Aquaculture, 25 (1981) 153-159 153 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

INDUCTION OF SPAWNING IN FOUR SPECIES OF BIVALVES OF THE INDIAN COASTAL WATERS

DAVID STEPHEN* and H.P.C. SHETTY

Department of Fishery Biology, College of Fisheries, University of Agricultural Sciences, Mangalore - 575 002 (India)

*Present address: East-West Center, Box 1975, 1777 East-West Road, Honolulu, HI 96848 (U.S.A.)

(Accepted 23 January 1981)

ABSTRACT

Stephen, D. and Shetty, H.P.C., 1981. Induction of spawning in four species of bivalves of the Indian coastal waters. Aquaculture, 25: 153-159.

Spawning was successfully induced in four bivalves, Saccostrea cuccullata, S. commer- cialis, Perna uiridis, and P. indica, by osmotic and biological stimulation. The response to rapid salinity change is considered to have an adaptive value. The ecological implica- tions of this adaptive response are indicated.

INTRODUCTION

In recent years it has become increasingly apparent that salinity is the most proximate environmental factor influencing reproduction in coastal marine and estuarine invertebrates of the southern coast of India. Correlative evidences indicate that (1) long range seasonal fluctuations in salinity are important to the synchronisation of the gametogenic cycle (Stephen, 1980a, b), (2) extreme levels of salinity inhibit gametogenic activity and therefore influence the reproductive pattern (Rao, 1956; Durve, 1964; Stephen, 1980a, b) and (3) rapid salinity changes may stimulate spawning (Homell, 1910; Rao, 1951; Panikkar and Aiyar, 1939; Paul, 1942; Durve, 1965; Stephen, 1978). In a recent report the senior author (Stephen, 1980a) has provided a new status for the salinity-reproduction relationship by terming it Hornell’s rule. It was, therefore, thought that experimental manipulation of reproduc- tion, both gamete maturation and gamete release, could be attempted as a natural corollary to the earlier finding.

MATERIALS AND METHODS

Collections of animals were made along the Mangalore coast. Two species of mussels, Perna viridis (Kuriakose and Nair, 1976) and P. indica (Kuriakose

0044-8486/81/0000~000/$02.50 o 1981 Elsevier Scientific Publishing Company

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and Nair, 1976) and the rock oyster Saccostrea cuccullatu (Born) were taken from rock stacks on the sea shore; another species of oyster S. commercialis (Iredale and Roughley) was obtained from the Mulki estuary. After examin- ing the gonad condition of a few specimens, the rest of the sample was placed in seawater for 24 h conditioning. All experiments were carried out in singly aerated 31 glass troughs. For every experiment with a species, 5 ex- perimental troughs with 1 or 2 controls were used. In each trough (a unit) 4-6 individuals were placed. Water was changed daily and no feed was added. During the night the troughs were drained or the animals were removed in order to prevent spawning. Experiments were carried out at room temperature (27-29°C).

Techniques

1. Rapid salinity changes All salinity changes consisted of a linear fall from 34°/oo to 24 f lo/,, S

over a period of three days. Thereafter the animals were continuously main- tained in the lower salinity. Dilutions were made with stored rainwater or distilled water.

2. Rapid salinity changes in combination with gametes The salinity changes were effected as described above. Live gametes were

obtained by stripping and a thick suspension of the eggs and sperm was made separately in seawater (Loosanoff and Davis, 1963) and 1 ml of this suspen- sion was added (conspecifically) once or twice daily.

3. Stable salinity in combination with gametes Salinity was maintained at ambient field level and gametes were added as

described above.

Rearing

The main reason for rearing the larvae was to observe ontogenic develop- ment and viability of offspring. A small quantity of fertilized eggs was re- moved from spawning units, or eggs were collected from isolated females and fertilized by adding sufficient quantity of sperm. The fertilized eggs were placed in UV sterilised seawater for further development. Rearing of the larvae was continued for about 5 days, during which time they were fed with a culture of Isochrysis galbana. The water in the rearing tanks was changed daily.

