live specimen reported was from 1975) (Clench and Abbott, 1943; Abbott, 1976); it is likely that its absence is contributed to by the limited larval dispersal of the species. Attempted reintrod- uctions of C. pica to Bermuda and the Florida Keys were unsuccessful (Verrill, 1901; Clench and Abbott, 1943; Abbott, 1974), however a successful reintroduction is presently underway in Bermuda (Wingate, 1989; 1990). Management of C. pica fisheries in the Caribbean is not common. Presently only the U.S. Virgin Islands have a designated collecting season and minimum size for C.pica (Govt. U.S. Virgin Isl., 1991). The short larval life and concomitant limited larval dispersal ability imply C. pica populations are self-recruiting and strict regulations are therefore needed to prevent local overfishing. An alternate approach to reintroduction is mariculture, which could provide large numbers of spat, or juveniles, for release in the field. The present study suggests that mariculture of C. pica could be feasible (see also Bell, submitted).1 The need for mariculture of the West Indian topshell will grow as overexploitation persists and the natural stocks continue to dwindle. Trochus niloticus, one of the common intertidal trochids throughout the tropical Pacific, has been successfully cultured for many years (Heslinga and Hillmann, 1981; G. Heslinga, pers. comm.) BELL: TOPSHELL REPRODUCTION AND LARVAL DEVELOPMENT 265 and there are no reasons to suggest that the same cannot be accomplished for C. pica.

ACKNOWLEDGMENTS

I would like to thank the Caribbean Marine Research Center and its staff for support of this project. The following persons are thanked for help with various aspects of the work: C. Bowling, J. Elliott, C. Kammiro, E. Maddox, D. Mansfield, E. Martin, S. Pearl, D. Prescott, B. Sabol, V. Sandt, and A. Stoner. D. Watson and A. Black are gratefully acknowledged for their generous help with the histology, and I would like to thank R. Wicklund (CMRC) and A. Debrot for providing the temperature data. I would also like to thank Drs. W. Heard, C. Young and W. Herrnkind for their time and input, and to Dr. Heard again for some financial support. Comments of two anonymous reviewers are greatly appreciated. Finally, I would like to thank P. Colin for his unlimited support.

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DATEACCEPTED: April 6, 1992.

ADDRESS:Department of Biological Science, Florida State University, Tallahassee. Florida 32306; PRESENTADDRESS:Coral Reef Research Foundation. P.O. Box 70, Weno, Chuuk State, Fed. States of Micronesia 96942.