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Induction of spawning by temperature and serotonin in the hermaphroditic tropical scallop, Pecten ziczac
84 (1990) 307-313 Elsevier Science Publishers B.V., Amsterdam
307
Aquaculture,
-
Printed
in The Netherlands
Induction of Spawning by Temperature and Serotonin in the Hermaphroditic Tropical Scallop, Pecten xicxac ANIBAL VELEZ”, ELIZABETH aZnstituto Oceanogrbfico
de Venezuela,
bZnstituto de Zoologia Tropical, ‘Consejo
National
Universidad
Universidad
de Inuestigaciones
PO 70617, Los Ruices,
(Accepted
ALIFAb and ORLANDO
AZUAJE
de Oriente,
Cumand
Central de Venezuela,
Cientificas
y Tecnoldgicas
(Venezuela)
Caracas (Venezuela)
de Venezuela,
CONICIT,
Caracas (Venezuela)
19 July 1989)
ABSTRACT Velez, A., Alifa, E. and Azuaje, O., 1990. Induction of spawning by temperature the hermaphroditic tropical scallop, Pecten ziczac. Aquaculture, 84: 307-313.
and serotonin
in
Intragonadal injection of serotinin, alone or in combination with thermal shock, was effective in inducing the release of spermatozoa, but no eggs were released in the ripe tropical hermaphroditic scallop, Pecten ziczac. Dopamine, epinephrine, octopamine and norepinephrine were not effective with either sex. The effective dose of serotonin appeared to be a 0.4-ml injection of a 2 m&f solution. Thermal stimulation was an effective method for inducing the release of both gametes of mature animals previously conditioned in high temperature water.
INTRODUCTION
As a result of the progress achieved in commercial shellfish aquaculture, different methods of artificial induction of spawning of bivalves have been developed and applied to various species (Ino, 1972, review ) . The most common is thermal stimulation with addition of live sperm or ova (Loosanoff and Davis, 1963). Limited success has been attained with tropical species, using thermal shocks when induced spawning was tried out of season (Berg and Alatalo, 1985; Belda and Del Norte, 1988). However, some authors have been successful, placing broodstock in running seawater at 5-10’ C above ambient water temperature combined with addition of gamete suspensions or peroxide (VBlez and Martinez, 1967; Costello et al., 1973; Siddall, 1978; Berg and Alatalo, 1985 ) . Recently, it has been reported that injections of serotonin (hydroxytryptamine; 5-HT) induce spawning in Patimpecten yessoensis, Argopecten irradi-
0044-8486/90/$03.50
0 1990 Elsevier Science Publishers
B.V.
308
A. VELEZ ET AL.
urn, Pecten albicans and other marine bivalves (Matsutani and Nomura, 1982, 1987; Gibbons et al., 1983; Gibbons and Castagna, 1984, 1985; Braley, 1985; Tanaka and Murakoshi, 1985; Hirai et al., 1988). Matsutani and Nomura (1982) found that intragonadal injection of serotonin induced spawning of males and females of the dioecious scallop P. yessoensis. However, serotonin has been inefficient for egg release in the hermaphroditic scallops P. &cans and A. irradiuns. Moreover, Hirai et al. (1988) showed that serotonin induces gamete spawning and initiates oocyte maturation in the surf clams Spisulu solidissima and S. sachalinensis. Previous attempts to induce spawning in the tropical scallop, Pecten ziczuc, out of breeding season in our laboratory have been unsuccessful. Various stimuli, such as thermal shock, addition of gonadal products, salinity and pH changes, injections and exposure to Hz02, NH,OH and KCl, were ineffective in inducing spawning and obtaining viable gametes. This study evaluates the effectiveness of thermal stimulation and intragonadal injections of serotonin, dopamine, epinephrine, and norepinephrine. MATERIALS
AND METHODS
