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Epinephrine and l -DOPA promote larval settlement and metamorphosis of the tropical oyster, Crassostrea iredalei (Faustino, 1932): An oyster hatchery perspective
Aquaculture 338–341 (2012) 260–263
Contents lists available at SciVerse ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Epinephrine and l-DOPA promote larval settlement and metamorphosis of the tropical oyster, Crassostrea iredalei (Faustino, 1932): An oyster hatchery perspective C.P. Teh ⁎, Y. Zulfigar, S.H. Tan School of Biological Science, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia
a r t i c l e
i n f o
Article history: Received 18 August 2011 Received in revised form 17 January 2012 Accepted 17 January 2012 Available online 25 January 2012 Keywords: Oyster Spat Cemented larvae Neuroactive compounds Mortality
a b s t r a c t The critical and foundation stage of most invertebrates is during the larval settlement and metamorphosis period. Neuroactive compounds at different levels of concentration and exposure time are frequently used as an inducer in the settlement of most bivalve larvae. The larval settlement of the tropical oyster Crassostrea iredalei was investigated by exposing competent larvae to epinephrine (EPI) and L-3,4-dihydroxyphenylalanine (L-DOPA) at five different concentrations (10− 3, 10− 4, 10 − 5, 10 − 6 and 10 − 7 M) for 1 and 24 h. The control group consisted of the larvae exposed to 1 μm filtered seawater. The optimum yield, 60% of cemented spat, was recorded when larvae were exposed to 10 − 6 M EPI for 1 and 24 h. Larvae exposed to all concentrations of L-DOPA yielded lower percentages of cemented spats (b 50%) compared to the larvae exposed to EPI. In this study, it was shown that the use of EPI effectively induced cemented spats compared to L-DOPA. After the larvae had cemented onto the substrate, the larvae started to metamorphose. A delay in the larvae settlement caused the mortality of some larvae. The results showed significant differences (p b 0.05) between the control and the treatment groups and they provide useful information on the technique of seed production in hatcheries where the chemicals are able to promote the larval settlement and metamorphosis of the larvae of C. iredalei. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The oyster was one of the preferred shellfish and permanent element in the annual food cycle of Malaysians after they became aware of the value of this shellfish. Malaysia is one of the first few countries in the world to produce oyster seeds using hatchery technology (Kechik, 1995). Besides Malaysia, United States, Canada and Australia are the only countries which are currently producing hatchery oyster seeds commercially; while other countries like China, Thailand and the Philippines are still relying on spat from the wild (Szuster et al., 2008). The harvesting of oysters has resulted in economic opportunities and created new wealth, resulting in the overharvesting of oysters on a global scale. The major global market for the hatchery produced oysters and other shellfish is in Asia. There is a big potential market for oyster seeds in the region due to rapid environmental degradation that destroys the natural habitats of oysters. The tropical oyster, Crassostrea iredalei and Crassostrea belcheri are the oyster species that are commercially cultured in Malaysia. Some taxonomists had considered C. iredalei as the synonymous species of C. belcheri. However, the recent genetic work done by Klinbunga et al. (2003) had shown that these two species are indeed distinct. The ⁎ Corresponding author at: School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia. Tel./fax: + 60 4 653 3500. E-mail addresses: [email protected] (C.P. Teh), zulfi[email protected] (Y. Zulfigar), [email protected] (S.H. Tan). 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.01.014
Malaysia Fisheries Statistic (Department of Fisheries Malaysia, 2010) reported that oyster production from aquaculture in Malaysia was at approximately 869.72 MT in 2007 but decreased to 275.47 MT in 2008 probably due to insufficient good quality oyster seeds to support the industry. Seeds are mainly collected from the wild but the supply is inconsistent and often inadequate (Kechik, 1995). Producers are exposed to the vagaries of the seasons, seed quality is poor and the seeds are definitely of variable sizes and ages. The solution to overcome the insufficient and poor quality seed supply is hatcheryproduced high quality oyster seeds. However, although oyster seeds can be produced from the hatchery; some problems continue to exist at certain stages of the oyster production such as high mortality and low larvae settlement and metamorphosis. The percentage of larval settlement is considered to be important and critical for the cultivation. Delay of the larvae settlement in oysters may inhibit the growth of these oysters and cause the mortality of the larvae (Faimali et al., 2004; Najiah et al., 2008). The capacity to increase the attraction of the pediveliger larvae to the settling surfaces and metamorphosis will definitely increase seed production (Li et al., 2006). The higher the percentage of settlement, the higher will be the numbers of spats and juveniles which can be obtained per spawning batch. Therefore, there is a need to investigate the effect of endogenous and exogenous chemical cues for settlement and metamorphosis of the tropical pediveliger oyster larvae to ensure optimal settlement and metamorphosis rates. In previous studies (Bonar et al., 1990; Fang et al., 2001; Gao and Liu, 2006), neuroactive compounds such as Epinephrine (EPI), L-3,4-dihydroxyphenylalanine (L-
C.P. Teh et al. / Aquaculture 338–341 (2012) 260–263
DOPA), γ-aminobutyric acid (GABA) and Serotonin (5-HT) had induced larval settlement in oyster and clams. EPI is the powerful catabolic stimulant where it plays an important role in settlement and metamorphosis for most of the invertebrate larvae such as scallop, Argopecten purpuratus (Martinez et al., 1999) and Crassostrea gigas (Coon et al., 1990a,b). Based on the previous studies from Coon et al. (1990a,b) and Walch et al. (1999), the presence of L-DOPA has enhanced the larvae settlement and metamorphosis of Pacific oyster C. gigas and Crassostrea virginica. EPI and L-DOPA are generally used as artificial invertebrate larvae inducers. However, no reports were found on the effect of these inducers on tropical oysters. Therefore, in this paper we focused on the effects of neuroactive compounds (EPI and L-DOPA) on the larval settlement and metamorphosis of tropical oyster (C. iredalei), and to identify the optimal concentration and exposure duration to yield the optimum settlement and metamorphosis rate.
