The Effect of Chemical Cues on the Settlement of Sea Cucumber (Apostichopus japonicus) Larvae

SUN Xiujun, LI Qi, YU Hong, and KONG Lingfeng

Key Laboratory of Mariculture of Ministry of Education of China, Ocean University of China, Qingdao 266003, P. R. China

The Effect of Chemical Cues on the Settlement of Sea Cucumber (Apostichopus japonicus) Larvae

SUN Xiujun, LI Qi*, YU Hong, and KONG Lingfeng

Key Laboratory of Mariculture of Ministry of Education of China, Ocean University of China, Qingdao 266003, P. R. China

The effects of four ions and eight neuroactive compounds on inducing larval settlement of A. japonicus were assessed in the present study. All bioassays were conducted in 60 × 9 mm Petri dishes, each contained 10 mL of the test solution and 10 doliolaria larvae. There were significant inductive effects of K+(10 mmol L−1), NH4+(0.1 mmol L−1), GABA (10−3mol L−1), acetylcholine (10−5mol L−1), L-DOPA (10−5mol L−1), norepinephrine (10−5mol L−1) and dopamine (10−7mol L−1and 10-5mol L−1) on the settlement of sea cucumber larvae. L-DOPA and dopamine are the most efficient chemical cues to induce A. japonicus larvae to settle. The highest percentage of larval settlement was induced by 10−5mol L−1L-DOPA and dopamine (33% and 40%) compared to the control (7%). However, Ca2+, Mg2+, choline, serotonin, and epinephrine were less effective on larval settlement at all tested concentrations. This study evaluated the stability and feasibility of chemical cues for larval settlement in different culture systems, which can be applied to improve the hatchery production of this valuable species.

Apostichopus japonicus; sea cucumber; chemical cue; settlement; larva

1 Introduction

Larval settlement and metamorphosis are critical events in the life cycle of most benthic marine invertebrates. Planktonic marine invertebrate larvae must develop competence and recognize appropriate exogenous morphogenetic cues before they can settle and metamorphose (Crisp, 1974, 1976). A variety of biological, physical, and chemical factors have been shown to induce the settlement of planktonic larvae (Rodriguez et al., 1993). Chemical cues associated with substrata in the environment are considered as the primary stimuli initiating settlement of many marine invertebrate larvae (Morse et al., 1979; Pawlik and Hadfield, 1990; Pawlik, 1992). Applying artificial chemical at appropriate concentrations have been proved to be useful and powerful to induce settlement of competent invertebrate larvae (Coon and Bonar, 1986; Lairnek et al., 2008).

The performance of KCl in promoting settlement of larvae of bryozoan (Wendt and Woollacott, 1995), pearl oyster (Zhao et al., 2003; Yu et al., 2008), and abalone (Kang et al., 2004) has been acknowledged. The optimal concentration of K+for inducing the settlement of larvae of these species varied between 10 mmol L−1and 30 mmol L−1. At 50 mmol L−1, CaCl2stimulated settlement behavior and induced metamorphosis of pearl oysters, Pinctada fucata martensii and P. maxima (Zhao et al., 2003; Yu et al., 2008). The obvious effect of NH4Cl (less than 10 mmol L−1) on settlement was observed in the larvae of oyster Crassostrea gigas (Coon et al., 1990) and pearl oyster P. martensii (Yu et al., 2008), but not in the larvae of goldlip pearl oyster P. maxima (Zhao et al., 2003). The settlement of abalone larvae was inhibited by elevated magnesium (Baloun and Morse, 1984; Yu et al., 2010). GABA at concentrations varying between 10−6mol L−1and 10−4mol L−1was the most effective in inducing the settlement of larvae of mussel (Garcia-Lavandeira et al., 2005), oyster (Garcia-Lavandeira et al., 2005), pearl oysters (Zhao et al., 2003; Yu et al., 2008), black chiton (Rumrill and Cameron, 1983), and abalones (Baloun and Morse, 1984; Li et al., 2006); while GABA at much higher concentrations (10−3−10−2mol L−1) was required to induce a high level of settlement of sea urchin larvae (Rahmani and Ueharai, 2001). There were significant effect of choline, acetylcholine and serotonin (10−5−10−3mol L−1) on the settlement of P. maxima and P. fucata martensii larvae (Zhao et al., 2003; Yu et al., 2008). The inductive effect of L-DOPA and dopamine at concentrations between 10−6mol L−1and 10−5mol L−1on settlement of larvae were reported in mussels (Cooper, 1982; Dobretsov and Qian, 2003), oysters (Cooper, 1982; Coon et al., 1985), and abalones (Akashige et a1., 1981; Yu et al., 2010). Norepinephrine at 10−4mol L−1was capable of inducing the highest percentage of metamorphosis in the pacific oyster (Coon et al., 1985). Exposure of larvae to 10−5mol L−1epinephrine induced significantly higher lev-els of settlement and metamorphosis in oyster, clam and mussel (Garcia-Lavandeira et al., 2005). Although larval metamorphosis inducers of sea cucumber were screened from physiologically active compounds (Matsuura et al., 2009), the effect of these chemical cues on settlement of larvae of sea cucumbers were still poorly understood.

