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Evaluation of the effects of serotonin (5-HT) on oocyte competence in Ruditapes decussatus (Bivalvia, Veneridae)
Aquaculture 239 (2004) 413 – 420 www.elsevier.com/locate/aqua-online
Evaluation of the effects of serotonin (5-HT) on oocyte competence in Ruditapes decussatus (Bivalvia, Veneridae) Leila Hamida a,*, Mohamed-Nejib Medhioub a,1, Jean Claude Cochard b,2, Marcel Le Pennec c,3 a
Centre De Monastir, Institute of National Des Sciences Et Technologie De la Mer, 5000 Route De Khniss, Tunisia b Institut Francßais pour la Recherche et l’Exploitation de la Mer-Centre de Brest, BP 70, 29280 Plouzane´, France c Institut Universitaire Europe´en de la Mer, Universite´ de Bretagne Occidentale, Place Nicolas Copernic, Technopole Brest-Iroise, 29280 Plouzane´, France Received 1 October 2003; received in revised form 30 April 2004; accepted 2 May 2004 Available online
Abstract In bivalves, the blockage of oocytes at prophase I is released only during the egg-laying period. The second blockage occurs during metaphase I and is released by fertilization. In the case of the venerid Ruditapes decussatus, when we dissect the gonad we obtain oocytes which are blocked at prophase I and so cannot be fertilized. In order to release this blockage, we used serotonin (5-HT). This study is the first to scrutinise the maturation of oocytes induced by serotonin in R. decussatus. We have thus demonstrated that adding 20 AM of serotonin in an external application allows the recovery of meiosis and the passage of oocytes from germinal vesicle (G.V.) to germinal vesicle breakdown (GVBD) in approximately 90 min at a temperature of 20 jC. The highest percentage of oocytes obtained in GVBD was 67%. We have also evaluated the competence of oocytes in GVBD during fertilization, and found the greater the degree of reproductive condition, the higher is the percentage of oocytes that can undergo GVBD. From an estimated threshold of 53% of oocytes
* Corresponding author. Tel.: +216-98-68-41-41; fax: +216-73-46-00-34. E-mail addresses: [email protected] (L. Hamida), [email protected] (M.-N. Medhioub), [email protected] (J.C. Cochard), [email protected] (M.L. Pennec). 1 Tel.: +216-73-53-18-67; fax: +216-73-53-16-20. 2 Tel.: +33 2 98 22 43 96; fax: +33 2 98 22 46 53. 3 Tel.: +33-2-98-49-86-41; fax: +33-2-98-49-87-90. 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.05.010
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L. Hamida et al. / Aquaculture 239 (2004) 413–420
capable of GVBD, broodstock placed in a controlled environment are liable to spawn when they undergo thermal shocks. This allows us to consider this percentage as a criterion for the evaluation of oocytes during fertilization. D 2004 Elsevier B.V. All rights reserved. Keywords: Bivalve; Ruditapes decussatus; Oocyte maturation; Fertilization; Serotonin
1. Introduction In the last decade, Tunisia set up an aquacultural development scheme along the coastline, and the Venerid, Ruditapes decussatus, is one of the species of major interest. Economically, R. decussatus is the most important shellfish species in terms of both employment and value, with an estimated 1200 tonnes, worth US$7 million, exported to Europe annually (EL Abed, INSTM, personal communication). Two approaches are followed: the preservation of the wild resource by setting up fishing seasons and, in parallel, aquaculture development emphasizing restocking with spat produced in hatcheries. Development of production techniques in hatcheries is needed as was the case in France with hatcheries for Ruditapes philippinarum and in Chile for Venus antigua (Bustos and Olavarria, 2000). In hatcheries, gametes are usually obtained by applying thermal shocks to adult clams (Le Pennec, 1987), although an alternative method of ‘‘stripping’’ exists, which allows us to obtain mature gametes easier and faster, by dissecting the gonad. However, in stripping R. decussatus, we only obtain oocytes which are blocked at prophase I and which cannot be fertilized. Generally, bivalve oocytes are held in the ovaries at prophase of the first division of meiosis, and development reinitiates during spawning. A second barrier to development occurs at metaphase I, and it is released by fertilization. In order to initiate meiosis and have the oocytes pass through to metaphase I, serotonin can be applied. Serotonin is a regulator (5-hydroxytryptamine) synthesized from L-tryptophan that allows germinal vesicle breakdown (GVBD) and the completion of meiosis. Its intervention prompts an increase in intracellular pH and the expulsion of the first polar body (Kyosuka et al., 1997; Colas and Dube´, 1998). The aim of this research is to evaluate the competence of R. decussatus oocytes treated with serotonin before fertilization rather than the histological methods frequently used with bivalves (Laruelle et al., 1994; Lango-Reynoso et al., 2000; Chavez-Villalba et al., 2002a,b). This is achieved by determining the optimal dose of serotonin for oocyte GVBD, and successful fertilization by oocytes obtained through dissecting the gonad. For the first time, this research aims at establishing a relationship between the percentage of oocytes during GVBD and the reproductive maturation of R. decussatus.