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settlement. The control group was kept in 18 ppt of 1 μm filtered seawater. Other treatments were also diluted by 18 ppt filtered seawater. For all the experiments, the larvae settlement was examined regularly under the dissecting microscope to monitor the behavior patterns. The total number of the settled larvae for each treatment was recorded (Tan and Wong, 1995). 2.5. Statistical analyses Data obtained from this study were analyzed using the one-way analysis of variance (ANOVA) from SPSS 11.0. The Least Significant Different Test (LSD) was conducted on the percentage of the total number of larvae that settled and metamorphosed in order to compare the differences at 95% levels of significance (p b 0.05). All the percentage values were transformed to arcsine values before undergoing statistical analyses.
2. Materials and methods 3. Results 2.1. Test animal 3.1. Visual observation of larval settlement Tropical oyster C. iredalei is one of the commercial species cultured in the hatchery due to its high demand. The broodstock were collected from wild (Penang) and acclimated in sand filtered seawater with 30 ppt salinity in the experimental hatchery at Penang (Northern Peninsular Malaysia). Currently only one commercial hatchery is found in this region which is located at Penang Island. Furthermore, this hatchery was still in its infant stage. 2.2. Larval culture The broodstock of C. iredalei were induced to spawn and fertilization occurred in the fiber tank in the hatchery. For this tropical oyster, there is no seasonal restriction on the spawning process. Spawning was done once every month to ensure that sufficient larvae were supplied. Larvae with well developed eyes (competent larvae) and ready to settle were collected for this study. These competent larvae were collected by using 253 μm Nitex sieves and transported back to the laboratory for further investigation. Experiments were carried out using larvae from the same cohort. The larvae, which swam actively with a well-developed eyespot, were used in the experiments. The competent larvae used exhibited the shell length of 242–304 μm. 2.3. Settlement cues The neuroactive compounds, epinephrine (EPI) and L-3,4dihydroxyphenylalanine (L-DOPA), were tested at five different concentrations (10 − 3, 10 − 4, 10 − 5, 10 − 6 and 10 − 7 M) in triplicates against the control group. EPI and L-DOPA were dissolved by using 0.0005 N of hydrochloric acid (HCl) and 1 μm filtered seawater (salinity: 18 ppt) was used to dilute the solution to reach final concentrations. Besides that, 1 μm of filtered seawater was used in the control group. All the neuroactive compounds were freshly prepared to avoid oxidation of the chemicals prior to the experiment. 2.4. Effects of EPI and L-DOPA on the oyster larvae settlement The experiments were conducted in tissue culture plates (24-well Multiwell Plat, BD Falcon™). A total number of 30–50 larvae were placed in 24 wells of the tissue culture plates each filled with 1.5 ml of test solution. A few washed marble chips were placed in the tissue culture plates as cultch. The larvae were exposed to different concentrations of test solutions for 1 h and 24 h. Food and aeration were not provided during the experiments. After the exposure duration, the larvae were removed from the test solution, rinsed with 1 μm filtered seawater and placed back into the wells filled with clean filtered seawater for another 24 h for observation and calculation of survival
The competent larvae of C. iredalei were observed to be moving downward in the water column, then swimming in slow curved paths when exposed to the neuroactive compounds. The competent larvae (larvae with well developed eye-spots) swam up and down along the water column and began rotating abnormally in the solution where the velum and cilia were extended. After being exposed to the chemicals for about 15 to 20 min, some of the larvae started to sink to the bottom of the wells where the velum and cilia were contracted and feet extended for crawling along the walls and substrate. Some of the larvae returned to the free-swimming stage when the substrate or the condition was not suitable. The larvae kept crawling around and reversing their steps until they found a suitable substrate for settlement. Once the environment and substrates were fit for the larvae to settle, they would attach themselves to the flume bottom by using their left valves. This observation was similar to that reported by Bonar et al. (1990). In this study, the larval settlement was indicated when the free-swimming larvae underwent the swimming, crawling process and finally cemented onto the substrate. 3.2. Settlement of larvae exposed to various concentrations of epinephrine (EPI) As a result of investigation of the larvae settlement of the tropical oyster C. iredalei exposed to neuroactive compounds, it was found that epinephrine (EPI) was an active inducer of settlement for the oyster. The settlement percentage was highest where the larvae were exposed to EPI at 10− 6 M and 10 − 5 M concentrations for 1 h with 60.87 ± 13.14% and 38.79 ± 4.83%, respectively. These were significantly higher than those recorded in the control group (15.10 ± 3.36%) (pb 0.05). In the larvae exposed to EPI for 24 h, exposure to 10 − 6 M of EPI displayed the highest settlement percentage of 59.13 ± 5.53%, which was higher than that of the control group (36.26± 33.72%) (Fig. 1). The maximum percentages of larvae settlement were induced by 10− 6 M of EPI in both exposure durations (1 h and 24 h), yielding percentages of larvae settlement of approximately 60%. The settlement percentage of larvae exposed to higher concentrations of EPI (10 − 4 and 10 − 3 M) showed no significant differences compared to that of the control group (p> 0.05). 3.3. Settlement of larvae exposed to various concentrations of L-DOPA Besides EPI, this study also found that L-DOPA was an active inducer of settlement of the tropical oyster C. iredalei larvae. The differences between EPI and L-DOPA were that larvae exposed to the higher concentrations of L-DOPA did not show any settlement. The
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C.P. Teh et al. / Aquaculture 338–341 (2012) 260–263
4. Discussion
Fig. 1. Settlement percentages of competent larvae of Crassostrea iredalei exposed to different concentrations of EPI for 1 h and 24 h (* indicated significant level at 95%; p b 0.05).