The sea cucumber (Apostichopus japonicus Selenka) is naturally distributed along the coast of China, Korea, Japan and far eastern Russia, which is a commercially important species in China. In response to over-exploitation of most wild stocks and an increasing market demand, farming A. japonicus has become widespread in northern coast of China and the production is increasing rapidly (Chen et al., 2008; Li et al., 2010). In 2010, the production of sea cucumber in China reached 130303 t, and over 95% were from Shandong and Liaoning Province (DOF, 2011). Similar to other benthic marine invertebrate species, A. japonicus has a planktonic larval stage in its life history. The fertilized eggs develop through early auricularia, mid auricularia, late auricularia, doliolaria, and pentactula larvae that are competent to settle (Ito and Kitamura, 1997; Yu et al., 2005). The metamorphosis and settlement of larvae of the sea cucumbers Cucumaria frondosa, Stichopus sp., Holothuria spinifera Theel, H. scabra and Australostichopus mollis have also been investigated (Hamel and Mercier, 1996; Mercier et al., 2000; Asha and Muthiah, 2002; Morgan, 2008; Hu et al., 2010). Studies on the induction of larval metamorphosis and settlement of sea cucumber are comparatively scarce (Ito and Kitamura, 1997; Mercier et al., 2000; Ivy and Giraspy, 2006; Asha and Muthiah, 2007; Li et al., 2010). Previous studies have focused on larval settlement of A. japonicus in response to periphitic diatom, microbial films, and conspecific adults (Ito and Kitamura, 1997; Li et al., 2010). Using chemical stimulus for settlement may produce more rapid, greater degree of settlement and improve the synchrony of settlement, which would provide significant improvement in hatchery culture of this valuable species. Matsuura et al. (2009) indicated that dopamine and L-DOPA are endogenous larval metamorphosis inducer of the sea cucumber A. japonicus. However, further experiments are needed to evaluate their stability and feasibility in different culture systems before the practical use of the chemical inducer in sea cucumber hatcheries. In this study, we investigated the effect of four ions and eight neuroactive compounds on larval settlement of sea cucumber A. japonicus in order to improve larval settlement in the sea cucumber industry.

2 Materials and Methods

2.1 Larval Culture and General Bioassay Procedure

Adult A. japonicus, body weight of ≥ 200 g, were obtained from Xintai Hatchery, Haiyang, China. Mature A. japonicus (n = 20) were induced to spawn by air-drying in the dark for 1 h and then thermal stimulated (water temperature raised by 2–3℃) with flowing sea water for 1 h. Approximately 36 h after fertilization, newly hatched early auricularia were siphoned into a larval culture tank containing 10 m3sand filtered seawater (SFSW). Culture water temperature was maintained at 20℃ ± 1℃ (mean ± SD), with salinity at 33 ± 1 (mean ± SD). The initial density was 0.2 larvae mL−1. During the rearing period, 2/3 of the seawater was replaced with fresh SFSW every day. The algal diet of Isochrysis galbana and Nitzschia closterium minutissima (1:1) was provided at an initial density of 5000 cells mL−1, and progressively increased to 30000 cells mL−1as the larvae grew. After 11 days post fertilization, more than 50% of the planktonic larvae had reached non-feeding doliolaria stage. These doliolaria larvae were selected for settlement bioassays.