2. Materials and methods Adult R. decussatus were brought into reproductive condition using the method described by Medhioub et al. (2000). Temperature was maintained at 21 F 1 jC, salinity at 30 F 1 ppm, and lighting was continuous. Clams were fed a mix of unicellular algae:
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Chaetoceros calcitrans and Isochrysis galbana, at a rate of 1 109 cells per individual per day. The seawater was renewed 1.7 times/24 h. A summer conditioning experiment carried out from July to September 2001, while a winter experiment was carried out from February to May 2002. In both experiments, gametogenesis was followed using histological examination of the gonad every 2 weeks throughout the conditioning period to determine the best period for the induction tests of the oocyte maturation by serotonin. Induction tests of GVBD started after 1 week for summer conditioning and 7 weeks later for winter conditioning. This gap is due to the fact that for summer conditioning broodstock had already commenced gonadal development on their arrival to the hatchery, whereas for winter conditioning the clams were only at the beginning of gametogenesis. Gametes were obtained by dissecting the gonads of 10 broodstock. Oocytes were first filtered through a 100-Am filter to eliminate tissue debris, and then retained on a 30-Am filter and kept in suspension in a volume of 250 ml. Three aliquots were recovered from this suspension to calculate the total number of oocytes. Spermatozoa were also filtered through a 100-Am filter to eliminate tissue debris and kept in suspension in a volume of 100 ml at a temperature of 4 jC until fertilization. To determine the concentration of serotonin permitting the highest percentage of oocyte in GVBD, we tested different concentrations, from 2 to 100 AM, by putting about 2000 oocytes in contact with those concentrations in wells of 2 ml of microtitration plates (1 volume serotonin/1 volume oocytes) in triplicate and placing them in a temperature of 20 jC. These concentrations of serotonin were obtained from an initial solution of 200 AM derived from 2 g of serotonin creatinine sulfate (Sigma) dissolved in distilled water. Oocytes GVBD were evaluated after 90 min by microscopic observation. Those oocytes that had undergone GVBD were spherical and had a nucleus that could not be differentiated from the cytoplasm (Fig. 1). After determining the concentration of serotonin permitting the highest percentage of oocytes to undergo GVBD, we administered this concentration to the gametes obtained by dissection of the gonad. In order to relieve the second blockage and obtain fertilization, we added sperm suspension. The fertilized oocytes were then put in cylindroconical buckets of 30 l of seawater with aeration at the bottom to avoid settlement of the eggs.
Fig. 1. Oocytes in GV (visible nucleus compared to cellular continuity) and in GVBD (invisible nucleus) observed in a microscope.
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Twenty-four hours later, we estimated the percentage of developing to D larvae (Ge´rard et al., 1989) and the percentage of eggs developing abnormally. (Salaum et al., 1991) Percentage of development to D larvae ¼ total number of larvae=total number of oocytes: Percentage of eggs developing abnormally ¼ number of abnormal larvae=total number of larvae: All experiments on the fertilization of oocytes obtained by dissection were carried out in parallel with experiments on the fertilization of oocytes released following thermal shocks. These oocytes were obtained from 10 broodstock and used as controls. Oocyte fertilization and larval culture were carried out at the same time and in the same conditions for both experiments. 2.1. Statistical analyses The results obtained were analyzed by the software XSTAT; nonparametric comparisons (Kruskal – Wallis’ test) were used to determine whether the difference between the averages is significant or not.
3. Results 3.1. The effect of serotonin concentration on GVBD in stripped oocytes The percentage of eggs undergoing GVBD differed significantly ( P < 0.05) with the concentration of serotonin in stripped oocyte suspensions (Fig. 2). In the absence of serotonin, GVBD occurred in approximately 22% of oocytes. With increasing serotonin
Fig. 2. Variation in the percentage of oocytes in GVBD according to the concentration of serotonin (5-HT) (v F S.E.).