optimum larvae settlement of C. iredalei was observed when they were exposed to L-DOPA at 10 − 6 M for 1 h; however, the percentage of larvae settlement (%) showed no significant difference compared to larvae exposed to EPI at 10 − 6 M (60.87 ± 13.14%). Nevertheless, this result was significantly higher than the larvae settlement recorded in the control group (15.10 ± 3.36%) (p b 0.05). Even though L-DOPA at lower concentrations could have induced optimum larvae settlement, the inducement of larvae at higher concentrations of LDOPA was not successful. In addition, L-DOPA also failed to induce settlement of C. iredalei larvae when exposed for 1 or 24 h at higher concentrations (Fig. 2).
3.4. Mortality of the oyster larvae exposed to EPI and L-DOPA The mortality of the larvae exposed to EPI (maximum: 38 ± 14.35%) was lower than that of larvae exposed to all concentrations of L-DOPA (100.00 ± 0.00%). Mortalities of larvae exposed to EPI and L-DOPA increased when the concentrations of the neuroactive compounds increased. Thus the higher concentrations of neuroactive compounds caused the mortality of larvae especially those exposed to EPI and LDOPA for a longer duration (24 h). There was no settlement at higher concentrations of L-DOPA and the larval mortality was 100% after exposure for 24 h (Fig. 3). The results were significantly different compared to those for the control group.
Fig. 2. Settlement percentages of competent larvae of Crassostrea iredalei exposed to different concentrations of L-DOPA for 1 h and 24 h (* indicated the 95% significant level; p b 0.05).
According to Bonar et al. (1990), the process of settlement and metamorphosis is generally known as settling. Settlement behavior in most of the invertebrates is considered a reversible process except for oysters. The settlement process includes foot extension, crawling on the substrate and finally cementing onto the substratum. Abundant literature has shown that the settlement of marine invertebrate larvae is influenced by specific physical, biological and chemical cues. Studies have shown that some neuroactive compounds, such as EPI, L-DOPA, GABA and Serotonin (5-HT) can actively induce the larval settlement (Bonar et al., 1990; Fang et al., 2001; Gao and Liu, 2006). Chemical cues represent one of the factors that affect the larval settlement and they are easy to monitor for larval settlements in hatchery-scale production under natural conditions. The exposure of larvae to an appropriate chemical cue at suitable concentrations may bring about more rapid or greater degrees of settlement. Using chemical cues is a convenient and effective technique to increase the percentage of larvae settlement under hatchery conditions. In this present study, the concentration of EPI and L-DOPA at 10 − 7 M was not effective in inducing the larval settlement. The optimum larval settlements of the tropical oyster C. iredalei were achieved at 10 − 6 M of EPI and L-DOPA. Higher concentrations of EPI and L-DOPA (10 − 5, 10 − 4 and 10 − 3 M) gave low percentages of larval settlement and also caused higher mortalities. The results obtained in the experiments were very much dependent on the species studied. In the present study, inductive behavior was observed when the neuroactive compounds EPI and L-DOPA were added. Similar results were shown in the blue mussel Mytilus edulis (Dobretsov and Qian, 2003); the tropical oyster C. belcheri (Tan and Wong, 1995) and the Pacific oyster C. gigas (Beiras and Widdows, 1995; Bonar et al., 1990; Coon et al., 1990a,b; Fang et al., 2001) whereas pearl oyster larvae (Pinctada margaritifera and Pinctada fucata) did not show any inductive responses (Doroudi and Southgate, 2002; Yu et al., 2008). Larvae of different species of invertebrates may respond to the same cues in a different manner. In this study, the effectiveness of the neuroactive compounds, such as EPI and L-DOPA, were specific to the larvae settlement of the oyster C. iredalei. Results obtained in this study might be different from those of previous studies because bioassay procedure used to test the effects of EPI and L-DOPA in different species of larvae might have been different (Bonar et al., 1990; Fang et al., 2001). Moreover, the physiological and molecular mechanism of how EPI and L-DOPA induce the larvae settlement of this tropical oyster C. iredalei has never been conducted. Further studies on the similarities and differences between the temperate and tropical species on the mechanism of induced settlement and metamorphosis may need to be addressed in the future. Alterations in the development sequence of oyster larvae can occur when neuroactive compounds were added to induce settlement and metamorphosis process. However, no differences were observed in the early development of the larvae in this research and in the study done by Ver (1986). Between the two neuroactive compounds, EPI has been found to be more efficient in inducing larval settlement compared to L-DOPA due to its low toxicity.