Specimens of Pecten ziczuc were collected during the season of gametogenesis (February, March and April) by diving on natural beds located in the Cariaco Gulf, State of Sucre, in eastern Venezuela. The animals were conditioned by holding them in shallow water where the temperature was 3-5°C above ambient for the natural beds (VBlez et al., 1988a,b). Gonadal condition was checked in live animals using the method described by Castagna and Duggan (1971) for Argopecten irradzims. P. ziczac is a functional hermaphrodite with a reddish-orange ovarian portion and a white testis; when ripe, the ovarian portion becomes rose-colored. Ripe animals with a shell length 7 to 8 cm were selected for different trials. The number of animals per trial varied from a minimum of 10 for individual stimuli to a maximum of 20 when comparing thermal stimulation and serotonin injection. The spawning procedures used for thermal stimulation followed those described by Loosanoff and Davis (1963). Spawning was induced by rapidly raising the water temperature from 20’ C to 29’ C; the animals had been previously kept at 20°C for 12 h. A batch of 60 animals (n= 10 per trial) was selected for treatment with serotonin, dopamine, epinephrine, octapamine, norepinephrine and controls. In each case, 0.4 ml of a 2 mA4 solution was injected into the anterior portion of the adductor muscle and in the male and female portions of the gonad. Controls were injected with filtered seawater. New needles were used for each injection to prevent transfer of chemicals. Based on initial success with serotonin, another batch of 60 animals (10 for each dose) was treated with 0.0,0.1, 1,2,4 and 6 mM serotonin solutions to determine the dose response.
INDUCTION OF SPAWNING IN PECTEN
309
ZICZAC
Finally, a total of 135 animals was selected for three treatments to compare the effects of temperature stimulation (n = 45)) serotonin injections (n = 45 ) and the combined effect of temperature and serotonin (n=45). Three trials were run in February, March and April, preceding the first annual spawning season (VQlez et al., 1988a). For all treatments, animals were placed individually in glass dishes containing 11 of 5-pm filtered seawater (37%0 ) for spawning. RESULTS
Of all amines tested, only serotonin was effective in inducing sperm release in ripe individuals, but no eggs were released (Table 1) . The animals reacted immediately to the serotonin dose by increasing adduction of the valves and by swimming. The release of gametes began within 20 min after serotonin injection. The control animals did not exhibit this behavior and did not release any gametes. The dosage of 0.4 ml of 0.1 to 6.0 mikf serotonin solutions stimulated spawning in the male phase, releasing active spermatozoa; however, none of these concentrations stimulated the female phase. Percentage of male phase spawning increased significantly from 5% to 55% as the concentration of serotonin increased from 0.1 to 2.0 mM, but serotonin efficiency in sperm release decreased at higher concentrations. Furthermore, animal survival was inversely related to serotonin concentration (Fig. 1) . TABLE 1 Spawning induction of the tropical scallop P. ticruc by thermal.shock and injection of 0.4 ml (2 mM) of serotonin in the gonad and the adductor muscle Trials
Treatment
No. of animals tested
1 (February 1985)
Temperature Serotonin Controls
20 20 20
5 0 0
5 55 0
2 (March 1985)
Temperature Serotonin Serot./Temp. Control
15 15 15 15
33 0 0 0
25 100 100 0
3 (April 1985)
Temperature Serotonin SerotJTemp. Control
10 10 10 10
50 0 0 0
50 90 50 0
Females spawned (%)
Males spawned (%)
A. VELEZ ET AL.
310
0
’ co
I 01
I SEROTONIN
2
4
I 6
,
(mM)
Fig. 1. Survival of P. ziczac 24 h after treatment (n=lO).