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Fig. 3. (A) Variation in the percentage of oocytes in GVBD according to time for summer conditioning in 2001. (B) Variation in the percentage of oocytes in GVBD according to time for winter conditioning in 2002 (v F S.E.).
concentration, GVBD percentage increased to reach a maximum of 67% at 20 AM serotonin. Beyond this concentration, GVBD percentage decreased. At 40 AM serotonin, GVBD had fallen to the same level observed in the absence of serotonin (22%), and at serotonin concentration of 100 AM, GVBD did not occur. 3.2. Variation in percentage of oocytes in GVBD according to time For summer conditioning, GVBD percentage increased significantly ( P < 0.05) from 20% to 77% after 4 weeks of conditioning (Fig. 3). From 61 F 5.1% of GVBD, the clams spontaneously excreted their gametes in the buckets. With regards to winter conditioning, GVBD percentage increased significantly ( P < 0.05) from 25% to 68% after 15 weeks of conditioning (Fig. 3). From 46 F 0.4%, the clams had a positive response to thermal shocks leading to the emission of gametes. 3.3. Fertilization of oocytes obtained through dissection of the gonad in comparison with those obtained by thermal shocks The percentage of fertilized oocytes obtained through stripping is low compared with that obtained by thermal shocks 26 F 5.9% and 92 F 0.5%, respectively. Similarly, the percentage of development to D larvae is low compared with that obtained by thermal shocks 7 F 5.7% and 65 F 3%, respectively. In addition, the level of anomaly is very high for oocytes in GVBD obtained by stripping (93%).
4. Discussion Obtaining mature gametes by dissecting the gonad is a fast and efficient means to achieve larval breeding. This technique allows the evaluation of the level of reproductive maturity, which assists successful larval production.
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Stripping is used with species where oocytes can be fertilized directly after dissecting the gonad, such as the oyster Crassostrea gigas. In species where barriers exist to the fertilization of oocytes after dissection from the gonad, such as the clams R. philippinarum (Osanai and Kuraishi, 1988; Guerrier et al., 1993) and Spisula solidissima (Masseau et al., 1998; Yi et al., 1998), serotonin can be used to reinitiate meiosis and allow fertilization. According to Osanai (1985), the percentage of oocytes in GVBD for C. gigas, increases dramatically in seawater containing 1 AM of serotonin. GVBD is noticed from the 10th minute of incubation, and a maximum of 100% is reached within 20 –30 min. According to Moreau and Leclerc (1999), adding 10 AM of serotonin to an oocyte suspension allows the reinitiation of meiosis in about 20 min at a temperature of 20 jC. The maximum number of oocytes in GVBD 90% is reached after 45 min. In order to inhibit the spontaneous passage of oocytes in GVBD, acidic seawater (pH 4 –6.5) is used during the dissection of the gonad. For R. philippinarum, the recommencement of meiosis occurs after adding 10 AM of serotonin into an oocyte suspension at 21 jC. A maximum of 91.7% of oocytes in GVBD is obtained after 87 min by adding 10 AM of serotonin (Osanai and Kuraishi, 1988). On the other hand, stripped Mytilus galloprovincialis oocytes cannot be fertilized in seawater with or without serotonin. This is not the case for another mytilid, Dreissena polymorpha, a freshwater mussel. In D. polymorpha, meiosis recommences 5– 10 min after the addition of serotonin and GVBD are possible within 30 min after adding 10 3 M of serotonin at 23 jC (Fong et al., 1994a). However, the intervention of methiothepin (10 6 M) and metergolin (10 4 M), known by their high affinity with serotonin receptors, can cause of the blockage of spawning for this species (Fong et al., 1994b). Ram et al. (1993) demonstrated that the intervention of serotonin through injection or external application equally triggers the emission of gametes with male or female D. polymorpha. For male zebra mussels, morphological criteria for judging gonadal maturity were well-correlated with probability of spawning in response to serotonin. For females, the likelihood of spawning in response to serotonin was not tightly coupled to morphological maturity of the gonad. Many morphologically ripe females failed to spawn and some apparently immature animals released oocytes. Indeed, serotonin plays an important role in controlling reproduction with bivalves. Injecting serotonin leads to the oocyte maturation and the release of gametes (O’Connor and Heasman, 1995; Madrones-Ladja, 1997; Fong et al., 2003; Louro et al., 2003). Haneji and Koide (1988) and Varaksin et al. (1992) demonstrated that the maturation of