Fig. 3. Mortality of Crassostrea iredalei larvae exposed to various concentrations of EPI and L-DOPA (* indicated the 95% significant level; p b 0.05).
C.P. Teh et al. / Aquaculture 338–341 (2012) 260–263
In the absence of an appropriate chemical inducer, larval settlement would still occur, but at slower rates and percentages. Previous studies have shown that the larvae that prolong the freeswimming stage will give negative effects. The larvae that delay their settlement and metamorphosis stage have been reported to be retarded in growth, affecting the survival rate in the post-larvae life (Pechenik and Cerulli, 1991). Mortalities of oyster larvae exposed to different concentrations of EPI were lower compared to those exposed to L-DOPA for both exposure times (1 h and 24 h). Although some neuroactive compounds can induce larval settlement, the larvae of C. iredalei cannot adapt to the high concentrations of the compounds. The chemicals at high concentrations or overdoses were either toxic to the larvae or would retard the growth of the larvae especially when exposed to L-DOPA. Prolonging the exposure time of the tropical oyster larvae to the neuroactive compounds at higher concentrations appears to reduce the larval settlement and metamorphosis and hence, increases mortalities. According to Nicolas et al. (1998), the toxic effect was not noted in the larvae of C. gigas when EPI was used and less mortality was observed in the C. gigas larvae. There was no acute toxicity observed in the larvae of C. gigas and Pecten maximus when exposed to L-DOPA.
5. Conclusion From the results obtained on the effects of chemical cues on larval settlement induction, it has been shown that the onset of larvae settlement can be accelerated from 15% (control) to 60% by the addition of EPI and L-DOPA. The settlement was higher in larvae exposed over 1 h compared to larvae exposed for 24 h for both of the neuroactive compounds (EPI and L-DOPA). In the case of the tropical oyster C. iredalei, EPI was able to induce about 61% of larval settlement and L-DOPA was able to induce about 54% of the larval settlement. These results can be applied to the commercial tropical hatchery and may increase the oyster seed production effectively. For commercial purposes, both chemicals EPI and L-DOPA applied at lower concentrations and over a shorter exposure duration can reduce investment costs. By investing about 3 to 4 USD in 450,000 larvae in 10 l of filtered seawater, it can increase the seed production by four folds if compared to normal condition. It is important to introduce these two compounds in this region because this will definitely increase oyster seed production at least by four folds.
Acknowledgments This study was financially supported by Universiti Sains Malaysia USM-RU-PRGS (1001/PBIOLOGI/833029). The authors are very grateful to the local commercial oyster farms for providing the healthy oyster larvae throughout the study. Without their support, this work would not have been possible. We also express our appreciation to Universiti Sains Malaysia for allowing us to conduct our work in the laboratory.
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References Beiras, R., Widdows, J., 1995. Induction of metamorphosis in larvae of the oyster Crassostrea gigas using neuroactive compounds. Marine Biology 123 (2), 327–334. Bonar, D.B., Coon, S.L., Walch, M., Weiner, R.M., Fitt, W., 1990. Control of oyster settlement and metamorphosis by endogeneous and exogenous chemical cues. Bulletin of Marine Science 46 (2), 484–498. Coon, S.L., Fitt, W.K., Bonar, D.B., 1990a. Competence and delay of metamorphosis in the Pacific oyster Crassostrea gigas. Marine Biology 106 (3), 379–387. Coon, S.L., Walch, M., Fitt, W.K., Weiner, R.M., Bonar, D.B., 1990b. Ammonia induces settlement behavior in oyster larvae. The Biological Bulletin 179 (3), 297–303. Department of Fisheries Malaysia, 2010. Annual Fisheries Statistic 2008. Retrieved from http://www.dof.gov.my/html/themes/moa_dof/documents/Jadual_akuakultur_V_ii. pdf. Dobretsov, S.V., Qian, P.Y., 2003. Pharmacological induction of larval settlement and metamorphosis in the blue mussel Mytilus edulis L. Biofouling 19 (1), 57–63. Doroudi, M.S., Southgate, P.C., 2002. The effect of chemical cues on settlement behavior of blacklip pearl oyster (Pinctada margaritifera) larvae. Aquaculture 209 (1–4), 117–124. Faimali, M., Garaventa, F., Terlizzi, A., Chiantore, M., Cattaneo-Vietti, R., 2004. The interplay of substrate nature and biofilm formation in regulating Balanus amphitire Darwin, 1854 larval settlement. Journal of Experimental Marine Biology and Ecology 306, 37–50. Fang, Q., Lin, B.S., Fang, Y.Q., 2001. Induction of larval settlement and metamorphosis of two oysters Crassostrea gigas and Ostrea cucullata by some chemicals. Journal of Oceanography Taiwan Strait 20 (1), 20–26. Gao, R.C., Liu, W.B., 2006. Induction of larval settlement and metamorphosis of Coelomactra antiquata using some chemicals. Journal of Fisheries China 30 (5), 597–602. Kechik, I.A., 