with serotonin
injection
at five concentrations
The efficiency of serotonin injection was compared with that of thermal stimulation at three periods during the breeding season (Table 1) . Serotonin injection gave similar results to those obtained in previous trials. Thermal shock caused P. ziczac to release spermatozoa initially followed by eggs in all trials. During February, 5% of the animals released eggs, in March 33% and in April 50%. In spite of this, the combined treatment of serotonin injection and thermal stimulation was unsuccessful in inducing egg release and also decreased male response from 90% to 50% in April (Table 1). DISCUSSION
Serotonin and other amines like norepinephrine and dopamine have been found in the central nervous systems of Myths edulis and P. yessoensis (Stefano and Aiello, 1975; Stefano and Catapane, 1977; Matsutani and Nomura, 1984,1986). Localization of serotonin in neurons of the central nervous system and the gonad of the scallop, Putinopecten yessoensis, and its direct action on oocytes in inducing germinal breakdown have indicated that serotonin plays an important role in reproduction or marine bivalves (Matsutani and Nomura, 1984,1986,1987). The present results show that thermal shock and the injection of serotonin are effective in inducing gamete release in the hermaphroditic tropical scallop P. ziczac. The use of thermal shock induces the release of both gametes in mature animals previously conditioned in high water temperatures (VQlez et al., 1988a). Serotonin injection alone or in combination with temperature shock is effective in inducing release of spermatozoa, but not eggs. The fact that individuals of P. Z&UC, subjected to temperature shock, released both sper-
INDUCTION OF SPAWNING IN PECTEN ZICZAC
311
matozoa and eggs, while animals treated with a combination of temperature and serotonin released only spermatozoa, suggests that serotonin injection inhibits thermal stimulation. The thermal and serotoninergic induction of spawning of P. ziczac implies that temperature may act as a trigger of male gamete release underlied by the neurotransmitter, serotonin. The role of serotonin is not yet understood clearly; however, evidence has been accumulated regarding the importance of prostaglandins in spawning of molluscs. Matsutani and Nomura (1987) reported that serotonin induces spawning of female P. yessoensis through specific serotonin receptors in the ovary. They found that the effect of serotonin on egg release was inhibited significantly in the presence of aspirin or indomethacin, which are well known inhibitors of prostaglandin biosynthesis via cyclooxygenase. This result suggests that serotonin injection stimulates biosynthesis of prostaglandins. They also have shown that prostaglandins type PGE, tended to increase the stimulatory effect of serotonin on egg release, whereas prostaglandins type PGF,, significantly reduced it. Furthermore, the PGF,, content in the ovary of P. yessoensis (Mori et al., 1984) as well as that of oyster Crassostrea gigas (Ono et al., 1982), increases during sexual maturation. This suggests that serotonin injection in P. yessoensis induces release of spermatozoa and also stimulates biosynthesis of PGEz to counterbalance the PGFza present in mature animals and to stimulate release of eggs. Similar situations may arise in Mercenaria mercenariu, Spisulu solidissima and giant clams that release spermatozoa and eggs with serotonin (Gibbons and Castagna, 1984; Braley, 1985; Alcazar et al., 1987). It is possible that serotonin injection in P. ziczac inhibited the thermal shock effect on egg release by stimulating biosynthesis of prostaglandins type PGF2,. There may be an inverse relationship between PGE and PGF levels which is critical to the whole process of maturation and ovulation underlied by the control of the neurotransmitter. Further research on a variety of species is necessary to establish the precise role of these compounds and their modulation by ambient factors. In conclusion, the results show that thermal shocks are effective in spawning of P. ziczac. In our routine aquaculture program, we are successfully using this method to obtain viable gametes for fertilization, larval development and setting, with animals previously conditioned in high-temperature waters. ACKNOWLEDGMENTS
This work was sponsored by the Instituto Oceanografico de Venezuela and by the Consejo de Investigaciones Cientificas y Tecnologicas de Venezuela, CONICIT, Proyect No. SI-1721. The authors are grateful to Drs. Joseph Ewald and Osmar Nusetti of the Universidad de1 Zulia and Universidad de Oriente,
312
A. VELEZ ET AL.
Venezuela, for their helpful suggestions, Antonio Sotillet and Pedro Mata for technical assistance and Ms. Dilia Mirquez for typing the manuscript.