oocytes in Spisula sp. could also be incited by adding serotonin through phosphoprotein. To date, no studies have been done on the maturation of oocytes induced by serotonin with R. decussatus. We have shown that adding 20 AM of serotonin to oocyte suspensions allows the reinitiation of meiosis and oocyte GVBD in approximately 90 min at 20 jC. The highest GVBD percentage achieved was 67%. We have also evaluated the competence of oocytes in GVBD during fertilization. The greater the degree of reproductive maturity, the higher the percentage of oocytes that can achieve GVBD. When GVBD of stripped eggs reaches 53%, broodstock exposed to thermal shocks will spawn naturally. This result allows us to consider this percentage as a criterion for the evaluation of oocytes during fertilization. Nevertheless, experiments to fertilize oocytes in which GVBD has been induced by serotonin do not provide satisfactory results. This urges us to search other
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interveners which increase the amount of serotonin within the animal before dissecting the gonad, in order to obtain oocytes which can be fertilized after stripping. Interveners such as prozac or fluoxetine hydrochloride, an inhibitor for the selective recapturing of serotonin, thus enables the increase in intrasynaptic concentration of serotonin (Gardier et al., 2001). Prozac is considered the most powerful spawning inducer in any bivalve, stimulating the serotonergic mechanisms without applying serotonin (Fong, 1998; Fong et al., 1998; Honkoop et al., 1999; Cunha and Machado, 2001). The results of this study suggest the use of serotonin in hatcheries to evaluate oocyte competence during fertilization of R. decussatus. Thus it will be possible, according to the percentage of oocytes in GVBD, to know whether to continue broodstock conditioning before the application of thermal shocks.
Acknowledgements This research was carried out within the framework of the «Programme Aquaculture 2001» between «Institut National des Sciences et Technologie de la Mer, centre de Monastir, Tunisie» and «Institut Francß ais pour la Recherche et l’exploitation de la Mer, centre de Brest, France». The authors would like to thank the teams at the hatchery of Monastir and Argenton (France) who contributed to the achievement of this work.
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Ge´rard, A., Salaun, M., Tritar, S., 1989. Crite`res de compe´tence des larves a` la me´tamorphose chez Pecten maximus. Haliotis 19, 373 – 380. Guerrier, P., Leclerc, D.C., Moreau, M., 1993. Evidence for the involvement of internal stores during serotonininduced meiosis reinitiation in oocytes of the bivalve mollusc Ruditapes philippinarum. Dev. Biol. 159 (2), 474 – 484. Haneji, T., Koide, S.S., 1988. Protein phosphorylation during 5-hydroxytryptamine-induced maturation of Spisula oocytes. Exp. Cell Res. 177 (1), 227 – 231. Honkoop, P.J.C., Luttikhuizen, P.C., Piersma, T., 1999. Experimentally extending the spawning season of a marine bivalve using temperature change and fluoxetine as a synergistic triggers. Mar. Ecol., Prog. Ser. 180, 297 – 300. Kyosuka, K., Deguchi, R., Yoshida, N., Yamashita, M., 1997. Change in intracellular Ca2 + is not involved in serotonin-induced meiosis reinitation from the first prophase in oocytes of the marine bivalve Crassostrea gigas. Dev. Biol. 182 (1), 33 – 41. Lango-Reynoso, F., Chavez-Villalba, J., Cochard, J.C., Le Pennec, M., 2000. Oocyte size, a means to evaluate the gametogenic development of the pacific oyster, Crassostrea gigas (Thunberg). Aquaculture 190, 183 – 199. Laruelle, F., Guillou, J., Paulet, Y.M., 1994. Reproductive pattern of the clams, Ruditapes decussatus and R. philippinarum on the intertidal flats in Britany. J. Mar. Biol. Assoc. U.K. 74, 351 – 366. Le Pennec, M., 1987. Gene`se de la coquille larvaire et post larvaire chez divers bivalves marins. The`se d’e´tat. Universite´ de Bretagne Occidentale. 229 p. Louro, A., De La Roche, J.P., Campos, M.J., Roman, G., 2003. Hatchery rearing of the black scallop, Chlamys varia. J. Shellfish Res. 22 (1), 95 – 99. Madrones-Ladja, J.A., 1997. Notes on the induced spawning, embryonic and larval development of the windowpane shell, Placuna placenta (Linnaeus, 1758), in the laboratory. Aquaculture (Amst.) 157 (1 – 2), 135 – 144. Masseau, I., Bannon, P., Anctil, M., Dube´, F., 1998. Localisation et quantification de la se´rotonine au cours de la game´togene`se et du de´veloppement embryonnaire de la palourde. Congre`s Acfas. Session: S-157 Reproduction. Medhioub, M.N., Lymeam, Y., Medhioub, A., Abed, E.L., 2000. De´veloppement gonadique de la palourde Ruditapes decussatus en milieu controˆle´. Quatrie`mes journe´es tunisiennes