1995. Aquaculture in Malaysia. Towards sustainable aquaculture in Southeast Asia and Japan. Proceedings of the Seminar–Workshop on Aquaculture Development in Southeast Asia, Held 26–28 July 1994 in Iloilo City, Philippines. SEAFDEC Aquaculture Department, Iloilo, Philippines, pp. 125–135. Klinbunga, S., Khamnamtong, N., Tassanakajon, A., Puanglarp, N., Jarayabhand, P., Yoosukh, W., 2003. Molecular genetic identification tools for three commercially cultured oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand. Marine Biotechnology 5 (1), 27–36. Li, H.F., Lin, W., Zhang, G., Cai, Z.H., Cai, G.P., Chang, Y.Q., Xing, K.Z., 2006. Enhancement of larval settlement and metamorphosis through biological and chemical cues in the abalone Haliotis diversicolor suertexta. Aquaculture 258 (1–4), 416–423. Martinez, G., Aguilera, C., Campos, E.O., 1999. Induction of settlement and metamorphosis of the scallop Argopecten purpuratus Lamarck by excess K + and epinephrine: energetic cost. Journal of Shellfish Research 18 (1), 41–46. Najiah, M., Nadirah, M., Lee, K.L., Lee, S.W., Wendy, W., Ruhil, H.H., Nurul, F.A., 2008. Bacteria flora and heavy metals in cultivated oysters Crassostrea iredalei of Setiu Wetland, East Coast Penincular Malaysia. Veterinary Research Communications 32, 377–381. Nicolas, L., Robert, R., Chevolot, L., 1998. Comparative effects of inducers on metamorphosis of the Japanese oysters Crassostrea gigas and the great scallop Pecten maximus. Biofouling 12 (1), 189–203. Pechenik, J., Cerulli, T., 1991. Influence of delayed metamorphosis on survival, growth, and reproduction of the marine polychaete Capitella sp. I. Journal of Experimental Marine Biology and Ecology 151, 17–27. Szuster, B.W., Chalermwat, K., Flaherty, M., Intacharoen, P., 2008. Peri-urban oyster farming in the upper Gulf of Thailand. Aquaculture Economics and Management 12 (4), 268–288. Tan, S.H., Wong, T.M., 1995. Induction of settlement and metamorphosis in the tropical oyster, Crassostrea belcheri (Sowerby), by neuroactive compounds. Journal of Shellfish Research 14 (2), 435–438. Ver, L.M.M., 1986. Early development of Crassostrea iredalei (Faustino, 1932) (Bivalvia: Ostreidae), with notes on the structure of the larval hinge. Veliger 29 (1), 78–85. Walch, M., Weiner, R.M., Colwell, R.R., Coon, S.L., 1999. Use of L-DOPA and soluble bacterial products to improve set of Crassostrea virginica (Gmelin, 1791) and C. gigas (Thunberg, 1793). Journal of Shellfish Research 18 (1), 133–138. Yu, X.J., He, W.H., Gu, J.D., He, M.X., Yan, Y., 2008. The effect of chemical cues on settlement of pearl oyster Pinctada fucata martensii (Dunker) larvae. Aquaculture 277 (1–2), 83–91.
Contents lists available at SciVerse ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Epinephrine and l-DOPA promote larval settlement and metamorphosis of the tropical oyster, Crassostrea iredalei (Faustino, 1932): An oyster hatchery perspective C.P. Teh ⁎, Y. Zulfigar, S.H. Tan School of Biological Science, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia
a r t i c l e
i n f o
Article history: Received 18 August 2011 Received in revised form 17 January 2012 Accepted 17 January 2012 Available online 25 January 2012 Keywords: Oyster Spat Cemented larvae Neuroactive compounds Mortality
a b s t r a c t The critical and foundation stage of most invertebrates is during the larval settlement and metamorphosis period. Neuroactive compounds at different levels of concentration and exposure time are frequently used as an inducer in the settlement of most bivalve larvae. The larval settlement of the tropical oyster Crassostrea iredalei was investigated by exposing competent larvae to epinephrine (EPI) and L-3,4-dihydroxyphenylalanine (L-DOPA) at five different concentrations (10− 3, 10− 4, 10 − 5, 10 − 6 and 10 − 7 M) for 1 and 24 h. The control group consisted of the larvae exposed to 1 μm filtered seawater. The optimum yield, 60% of cemented spat, was recorded when larvae were exposed to 10 − 6 M EPI for 1 and 24 h. Larvae exposed to all concentrations of L-DOPA yielded lower percentages of cemented spats (b 50%) compared to the larvae exposed to EPI. In this study, it was shown that the use of EPI effectively induced cemented spats compared to L-DOPA. After the larvae had cemented onto the substrate, the larvae started to metamorphose. A delay in the larvae settlement caused the mortality of some larvae. The results showed significant differences (p b 0.05) between the control and the treatment groups and they provide useful information on the technique of seed production in hatcheries where the chemicals are able to promote the larval settlement and metamorphosis of the larvae of C. iredalei. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The oyster was one of the preferred shellfish and permanent element in the annual food cycle of Malaysians after they became aware of the value of this shellfish. Malaysia is one of the first few countries in the world to produce oyster seeds using hatchery technology (Kechik, 1995). Besides Malaysia, United States, Canada and Australia are the only countries which are currently producing hatchery oyster seeds commercially; while other countries like China, Thailand and the Philippines are still relying on spat from the wild (Szuster et al., 2008). The harvesting of oysters has resulted in economic opportunities and created new wealth, resulting in the overharvesting of oysters on a global scale. The major global market for the hatchery produced oysters and other shellfish is in Asia. There is a big potential market for oyster seeds in the region due to rapid environmental degradation that destroys the natural habitats of oysters. The tropical oyster, Crassostrea iredalei and Crassostrea belcheri are the oyster species that are commercially cultured in Malaysia. Some taxonomists had considered C. iredalei as the synonymous species of C. belcheri. However, the recent genetic work done by Klinbunga et al. (2003) had shown that these two species are indeed distinct. The ⁎ Corresponding author at: School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia. Tel./fax: + 60 4 653 3500. E-mail addresses: [email protected] (C.P. Teh), zulfi[email protected] (Y. Zulfigar), [email protected] (S.H. Tan). 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.01.014
Malaysia Fisheries Statistic (Department of Fisheries Malaysia, 2010) reported that oyster production from aquaculture in Malaysia was at approximately 869.72 MT in 2007 but decreased to 275.47 MT in 2008 probably due to insufficient good quality oyster seeds to support the industry. Seeds are mainly collected from the wild but the supply is inconsistent and often inadequate (Kechik, 1995). Producers are exposed to the vagaries of the seasons, seed quality is poor and the seeds are definitely of variable sizes and ages. The solution to overcome the insufficient and poor quality seed supply is hatcheryproduced high quality oyster seeds. However, although oyster seeds can be produced from the hatchery; some problems continue to exist at certain stages of the oyster production such as high mortality and low larvae settlement and metamorphosis. The percentage of larval settlement is considered to be important and critical for the cultivation. Delay of the larvae settlement in oysters may inhibit the growth of these oysters and cause the mortality of the larvae (Faimali et al., 2004; Najiah et al., 2008). The capacity to increase the attraction of the pediveliger larvae to the settling surfaces and metamorphosis will definitely increase seed production (Li et al., 2006). The higher the percentage of settlement, the higher will be the numbers of spats and juveniles which can be obtained per spawning batch. Therefore, there is a need to investigate the effect of endogenous and exogenous chemical cues for settlement and metamorphosis of the tropical pediveliger oyster larvae to ensure optimal settlement and metamorphosis rates. In previous studies (Bonar et al., 1990; Fang et al., 2001; Gao and Liu, 2006), neuroactive compounds such as Epinephrine (EPI), L-3,4-dihydroxyphenylalanine (L-
C.P. Teh et al. / Aquaculture 338–341 (2012) 260–263
DOPA), γ-aminobutyric acid (GABA) and Serotonin (5-HT) had induced larval settlement in oyster and clams. EPI is the powerful catabolic stimulant where it plays an important role in settlement and metamorphosis for most of the invertebrate larvae such as scallop, Argopecten purpuratus (Martinez et al., 1999) and Crassostrea gigas (Coon et al., 1990a,b). Based on the previous studies from Coon et al. (1990a,b) and Walch et al. (1999), the presence of L-DOPA has enhanced the larvae settlement and metamorphosis of Pacific oyster C. gigas and Crassostrea virginica. EPI and L-DOPA are generally used as artificial invertebrate larvae inducers. However, no reports were found on the effect of these inducers on tropical oysters. Therefore, in this paper we focused on the effects of neuroactive compounds (EPI and L-DOPA) on the larval settlement and metamorphosis of tropical oyster (C. iredalei), and to identify the optimal concentration and exposure duration to yield the optimum settlement and metamorphosis rate.
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settlement. The control group was kept in 18 ppt of 1 μm filtered seawater. Other treatments were also diluted by 18 ppt filtered seawater. For all the experiments, the larvae settlement was examined regularly under the dissecting microscope to monitor the behavior patterns. The total number of the settled larvae for each treatment was recorded (Tan and Wong, 1995). 2.5. Statistical analyses Data obtained from this study were analyzed using the one-way analysis of variance (ANOVA) from SPSS 11.0. The Least Significant Different Test (LSD) was conducted on the percentage of the total number of larvae that settled and metamorphosed in order to compare the differences at 95% levels of significance (p b 0.05). All the percentage values were transformed to arcsine values before undergoing statistical analyses.