REFERENCES Alcazar, S.N., Solis, E.P. and Alcala, AC., 1987. Serotonin-induced spawning and larval rearing of the China clam, Hippopusporcellanus Rosewater (Bivalvia: Tridacnidae). Aquaculture, 66: 359-368. Belda, C.A. and Del Norte, A.G., 1988. Notes on the induced spawning and larval rearing of the Asian moon scallop, Amusium pleuronectes (Linnk), in the laboratory. Aquaculture, 72: 173179. Berg, C.J. and Alatalo, P., 1985. Biology of the tropical bivalve Asaphis deflorata (Linne, 1758). Bull. Mar. Sci., 37: 827-838. Braley, R.D., 1985. Serotonin-induced spawning in giant clams (Bivalvia, Tridacnidae). Aquaculture, 47: 321-325. Castagna, M. and Duggan, W., 1971. Rearing of bay scallop, Aequipecten h-radians. Proc. Natl. Shellfish. Assoc., 61: 80-85. Costello, J.J., Hudson, J.H., Dupuy, J.R. and Rivki, S., 1973. Larval culture of the calico scallop, Argopecten gibbus. Proc. Natl. Shellfish. Assoc., 63: 72-76. Gibbons, M. and Castagna, M., 1984. Serotonin as an inducer of spawning in six bivalve species. Aquaculture, 40: 189-191. Gibbons, M. and Castagna, M., 1985. Responses of hard clam hfercenaria mercenaria (Linne) to induction of spawning by serotonin. J. Shellfish Res., 5: 65-67. Gibbons, M., Goodsell, J.G., Castagna, M. and Luz, R., 1983. Chemical stimulation of spawning by serotonin in the ocean quahog Artica islandica (Linne). J. Shellfish Res., 3: 203-205. Hirai, S., Kishimoto, T., Kadam, A.L., Kanatani, H. and Koide, S.S., 1988. Induction of spawning and oocyte maturation by 5-hydroxytryptamine in the surf clam. J. Exp. Zoo]., 245: 318-321. Ino, T., 1972. Controlled breeding of molluscs. In: T.R. Pillay (Editor), Coastal Aquaculture in the Indo-Pacific Region. Fishing News (Books), London, pp.260-272. Loosanoff, V. and Davis, H., 1963. Rearing of bivalve molluscs. Adv. Mar. Biol., 1: l-136. Matsutani, T. and Nomura, T., 1982. Induction of spawning by serotonin in the scallop, Putinopecten yessoensis (Jay). Mar. Biol. Lett., 3: 353-358. Matsutani, T. and Nomura, T., 1984. Localization of monoamines in the central nervous system and the gonad of the scallop Patinopecten yessoensis. Bull. Jpn. Sot. Sci. Fish., 50: 425-230. Matsutani, T. and Nomura, T., 1986. Serotonin-like immunoreactivity in the central nervous system and the gonad of the scallop Patinopecten yessoensis. Cell Tissue Res., 244: 515-517. Matsutani, T. and Nomura, T., 1987. In vitro effects of serotonin and prostaglandins in release of eggs from the ovary of the scallop Putinopecten yessoensis. Gen. Comp. Endocrinol., 67: lll118. Mori, K., Takenaga, M., Ino, T. and Nomura, T., 1984. Prostaglandin PGF,, profile during the spawning season in the scallop Patinopecten yessoensis. Kyoto Conference on Prostaglandins, Kyoto, p. 250 (abstr. ). Ono, K., Osada, M., Matsutani, T., Mori, K. and Nomura, T., 1982. Gonadal prostaglandin PGF, profile during sexual maturation in the oyster, Crassostreo gigas Thunberg. Mar. Biol. Lett., 3: 223-230. Siddall, S.E., 1978. Temporal changes in the salinity and temperature requirements of tropical mussel larvae. In: J.W. Avault, Jr. (Editor), Ninth Annual Meeting World Mariculture Society, Louisiana State University, Baton Rouge, LA, pp. 549-566.