des sciences de la Mer-Mehdia 17 – 19 Nov, Tunisia, p. 39. Moreau, M., Leclerc, C., 1999. E´tude de la reprise de la me´iose et de la fe´condation dans l’ovocyte de Crassostrea gigas. Roˆle des canaux Ca2 +. Centre de biologie du de´veloppement, Universite´ Paul Sabatier, Toulouse. O’Connor, W.A., Heasman, M.P., 1995. Spawning induction and fertilization in the doughboy scallop Chlamys (Mimachlamys asperrima). Aquaculture 136 (1 – 2), 117 – 129. Osanai, K., 1985. In vitro induction of germinal vesicle breakdown in oyster oocytes. Bull. Mar. Biol. Stn. Asamushi, Tohoku Univ. 18 (1), 1 – 9. Osanai, K., Kuraishi, R., 1988. Response of oocytes to meiosis-inducing agents in pelecypods. Bull. Mar. Biol. Stn. Asamushi, Tohoku Univ. 18 (2), 45 – 56. Ram, J.L., Crawford, G.W., Walker, J.U., Mojares, J.J., Patel, N., Fong, P.P., Kyozuka, K., 1993. Spawning in the zebra mussel (Dreissena polymorpha): activation by internal or external application of serotonin. J. Exp. Zool. 265 (5), 587 – 598. Salaum, M., Boucher, J., Le Pennec, M., 1991. Prodissoconch shell characteristics as indicators of larval growth and viability in Pecten maximus (Linnaeus, 1758). J. Shellfish Res. 10 (1), 37 – 46. Varaksin, A.A., Varaksina, G.S., Reunova, O.V., Latyshe, N.A., 1992. Effect of serotonin, some fatty acids and their metabolites on reinitiation of meiotic maturation in oocytes of bivalve Spisula sachalinensis (Schrenk). Comp. Biochem. Physiol., C 101 (3), 627 – 630. Yi, J., Anctil, M., Dube`, F., 1998. L’implication de l’AMP cyclique dans la reprise de la me´iose chez les ovocytes de palourde. Congre`s Acfas. Session: S-157 Reproduction.
Evaluation of the effects of serotonin (5-HT) on oocyte competence in Ruditapes decussatus (Bivalvia, Veneridae) Leila Hamida a,*, Mohamed-Nejib Medhioub a,1, Jean Claude Cochard b,2, Marcel Le Pennec c,3 a
Centre De Monastir, Institute of National Des Sciences Et Technologie De la Mer, 5000 Route De Khniss, Tunisia b Institut Francßais pour la Recherche et l’Exploitation de la Mer-Centre de Brest, BP 70, 29280 Plouzane´, France c Institut Universitaire Europe´en de la Mer, Universite´ de Bretagne Occidentale, Place Nicolas Copernic, Technopole Brest-Iroise, 29280 Plouzane´, France Received 1 October 2003; received in revised form 30 April 2004; accepted 2 May 2004 Available online
Abstract In bivalves, the blockage of oocytes at prophase I is released only during the egg-laying period. The second blockage occurs during metaphase I and is released by fertilization. In the case of the venerid Ruditapes decussatus, when we dissect the gonad we obtain oocytes which are blocked at prophase I and so cannot be fertilized. In order to release this blockage, we used serotonin (5-HT). This study is the first to scrutinise the maturation of oocytes induced by serotonin in R. decussatus. We have thus demonstrated that adding 20 AM of serotonin in an external application allows the recovery of meiosis and the passage of oocytes from germinal vesicle (G.V.) to germinal vesicle breakdown (GVBD) in approximately 90 min at a temperature of 20 jC. The highest percentage of oocytes obtained in GVBD was 67%. We have also evaluated the competence of oocytes in GVBD during fertilization, and found the greater the degree of reproductive condition, the higher is the percentage of oocytes that can undergo GVBD. From an estimated threshold of 53% of oocytes
* Corresponding author. Tel.: +216-98-68-41-41; fax: +216-73-46-00-34. E-mail addresses: [email protected] (L. Hamida), [email protected] (M.-N. Medhioub), [email protected] (J.C. Cochard), [email protected] (M.L. Pennec). 1 Tel.: +216-73-53-18-67; fax: +216-73-53-16-20. 2 Tel.: +33 2 98 22 43 96; fax: +33 2 98 22 46 53. 3 Tel.: +33-2-98-49-86-41; fax: +33-2-98-49-87-90. 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.05.010
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capable of GVBD, broodstock placed in a controlled environment are liable to spawn when they undergo thermal shocks. This allows us to consider this percentage as a criterion for the evaluation of oocytes during fertilization. D 2004 Elsevier B.V. All rights reserved. Keywords: Bivalve; Ruditapes decussatus; Oocyte maturation; Fertilization; Serotonin