Experimental sequence

The major spawning season of these species coincides with rapidly decreas- ing ambient salinities; this salinity change is brought about by the southwest

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monsoon rainfall and drainage. The experiments were mainly intended to elucidate the gonad response by an approximation of this salinity change. Accordingly, the experiments were carried out before and during the onset of monsoon. Initially experiments were carried out during April 1979 to study the effect of salinity changes without the addition of gametes, along with a parallel series with gametes and in which the salinity was maintained at ambient field level (34%, S). Further experiments were conducted during May and consisted of salinity changes without gametes and salinity changes with gametes. An unusually late onset of southwest monsoon provided an opportunity to repeat the experiments. The animals collected during the first 3 days of June were subjected to rapid salinity changes, while animals collected during the weekend after a few initial showers of rainfall were stimulated by salinity change and gametes. A considerable decrease in salinity was noticed in the sampling areas during the 3rd week of June, when the spawning was found to have commenced. The animals collected during this period were kept at ambient field salinities and gametes were added.

RESULTS

The results of the experiments are summarised in Table I. Spawning was considered to have been successfully induced when at least one unit in each experimental set up of a species showed response to stimulation.

TABLE I

Summary of the gonad response to osmotic and biological stimulation

Experimental stimulation

Species April ‘79 May

~~~~~.. ___

June

1. Rapid salinity 1. S. cuccullata + + +

change 2. S. commercialis - + (a) + 3. P. virid is + + +

4. P. indica X X +

2. Rapid salinity 1. S. cuccullata X + +

change, with 2. S. commercialis x + (b) + addition of 3. P. viridis X + +

gametes 4. P. indica X X +

3. Stable salinity, 1. S. cuccullata - X +

with addition of 2. S. commercialis - X (cl +

gametes 3. P. viridis - X +

4. P. indica X X +

Field salinities: Stable at 34’/,, during April to May and first week of June (a); decreased

to 28 + 2’/,, during second week of June (b); decreased further to 26 i 2’/,, during third week of June (c). + = spawning; -= no spawning; x = no experiments.

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Spawning

Spawning was initiated by either of the two sexes and was followed by other individuals in the unit commencing sympathetic spawning. Male oysters spawned completely in about 40-60 min; the females took less time. Male mussels shed their gametes intermittently, producing each time two jets of white pasty milt which settled to the bottom and soon dispersed. The females also spawned in a similar fashion, producing two jets of orange coloured eggs. The eggs are spawned in the germinal vesicle stage; they soon dissociate and become round after fertilization. All 4 species responded equally well to both eggs and sperm of conspecifics. Spawning was generally observed after a latent period of 3-5 days.

Rapid salinity change experiment

In this experiment, made in April, spawning was observed in two units of Saccostrea cuccullata after 5 days of maintenance in depressed salinities. The transfer of a spawning male and a spawning female to two separate units led to spawning by the other oysters in these units within 60-90 min. The remaining unit maintained as such for the next 24 h did not show any

response. There was also no spawning in the control unit maintained at 34%0 salinity. In another set of experiments with P. uiridis spawning was observed

on the 4th day in a single unit. Another 3 units responded within an hour after spawning individuals had been transferred to each of them. The remain- ing unit and the control maintained for the next 24 h did not show any response. Experiments with S. commercialis were all unsuccessful, and the experiments were terminated on the 5th day. However, one female spawned in the control unit on the first day.

During May, all 3 species spawned in the laboratory. Spawning was ob- served in 2 units of S. cuccullata on the 4th day, during this time another 2 units responded when spawning individuals were transferred to them from the first 2 spawning units. A remaining unit and the control unit were kept undisturbed for further observations. S. commercialis and P. viridis spawned on the 5th day. Spawning was observed in one unit of each species. As before, another 2 units of both species responded to spawning conspecifics trans- ferred to them. The remaining 2 experimental units and control unit of both species were kept undisturbed for further observation. The experiments were terminated after 24 h when no spawning occurred in any of these remaining units. P. indica was included in the experiments during the first week of June. All 4 species spawned in the laboratory between the 3rd and 5th days of the experiments. Spawning was observed in one unit of each species. Subsequent- ly, 3 units of each species responded to spawning conspecifics. The remaining unit and the control unit of each species did not show any response.

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Rapid salinity change -gamete combination experiment In May, the two oysters and the mussel P. uiridis spawned in the laboratory