INDUCTION OF SPAWNING IN PECTEN ZICZAC
313
Stefano, G.B. and Aiello, E., 1975. Hi&fluorescence of serotonin in gill of Mytilus e&is (Bivalvia). Biol. Bull. (Woods Hole, Mass), 148: 141-156. Stefano, G.B. and Catapane, E.J., 1977. Seasonal monoamine changes in the central nervous system of Mytilus edulis (Bivalvia). Experientia, 33: 1341-1342. Tanaka, Y. and Murakoshi, M., 1985. Spawning induction of the hermaphroditic scallop, Pecten albicans, by injection with serotonin. Bull. Natl. Res. Inst. Aquacult., 7: 9-12. Velez, A. and Martinez, R., 1967. Reproduction y desarrollo larval experimental de1 mejillon comestible de Venezuela, Perna perna (Linnaeus, 1758). Bol. Inst. Oceanog. Univ. Oriente, Venezuela, 6: 266-285. Vklez, A., Alifa, E. and Freites, L., 1988a. Induction de la reproduction de la vieira Pecten ziczac (LinnC, 1758). Mem. Sot. Cien. Nat., La Salle (in press). Velez, A., Sotillo, F. and Perez, J., 1988b. Variation estacional de la composition quimica de 10s pectfnidos Pecten ziczac y Lyropecten nodosus. Bol. Inst. Oceanog. Univ. Oriente, Venezuela (in press).
307
Aquaculture,
-
Printed
in The Netherlands
Induction of Spawning by Temperature and Serotonin in the Hermaphroditic Tropical Scallop, Pecten xicxac ANIBAL VELEZ”, ELIZABETH aZnstituto Oceanogrbfico
de Venezuela,
bZnstituto de Zoologia Tropical, ‘Consejo
National
Universidad
Universidad
de Inuestigaciones
PO 70617, Los Ruices,
(Accepted
ALIFAb and ORLANDO
AZUAJE
de Oriente,
Cumand
Central de Venezuela,
Cientificas
y Tecnoldgicas
(Venezuela)
Caracas (Venezuela)
de Venezuela,
CONICIT,
Caracas (Venezuela)
19 July 1989)
ABSTRACT Velez, A., Alifa, E. and Azuaje, O., 1990. Induction of spawning by temperature the hermaphroditic tropical scallop, Pecten ziczac. Aquaculture, 84: 307-313.
and serotonin
in
Intragonadal injection of serotinin, alone or in combination with thermal shock, was effective in inducing the release of spermatozoa, but no eggs were released in the ripe tropical hermaphroditic scallop, Pecten ziczac. Dopamine, epinephrine, octopamine and norepinephrine were not effective with either sex. The effective dose of serotonin appeared to be a 0.4-ml injection of a 2 m&f solution. Thermal stimulation was an effective method for inducing the release of both gametes of mature animals previously conditioned in high temperature water.
INTRODUCTION
As a result of the progress achieved in commercial shellfish aquaculture, different methods of artificial induction of spawning of bivalves have been developed and applied to various species (Ino, 1972, review ) . The most common is thermal stimulation with addition of live sperm or ova (Loosanoff and Davis, 1963). Limited success has been attained with tropical species, using thermal shocks when induced spawning was tried out of season (Berg and Alatalo, 1985; Belda and Del Norte, 1988). However, some authors have been successful, placing broodstock in running seawater at 5-10’ C above ambient water temperature combined with addition of gamete suspensions or peroxide (VBlez and Martinez, 1967; Costello et al., 1973; Siddall, 1978; Berg and Alatalo, 1985 ) . Recently, it has been reported that injections of serotonin (hydroxytryptamine; 5-HT) induce spawning in Patimpecten yessoensis, Argopecten irradi-
0044-8486/90/$03.50
0 1990 Elsevier Science Publishers
B.V.