1. Introduction In the last decade, Tunisia set up an aquacultural development scheme along the coastline, and the Venerid, Ruditapes decussatus, is one of the species of major interest. Economically, R. decussatus is the most important shellfish species in terms of both employment and value, with an estimated 1200 tonnes, worth US$7 million, exported to Europe annually (EL Abed, INSTM, personal communication). Two approaches are followed: the preservation of the wild resource by setting up fishing seasons and, in parallel, aquaculture development emphasizing restocking with spat produced in hatcheries. Development of production techniques in hatcheries is needed as was the case in France with hatcheries for Ruditapes philippinarum and in Chile for Venus antigua (Bustos and Olavarria, 2000). In hatcheries, gametes are usually obtained by applying thermal shocks to adult clams (Le Pennec, 1987), although an alternative method of ‘‘stripping’’ exists, which allows us to obtain mature gametes easier and faster, by dissecting the gonad. However, in stripping R. decussatus, we only obtain oocytes which are blocked at prophase I and which cannot be fertilized. Generally, bivalve oocytes are held in the ovaries at prophase of the first division of meiosis, and development reinitiates during spawning. A second barrier to development occurs at metaphase I, and it is released by fertilization. In order to initiate meiosis and have the oocytes pass through to metaphase I, serotonin can be applied. Serotonin is a regulator (5-hydroxytryptamine) synthesized from L-tryptophan that allows germinal vesicle breakdown (GVBD) and the completion of meiosis. Its intervention prompts an increase in intracellular pH and the expulsion of the first polar body (Kyosuka et al., 1997; Colas and Dube´, 1998). The aim of this research is to evaluate the competence of R. decussatus oocytes treated with serotonin before fertilization rather than the histological methods frequently used with bivalves (Laruelle et al., 1994; Lango-Reynoso et al., 2000; Chavez-Villalba et al., 2002a,b). This is achieved by determining the optimal dose of serotonin for oocyte GVBD, and successful fertilization by oocytes obtained through dissecting the gonad. For the first time, this research aims at establishing a relationship between the percentage of oocytes during GVBD and the reproductive maturation of R. decussatus.
2. Materials and methods Adult R. decussatus were brought into reproductive condition using the method described by Medhioub et al. (2000). Temperature was maintained at 21 F 1 jC, salinity at 30 F 1 ppm, and lighting was continuous. Clams were fed a mix of unicellular algae:
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Chaetoceros calcitrans and Isochrysis galbana, at a rate of 1 109 cells per individual per day. The seawater was renewed 1.7 times/24 h. A summer conditioning experiment carried out from July to September 2001, while a winter experiment was carried out from February to May 2002. In both experiments, gametogenesis was followed using histological examination of the gonad every 2 weeks throughout the conditioning period to determine the best period for the induction tests of the oocyte maturation by serotonin. Induction tests of GVBD started after 1 week for summer conditioning and 7 weeks later for winter conditioning. This gap is due to the fact that for summer conditioning broodstock had already commenced gonadal development on their arrival to the hatchery, whereas for winter conditioning the clams were only at the beginning of gametogenesis. Gametes were obtained by dissecting the gonads of 10 broodstock. Oocytes were first filtered through a 100-Am filter to eliminate tissue debris, and then retained on a 30-Am filter and kept in suspension in a volume of 250 ml. Three aliquots were recovered from this suspension to calculate the total number of oocytes. Spermatozoa were also filtered through a 100-Am filter to eliminate tissue debris and kept in suspension in a volume of 100 ml at a temperature of 4 jC until fertilization. To determine the concentration of serotonin permitting the highest percentage of oocyte in GVBD, we tested different concentrations, from 2 to 100 AM, by putting about 2000 oocytes in contact with those concentrations in wells of 2 ml of microtitration plates (1 volume serotonin/1 volume oocytes) in triplicate and placing them in a temperature of 20 jC. These concentrations of serotonin were obtained from an initial solution of 200 AM derived from 2 g of serotonin creatinine sulfate (Sigma) dissolved in distilled water. Oocytes GVBD were evaluated after 90 min by microscopic observation. Those oocytes that had undergone GVBD were spherical and had a nucleus that could not be differentiated from the cytoplasm (Fig. 1). After determining the concentration of serotonin permitting the highest percentage of oocytes to undergo GVBD, we administered this concentration to the gametes obtained by dissection of the gonad. In order to relieve the second blockage and obtain fertilization, we added sperm suspension. The fertilized oocytes were then put in cylindroconical buckets of 30 l of seawater with aeration at the bottom to avoid settlement of the eggs.