between the 3rd and 4th days. Spawning occurred in all 3 species in about 3 h after the addition of gametes. Spawning was observed in all the experimental units. No spawning was observed in the control units, in which salinity changes were made and the addition of gametes was omitted. However, on the 5th day the control units of each species responded to the addition of gametes. From the results obtained in the previous experiments it was con- sidered desirable to add the gametes only on the 3rd day.

P. indica was included in the experiments in the 2nd week of June. All 4 species responded on the 3rd day within 3 h of the addition of gametes. The gonad response was evident in all the experimental units of the 4 species. At the end of the experiment, the control unit of each species also responded to gametes.

Stable salinity -gamete combination experiment During April the 3 species maintained in 34°/oo salinity showed no response

to the addition of gametes during a 5 day period. In May these experiments were discontinued due to the negative response observed. In late June the salinity of the coastal waters had considerably decreased to 26 + 2°/oo. All 4 species were maintained in the laboratory at 26°/oo S. There was no response to gametes on the first day. However sporadic spawning was observed be- tween the 2nd and 4th days. Although spawning was observed in all the 4

species, many individuals did not respond. These individuals were found to be spent, indicating that they had spawned earlier in their natural habitat.

Rearing

Fertilization was 100% in all the 4 species. The fertilized eggs of both the oysters and mussels developed to the first swimming stage by the end of 3 h (room temperature 28 f l”C), with the first cell division occurring within 40-60 minutes after fertilization. After 18-22 h they attained the first shelled stage. At this stage they were swimming mainly near the water surface. There was then a distinct difference in the shell dimensions of the two groups of bivalves. The oysters measured 70 pm along the hinge line and 60 pm perpendicular to this axis, while the mussels measured 80 pm and 60 urn respectively. At the end of 3 days of rearing there was more than 90% sur- vival, while after the 5th day there was about 75% survival.

DISCUSSION

The results of these experiments show that a rapid salinity change provides the stimulus for synchronised progress of ripe individuals to the prespawning condition. This view is further supported by the lack of response in animals maintained in stable salinity during April. The latent period indicates that ripe

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individuals probably undergo some physiological changes in preparation for the final spawning event. There is apparently a general tendency among in- dividuals in the prespawning condition to withhold spawning, and evidently conspecific gametes are the final stimulii. The adaptive value of spawning stimulants in the gametes is considered to be very high for species with epidemic spawning (Giese and Pearse, 1974). Those few individuals that initiate spawning are presumably more sensitive to the osmotic stimulation.

Rapid salinity changes have for long been considered to be the ‘natural’ spawning stimulus along the coast of India (e.g. Panikkar and Aiyar, 1939). Seasonal temperature changes in this region are insignificant; hence tem- perature changes have been considered to be of little importance in deter- mining spawning (Stephen, 1980a). To understand the adaptive value of spawning in response to salinity change, it is necessary to take ecological as- pects into consideration. A sudden decrease in salinity from stable condition provides an effective signal for individuals in a population to synchronise spawning and provide for maximum fertihzation. The major spawning ac- tivity of several invertebrates along this coast coincides with this salinity change, and appears to be so adapted. In estuaries especially, it is necessary that spawning be completed when salinities favour ontogenic development and setting (Rao, 1951; Stephen, 1980a).

Interestingly, the subsequent gametogenic activity of these species coin- cides with increasing salinities. It has been suggested that gradual changes in salinity synchronise the gametogenic cycle (Stephen, 1980a). The spawning activity during this period is known to be protracted in marine inshore popu- lations (Menon et al., 1977). However in estuaries a minor spawning peak is evident when salinities increase to about 20°/oo. There is apparently no sharp exogenous synchroniser to spawning during this period. It is tempting to speculate that the absence of an external synchroniser has indeed diminished the spawning activity.

Populations of euryhaline species (e.g. Crassostrea madrusensis) inhabiting different coastal ecosystems (of different salinity regimes) have revealed dif- ferences in their reproductive pattern (Stephen, 1980a). Moreover, consider- ing the adaptive response to salinity fluctuations, isolated populations may also show differences in their salinity requirements for spawning and larval development. These isolated populations may indeed constitute physiological races.

Further detailed experiments are certainly needed to elucidate fully the in- fluence of salinity on reproduction. It is hoped that the present report will provide a basis for future studies. The successful induction of spawning reported here should find application in both research and commercial culti- vation.

ACKNOWLEDGEMENT

The authors wish to thank Dr. M.B. Eldridge, Tiburon Laboratory, California for kindly providing a culture of the alga used as feed material in these experiments.

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