308
A. VELEZ ET AL.
urn, Pecten albicans and other marine bivalves (Matsutani and Nomura, 1982, 1987; Gibbons et al., 1983; Gibbons and Castagna, 1984, 1985; Braley, 1985; Tanaka and Murakoshi, 1985; Hirai et al., 1988). Matsutani and Nomura (1982) found that intragonadal injection of serotonin induced spawning of males and females of the dioecious scallop P. yessoensis. However, serotonin has been inefficient for egg release in the hermaphroditic scallops P. &cans and A. irradiuns. Moreover, Hirai et al. (1988) showed that serotonin induces gamete spawning and initiates oocyte maturation in the surf clams Spisulu solidissima and S. sachalinensis. Previous attempts to induce spawning in the tropical scallop, Pecten ziczuc, out of breeding season in our laboratory have been unsuccessful. Various stimuli, such as thermal shock, addition of gonadal products, salinity and pH changes, injections and exposure to Hz02, NH,OH and KCl, were ineffective in inducing spawning and obtaining viable gametes. This study evaluates the effectiveness of thermal stimulation and intragonadal injections of serotonin, dopamine, epinephrine, and norepinephrine. MATERIALS
AND METHODS
Specimens of Pecten ziczuc were collected during the season of gametogenesis (February, March and April) by diving on natural beds located in the Cariaco Gulf, State of Sucre, in eastern Venezuela. The animals were conditioned by holding them in shallow water where the temperature was 3-5°C above ambient for the natural beds (VBlez et al., 1988a,b). Gonadal condition was checked in live animals using the method described by Castagna and Duggan (1971) for Argopecten irradzims. P. ziczac is a functional hermaphrodite with a reddish-orange ovarian portion and a white testis; when ripe, the ovarian portion becomes rose-colored. Ripe animals with a shell length 7 to 8 cm were selected for different trials. The number of animals per trial varied from a minimum of 10 for individual stimuli to a maximum of 20 when comparing thermal stimulation and serotonin injection. The spawning procedures used for thermal stimulation followed those described by Loosanoff and Davis (1963). Spawning was induced by rapidly raising the water temperature from 20’ C to 29’ C; the animals had been previously kept at 20°C for 12 h. A batch of 60 animals (n= 10 per trial) was selected for treatment with serotonin, dopamine, epinephrine, octapamine, norepinephrine and controls. In each case, 0.4 ml of a 2 mA4 solution was injected into the anterior portion of the adductor muscle and in the male and female portions of the gonad. Controls were injected with filtered seawater. New needles were used for each injection to prevent transfer of chemicals. Based on initial success with serotonin, another batch of 60 animals (10 for each dose) was treated with 0.0,0.1, 1,2,4 and 6 mM serotonin solutions to determine the dose response.
INDUCTION OF SPAWNING IN PECTEN
309
ZICZAC
Finally, a total of 135 animals was selected for three treatments to compare the effects of temperature stimulation (n = 45)) serotonin injections (n = 45 ) and the combined effect of temperature and serotonin (n=45). Three trials were run in February, March and April, preceding the first annual spawning season (VQlez et al., 1988a). For all treatments, animals were placed individually in glass dishes containing 11 of 5-pm filtered seawater (37%0 ) for spawning. RESULTS
Of all amines tested, only serotonin was effective in inducing sperm release in ripe individuals, but no eggs were released (Table 1) . The animals reacted immediately to the serotonin dose by increasing adduction of the valves and by swimming. The release of gametes began within 20 min after serotonin injection. The control animals did not exhibit this behavior and did not release any gametes. The dosage of 0.4 ml of 0.1 to 6.0 mikf serotonin solutions stimulated spawning in the male phase, releasing active spermatozoa; however, none of these concentrations stimulated the female phase. Percentage of male phase spawning increased significantly from 5% to 55% as the concentration of serotonin increased from 0.1 to 2.0 mM, but serotonin efficiency in sperm release decreased at higher concentrations. Furthermore, animal survival was inversely related to serotonin concentration (Fig. 1) . TABLE 1 Spawning induction of the tropical scallop P. ticruc by thermal.shock and injection of 0.4 ml (2 mM) of serotonin in the gonad and the adductor muscle Trials
Treatment
No. of animals tested
1 (February 1985)
Temperature Serotonin Controls
20 20 20
5 0 0
5 55 0
2 (March 1985)
Temperature Serotonin Serot./Temp. Control
15 15 15 15
33 0 0 0
25 100 100 0
3 (April 1985)
Temperature Serotonin SerotJTemp. Control
10 10 10 10
50 0 0 0
50 90 50 0
Females spawned (%)
Males spawned (%)
A. VELEZ ET AL.
310
0
’ co
I 01
I SEROTONIN
2
4
I 6
,
(mM)
Fig. 1. Survival of P. ziczac 24 h after treatment (n=lO).