2. Materials and methods 3. Results 2.1. Test animal 3.1. Visual observation of larval settlement Tropical oyster C. iredalei is one of the commercial species cultured in the hatchery due to its high demand. The broodstock were collected from wild (Penang) and acclimated in sand filtered seawater with 30 ppt salinity in the experimental hatchery at Penang (Northern Peninsular Malaysia). Currently only one commercial hatchery is found in this region which is located at Penang Island. Furthermore, this hatchery was still in its infant stage. 2.2. Larval culture The broodstock of C. iredalei were induced to spawn and fertilization occurred in the fiber tank in the hatchery. For this tropical oyster, there is no seasonal restriction on the spawning process. Spawning was done once every month to ensure that sufficient larvae were supplied. Larvae with well developed eyes (competent larvae) and ready to settle were collected for this study. These competent larvae were collected by using 253 μm Nitex sieves and transported back to the laboratory for further investigation. Experiments were carried out using larvae from the same cohort. The larvae, which swam actively with a well-developed eyespot, were used in the experiments. The competent larvae used exhibited the shell length of 242–304 μm. 2.3. Settlement cues The neuroactive compounds, epinephrine (EPI) and L-3,4dihydroxyphenylalanine (L-DOPA), were tested at five different concentrations (10 − 3, 10 − 4, 10 − 5, 10 − 6 and 10 − 7 M) in triplicates against the control group. EPI and L-DOPA were dissolved by using 0.0005 N of hydrochloric acid (HCl) and 1 μm filtered seawater (salinity: 18 ppt) was used to dilute the solution to reach final concentrations. Besides that, 1 μm of filtered seawater was used in the control group. All the neuroactive compounds were freshly prepared to avoid oxidation of the chemicals prior to the experiment. 2.4. Effects of EPI and L-DOPA on the oyster larvae settlement The experiments were conducted in tissue culture plates (24-well Multiwell Plat, BD Falcon™). A total number of 30–50 larvae were placed in 24 wells of the tissue culture plates each filled with 1.5 ml of test solution. A few washed marble chips were placed in the tissue culture plates as cultch. The larvae were exposed to different concentrations of test solutions for 1 h and 24 h. Food and aeration were not provided during the experiments. After the exposure duration, the larvae were removed from the test solution, rinsed with 1 μm filtered seawater and placed back into the wells filled with clean filtered seawater for another 24 h for observation and calculation of survival
The competent larvae of C. iredalei were observed to be moving downward in the water column, then swimming in slow curved paths when exposed to the neuroactive compounds. The competent larvae (larvae with well developed eye-spots) swam up and down along the water column and began rotating abnormally in the solution where the velum and cilia were extended. After being exposed to the chemicals for about 15 to 20 min, some of the larvae started to sink to the bottom of the wells where the velum and cilia were contracted and feet extended for crawling along the walls and substrate. Some of the larvae returned to the free-swimming stage when the substrate or the condition was not suitable. The larvae kept crawling around and reversing their steps until they found a suitable substrate for settlement. Once the environment and substrates were fit for the larvae to settle, they would attach themselves to the flume bottom by using their left valves. This observation was similar to that reported by Bonar et al. (1990). In this study, the larval settlement was indicated when the free-swimming larvae underwent the swimming, crawling process and finally cemented onto the substrate. 3.2. Settlement of larvae exposed to various concentrations of epinephrine (EPI) As a result of investigation of the larvae settlement of the tropical oyster C. iredalei exposed to neuroactive compounds, it was found that epinephrine (EPI) was an active inducer of settlement for the oyster. The settlement percentage was highest where the larvae were exposed to EPI at 10− 6 M and 10 − 5 M concentrations for 1 h with 60.87 ± 13.14% and 38.79 ± 4.83%, respectively. These were significantly higher than those recorded in the control group (15.10 ± 3.36%) (pb 0.05). In the larvae exposed to EPI for 24 h, exposure to 10 − 6 M of EPI displayed the highest settlement percentage of 59.13 ± 5.53%, which was higher than that of the control group (36.26± 33.72%) (Fig. 1). The maximum percentages of larvae settlement were induced by 10− 6 M of EPI in both exposure durations (1 h and 24 h), yielding percentages of larvae settlement of approximately 60%. The settlement percentage of larvae exposed to higher concentrations of EPI (10 − 4 and 10 − 3 M) showed no significant differences compared to that of the control group (p> 0.05). 3.3. Settlement of larvae exposed to various concentrations of L-DOPA Besides EPI, this study also found that L-DOPA was an active inducer of settlement of the tropical oyster C. iredalei larvae. The differences between EPI and L-DOPA were that larvae exposed to the higher concentrations of L-DOPA did not show any settlement. The
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4. Discussion
Fig. 1. Settlement percentages of competent larvae of Crassostrea iredalei exposed to different concentrations of EPI for 1 h and 24 h (* indicated significant level at 95%; p b 0.05).
optimum larvae settlement of C. iredalei was observed when they were exposed to L-DOPA at 10 − 6 M for 1 h; however, the percentage of larvae settlement (%) showed no significant difference compared to larvae exposed to EPI at 10 − 6 M (60.87 ± 13.14%). Nevertheless, this result was significantly higher than the larvae settlement recorded in the control group (15.10 ± 3.36%) (p b 0.05). Even though L-DOPA at lower concentrations could have induced optimum larvae settlement, the inducement of larvae at higher concentrations of LDOPA was not successful. In addition, L-DOPA also failed to induce settlement of C. iredalei larvae when exposed for 1 or 24 h at higher concentrations (Fig. 2).
3.4. Mortality of the oyster larvae exposed to EPI and L-DOPA The mortality of the larvae exposed to EPI (maximum: 38 ± 14.35%) was lower than that of larvae exposed to all concentrations of L-DOPA (100.00 ± 0.00%). Mortalities of larvae exposed to EPI and L-DOPA increased when the concentrations of the neuroactive compounds increased. Thus the higher concentrations of neuroactive compounds caused the mortality of larvae especially those exposed to EPI and LDOPA for a longer duration (24 h). There was no settlement at higher concentrations of L-DOPA and the larval mortality was 100% after exposure for 24 h (Fig. 3). The results were significantly different compared to those for the control group.
Fig. 2. Settlement percentages of competent larvae of Crassostrea iredalei exposed to different concentrations of L-DOPA for 1 h and 24 h (* indicated the 95% significant level; p b 0.05).