Fig. 1. Oocytes in GV (visible nucleus compared to cellular continuity) and in GVBD (invisible nucleus) observed in a microscope.
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Twenty-four hours later, we estimated the percentage of developing to D larvae (Ge´rard et al., 1989) and the percentage of eggs developing abnormally. (Salaum et al., 1991) Percentage of development to D larvae ¼ total number of larvae=total number of oocytes: Percentage of eggs developing abnormally ¼ number of abnormal larvae=total number of larvae: All experiments on the fertilization of oocytes obtained by dissection were carried out in parallel with experiments on the fertilization of oocytes released following thermal shocks. These oocytes were obtained from 10 broodstock and used as controls. Oocyte fertilization and larval culture were carried out at the same time and in the same conditions for both experiments. 2.1. Statistical analyses The results obtained were analyzed by the software XSTAT; nonparametric comparisons (Kruskal – Wallis’ test) were used to determine whether the difference between the averages is significant or not.
3. Results 3.1. The effect of serotonin concentration on GVBD in stripped oocytes The percentage of eggs undergoing GVBD differed significantly ( P < 0.05) with the concentration of serotonin in stripped oocyte suspensions (Fig. 2). In the absence of serotonin, GVBD occurred in approximately 22% of oocytes. With increasing serotonin
Fig. 2. Variation in the percentage of oocytes in GVBD according to the concentration of serotonin (5-HT) (v F S.E.).
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Fig. 3. (A) Variation in the percentage of oocytes in GVBD according to time for summer conditioning in 2001. (B) Variation in the percentage of oocytes in GVBD according to time for winter conditioning in 2002 (v F S.E.).
concentration, GVBD percentage increased to reach a maximum of 67% at 20 AM serotonin. Beyond this concentration, GVBD percentage decreased. At 40 AM serotonin, GVBD had fallen to the same level observed in the absence of serotonin (22%), and at serotonin concentration of 100 AM, GVBD did not occur. 3.2. Variation in percentage of oocytes in GVBD according to time For summer conditioning, GVBD percentage increased significantly ( P < 0.05) from 20% to 77% after 4 weeks of conditioning (Fig. 3). From 61 F 5.1% of GVBD, the clams spontaneously excreted their gametes in the buckets. With regards to winter conditioning, GVBD percentage increased significantly ( P < 0.05) from 25% to 68% after 15 weeks of conditioning (Fig. 3). From 46 F 0.4%, the clams had a positive response to thermal shocks leading to the emission of gametes. 3.3. Fertilization of oocytes obtained through dissection of the gonad in comparison with those obtained by thermal shocks The percentage of fertilized oocytes obtained through stripping is low compared with that obtained by thermal shocks 26 F 5.9% and 92 F 0.5%, respectively. Similarly, the percentage of development to D larvae is low compared with that obtained by thermal shocks 7 F 5.7% and 65 F 3%, respectively. In addition, the level of anomaly is very high for oocytes in GVBD obtained by stripping (93%).
4. Discussion Obtaining mature gametes by dissecting the gonad is a fast and efficient means to achieve larval breeding. This technique allows the evaluation of the level of reproductive maturity, which assists successful larval production.