with serotonin
injection
at five concentrations
The efficiency of serotonin injection was compared with that of thermal stimulation at three periods during the breeding season (Table 1) . Serotonin injection gave similar results to those obtained in previous trials. Thermal shock caused P. ziczac to release spermatozoa initially followed by eggs in all trials. During February, 5% of the animals released eggs, in March 33% and in April 50%. In spite of this, the combined treatment of serotonin injection and thermal stimulation was unsuccessful in inducing egg release and also decreased male response from 90% to 50% in April (Table 1). DISCUSSION
Serotonin and other amines like norepinephrine and dopamine have been found in the central nervous systems of Myths edulis and P. yessoensis (Stefano and Aiello, 1975; Stefano and Catapane, 1977; Matsutani and Nomura, 1984,1986). Localization of serotonin in neurons of the central nervous system and the gonad of the scallop, Putinopecten yessoensis, and its direct action on oocytes in inducing germinal breakdown have indicated that serotonin plays an important role in reproduction or marine bivalves (Matsutani and Nomura, 1984,1986,1987). The present results show that thermal shock and the injection of serotonin are effective in inducing gamete release in the hermaphroditic tropical scallop P. ziczac. The use of thermal shock induces the release of both gametes in mature animals previously conditioned in high water temperatures (VQlez et al., 1988a). Serotonin injection alone or in combination with temperature shock is effective in inducing release of spermatozoa, but not eggs. The fact that individuals of P. Z&UC, subjected to temperature shock, released both sper-
INDUCTION OF SPAWNING IN PECTEN ZICZAC
311
matozoa and eggs, while animals treated with a combination of temperature and serotonin released only spermatozoa, suggests that serotonin injection inhibits thermal stimulation. The thermal and serotoninergic induction of spawning of P. ziczac implies that temperature may act as a trigger of male gamete release underlied by the neurotransmitter, serotonin. The role of serotonin is not yet understood clearly; however, evidence has been accumulated regarding the importance of prostaglandins in spawning of molluscs. Matsutani and Nomura (1987) reported that serotonin induces spawning of female P. yessoensis through specific serotonin receptors in the ovary. They found that the effect of serotonin on egg release was inhibited significantly in the presence of aspirin or indomethacin, which are well known inhibitors of prostaglandin biosynthesis via cyclooxygenase. This result suggests that serotonin injection stimulates biosynthesis of prostaglandins. They also have shown that prostaglandins type PGE, tended to increase the stimulatory effect of serotonin on egg release, whereas prostaglandins type PGF,, significantly reduced it. Furthermore, the PGF,, content in the ovary of P. yessoensis (Mori et al., 1984) as well as that of oyster Crassostrea gigas (Ono et al., 1982), increases during sexual maturation. This suggests that serotonin injection in P. yessoensis induces release of spermatozoa and also stimulates biosynthesis of PGEz to counterbalance the PGFza present in mature animals and to stimulate release of eggs. Similar situations may arise in Mercenaria mercenariu, Spisulu solidissima and giant clams that release spermatozoa and eggs with serotonin (Gibbons and Castagna, 1984; Braley, 1985; Alcazar et al., 1987). It is possible that serotonin injection in P. ziczac inhibited the thermal shock effect on egg release by stimulating biosynthesis of prostaglandins type PGF2,. There may be an inverse relationship between PGE and PGF levels which is critical to the whole process of maturation and ovulation underlied by the control of the neurotransmitter. Further research on a variety of species is necessary to establish the precise role of these compounds and their modulation by ambient factors. In conclusion, the results show that thermal shocks are effective in spawning of P. ziczac. In our routine aquaculture program, we are successfully using this method to obtain viable gametes for fertilization, larval development and setting, with animals previously conditioned in high-temperature waters. ACKNOWLEDGMENTS
This work was sponsored by the Instituto Oceanografico de Venezuela and by the Consejo de Investigaciones Cientificas y Tecnologicas de Venezuela, CONICIT, Proyect No. SI-1721. The authors are grateful to Drs. Joseph Ewald and Osmar Nusetti of the Universidad de1 Zulia and Universidad de Oriente,
312
A. VELEZ ET AL.
Venezuela, for their helpful suggestions, Antonio Sotillet and Pedro Mata for technical assistance and Ms. Dilia Mirquez for typing the manuscript.
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