According to Bonar et al. (1990), the process of settlement and metamorphosis is generally known as settling. Settlement behavior in most of the invertebrates is considered a reversible process except for oysters. The settlement process includes foot extension, crawling on the substrate and finally cementing onto the substratum. Abundant literature has shown that the settlement of marine invertebrate larvae is influenced by specific physical, biological and chemical cues. Studies have shown that some neuroactive compounds, such as EPI, L-DOPA, GABA and Serotonin (5-HT) can actively induce the larval settlement (Bonar et al., 1990; Fang et al., 2001; Gao and Liu, 2006). Chemical cues represent one of the factors that affect the larval settlement and they are easy to monitor for larval settlements in hatchery-scale production under natural conditions. The exposure of larvae to an appropriate chemical cue at suitable concentrations may bring about more rapid or greater degrees of settlement. Using chemical cues is a convenient and effective technique to increase the percentage of larvae settlement under hatchery conditions. In this present study, the concentration of EPI and L-DOPA at 10 − 7 M was not effective in inducing the larval settlement. The optimum larval settlements of the tropical oyster C. iredalei were achieved at 10 − 6 M of EPI and L-DOPA. Higher concentrations of EPI and L-DOPA (10 − 5, 10 − 4 and 10 − 3 M) gave low percentages of larval settlement and also caused higher mortalities. The results obtained in the experiments were very much dependent on the species studied. In the present study, inductive behavior was observed when the neuroactive compounds EPI and L-DOPA were added. Similar results were shown in the blue mussel Mytilus edulis (Dobretsov and Qian, 2003); the tropical oyster C. belcheri (Tan and Wong, 1995) and the Pacific oyster C. gigas (Beiras and Widdows, 1995; Bonar et al., 1990; Coon et al., 1990a,b; Fang et al., 2001) whereas pearl oyster larvae (Pinctada margaritifera and Pinctada fucata) did not show any inductive responses (Doroudi and Southgate, 2002; Yu et al., 2008). Larvae of different species of invertebrates may respond to the same cues in a different manner. In this study, the effectiveness of the neuroactive compounds, such as EPI and L-DOPA, were specific to the larvae settlement of the oyster C. iredalei. Results obtained in this study might be different from those of previous studies because bioassay procedure used to test the effects of EPI and L-DOPA in different species of larvae might have been different (Bonar et al., 1990; Fang et al., 2001). Moreover, the physiological and molecular mechanism of how EPI and L-DOPA induce the larvae settlement of this tropical oyster C. iredalei has never been conducted. Further studies on the similarities and differences between the temperate and tropical species on the mechanism of induced settlement and metamorphosis may need to be addressed in the future. Alterations in the development sequence of oyster larvae can occur when neuroactive compounds were added to induce settlement and metamorphosis process. However, no differences were observed in the early development of the larvae in this research and in the study done by Ver (1986). Between the two neuroactive compounds, EPI has been found to be more efficient in inducing larval settlement compared to L-DOPA due to its low toxicity.
Fig. 3. Mortality of Crassostrea iredalei larvae exposed to various concentrations of EPI and L-DOPA (* indicated the 95% significant level; p b 0.05).
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In the absence of an appropriate chemical inducer, larval settlement would still occur, but at slower rates and percentages. Previous studies have shown that the larvae that prolong the freeswimming stage will give negative effects. The larvae that delay their settlement and metamorphosis stage have been reported to be retarded in growth, affecting the survival rate in the post-larvae life (Pechenik and Cerulli, 1991). Mortalities of oyster larvae exposed to different concentrations of EPI were lower compared to those exposed to L-DOPA for both exposure times (1 h and 24 h). Although some neuroactive compounds can induce larval settlement, the larvae of C. iredalei cannot adapt to the high concentrations of the compounds. The chemicals at high concentrations or overdoses were either toxic to the larvae or would retard the growth of the larvae especially when exposed to L-DOPA. Prolonging the exposure time of the tropical oyster larvae to the neuroactive compounds at higher concentrations appears to reduce the larval settlement and metamorphosis and hence, increases mortalities. According to Nicolas et al. (1998), the toxic effect was not noted in the larvae of C. gigas when EPI was used and less mortality was observed in the C. gigas larvae. There was no acute toxicity observed in the larvae of C. gigas and Pecten maximus when exposed to L-DOPA.
5. Conclusion From the results obtained on the effects of chemical cues on larval settlement induction, it has been shown that the onset of larvae settlement can be accelerated from 15% (control) to 60% by the addition of EPI and L-DOPA. The settlement was higher in larvae exposed over 1 h compared to larvae exposed for 24 h for both of the neuroactive compounds (EPI and L-DOPA). In the case of the tropical oyster C. iredalei, EPI was able to induce about 61% of larval settlement and L-DOPA was able to induce about 54% of the larval settlement. These results can be applied to the commercial tropical hatchery and may increase the oyster seed production effectively. For commercial purposes, both chemicals EPI and L-DOPA applied at lower concentrations and over a shorter exposure duration can reduce investment costs. By investing about 3 to 4 USD in 450,000 larvae in 10 l of filtered seawater, it can increase the seed production by four folds if compared to normal condition. It is important to introduce these two compounds in this region because this will definitely increase oyster seed production at least by four folds.
Acknowledgments This study was financially supported by Universiti Sains Malaysia USM-RU-PRGS (1001/PBIOLOGI/833029). The authors are very grateful to the local commercial oyster farms for providing the healthy oyster larvae throughout the study. Without their support, this work would not have been possible. We also express our appreciation to Universiti Sains Malaysia for allowing us to conduct our work in the laboratory.
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