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Stripping is used with species where oocytes can be fertilized directly after dissecting the gonad, such as the oyster Crassostrea gigas. In species where barriers exist to the fertilization of oocytes after dissection from the gonad, such as the clams R. philippinarum (Osanai and Kuraishi, 1988; Guerrier et al., 1993) and Spisula solidissima (Masseau et al., 1998; Yi et al., 1998), serotonin can be used to reinitiate meiosis and allow fertilization. According to Osanai (1985), the percentage of oocytes in GVBD for C. gigas, increases dramatically in seawater containing 1 AM of serotonin. GVBD is noticed from the 10th minute of incubation, and a maximum of 100% is reached within 20 –30 min. According to Moreau and Leclerc (1999), adding 10 AM of serotonin to an oocyte suspension allows the reinitiation of meiosis in about 20 min at a temperature of 20 jC. The maximum number of oocytes in GVBD 90% is reached after 45 min. In order to inhibit the spontaneous passage of oocytes in GVBD, acidic seawater (pH 4 –6.5) is used during the dissection of the gonad. For R. philippinarum, the recommencement of meiosis occurs after adding 10 AM of serotonin into an oocyte suspension at 21 jC. A maximum of 91.7% of oocytes in GVBD is obtained after 87 min by adding 10 AM of serotonin (Osanai and Kuraishi, 1988). On the other hand, stripped Mytilus galloprovincialis oocytes cannot be fertilized in seawater with or without serotonin. This is not the case for another mytilid, Dreissena polymorpha, a freshwater mussel. In D. polymorpha, meiosis recommences 5– 10 min after the addition of serotonin and GVBD are possible within 30 min after adding 10 3 M of serotonin at 23 jC (Fong et al., 1994a). However, the intervention of methiothepin (10 6 M) and metergolin (10 4 M), known by their high affinity with serotonin receptors, can cause of the blockage of spawning for this species (Fong et al., 1994b). Ram et al. (1993) demonstrated that the intervention of serotonin through injection or external application equally triggers the emission of gametes with male or female D. polymorpha. For male zebra mussels, morphological criteria for judging gonadal maturity were well-correlated with probability of spawning in response to serotonin. For females, the likelihood of spawning in response to serotonin was not tightly coupled to morphological maturity of the gonad. Many morphologically ripe females failed to spawn and some apparently immature animals released oocytes. Indeed, serotonin plays an important role in controlling reproduction with bivalves. Injecting serotonin leads to the oocyte maturation and the release of gametes (O’Connor and Heasman, 1995; Madrones-Ladja, 1997; Fong et al., 2003; Louro et al., 2003). Haneji and Koide (1988) and Varaksin et al. (1992) demonstrated that the maturation of oocytes in Spisula sp. could also be incited by adding serotonin through phosphoprotein. To date, no studies have been done on the maturation of oocytes induced by serotonin with R. decussatus. We have shown that adding 20 AM of serotonin to oocyte suspensions allows the reinitiation of meiosis and oocyte GVBD in approximately 90 min at 20 jC. The highest GVBD percentage achieved was 67%. We have also evaluated the competence of oocytes in GVBD during fertilization. The greater the degree of reproductive maturity, the higher the percentage of oocytes that can achieve GVBD. When GVBD of stripped eggs reaches 53%, broodstock exposed to thermal shocks will spawn naturally. This result allows us to consider this percentage as a criterion for the evaluation of oocytes during fertilization. Nevertheless, experiments to fertilize oocytes in which GVBD has been induced by serotonin do not provide satisfactory results. This urges us to search other
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interveners which increase the amount of serotonin within the animal before dissecting the gonad, in order to obtain oocytes which can be fertilized after stripping. Interveners such as prozac or fluoxetine hydrochloride, an inhibitor for the selective recapturing of serotonin, thus enables the increase in intrasynaptic concentration of serotonin (Gardier et al., 2001). Prozac is considered the most powerful spawning inducer in any bivalve, stimulating the serotonergic mechanisms without applying serotonin (Fong, 1998; Fong et al., 1998; Honkoop et al., 1999; Cunha and Machado, 2001). The results of this study suggest the use of serotonin in hatcheries to evaluate oocyte competence during fertilization of R. decussatus. Thus it will be possible, according to the percentage of oocytes in GVBD, to know whether to continue broodstock conditioning before the application of thermal shocks.
Acknowledgements This research was carried out within the framework of the «Programme Aquaculture 2001» between «Institut National des Sciences et Technologie de la Mer, centre de Monastir, Tunisie» and «Institut Francß ais pour la Recherche et l’exploitation de la Mer, centre de Brest, France». The authors would like to thank the teams at the hatchery of Monastir and Argenton (France) who contributed to the achievement of this work.
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