All bioassays were conducted in the Petri dish (60 mm × 9 mm) containing 10 mL of the test solution and 10 doliolaria larvae. All larvae were not fed. All the assays were done in static water without exchange. The temperature was kept at 21℃ ± 2℃ under an ambient photoperiod regime with 14 h of light and 10 h of dark. The light intensity was 500–1000 lux. For all experiments, each treatment consisted of three replicates, which were arranged in a randomized block design. Larvae cultivated in Petri dishes containing 0.45 µm memberane filtered seawater (FSW) were used as the negative control. Larvae were assigned into four categories depending on their activities according to Hamel and Mercier (1996) and Mercier et al. (2000): 1) swimming (early doliolaria, active swimming behavior); 2) settlement behavior (late doliolaria and early pentactula, slowly losing buoyancy and exploring substrate); 3) settlement (late pentactula, with fully extended buccal podia and development of tube feet); and 4) dead (deformed). The number of larvae was recorded at 12, 24, 36, 60, 84 and 108 h. Dead larvae were not removed at each observation.

2.2 Larval Settlement in Response to Different Ions and Neuroactive Compounds

The following four ions and eight neuroactive compounds were tested for their activities in inducing the settlement of A. japonicus larvae: K+(potassium chloride), Ca2+(calcium chloride), NH4+(ammonium chloride) and Mg2+(magnesium chloride); GABA (γ-aminobutyric acid), choline chloride, acetylcholine chloride, L-DOPA [3-(3,4-Dihydroxyphenyl)-L-alanine], serotonin hydrochloride, dopamine, epinephrine, and norepinephrine. The condition of larvae and juveniles has been examined based on the assumption that there is no toxic damage in short or long period.

All larvae were obtained from the same batch of eggs except for epinephrine and norepinephrine trial. Two separate trials based on different batches of eggs were conducted, one with K+, Ca2+, NH4+, Mg2+, GABA, choline chloride, acetylcholine chloride, L-DOPA, serotonin hydrochloride and dopamine, and the other with epinephrine and norepinephrine. In each trial, the negative control was set up using the larvae from the same batch of eggs with chemical treatments.

2.3 Statistical Analysis

The data were described as the percentage of larvalswimming, settlement behavior, settlement, and mortality. The percentage of larvae in each treatment was arcsine-transformed before statistical analysis. The data presented in all figures were not transformed. In order to improve the arcsine transformation, the percentages were given a value of 1/4n or 1–1/4n (n = number of larvae in a replicate) when some replicates were zero or 100%, respectively. Normality was tested by Kolmogorov-Smirnov. Normal data were estimated by using parametrical tests: one-way ANOVA run on each chemical at each time point followed by Dunnett’s multiple comparison test, which has protection against false positives built in. Otherwise, the Kruskal-Wallis test was performed followed by the Mann-Whitney U test which was used for pairwise comparison of treatments with the control (Gebauer et al., 1998; Yu et al., 2008). Data were analyzed with SPSS 16.0 statistical package. The results were considered to be significantly different when P < 0.05.

3 Results

3.1 Larval Response to Different Ions

K+has little inductive effect on larval settlement behavior and larval settlement of A. japonicus. There was no significant effect of K+at all tested concentrations except for 10 and 50 mmol L−1on larval settlement behavior or larval settlement (Fig.1A and Fig.2A). The settlement behavior of larvae in 10 mmol L−1KCl for 60 h and 50 mmol L−1for 12 h increased significantly compared to the FSW control (Fig.1A). Larval settlement in 10 mmol L−1KCl after 60 h and 84 h was significantly higher than that of control (Fig.2A). Larval mortality was not affected by all concentrations of KCl (Fig.3A).

Ca2+has significant inductive effects on larval settlement behavior, but no effect on larval settlement. The significant inductive effect of Ca2+on larval settlement behavior was found at the concentrations of ≥ 30 mmol L−1compared to controls (Fig.1B). However, no obvious effect of CaCl2on larval settlement was observed at low tested concentrations (Fig.2B). Exposure of larvae to 1 mmol L−1CaCl2for 108 h and 50 mmol L−1for 84 h increased larval mortality significantly (Fig.3B).

NH4+has significant inductive effects on larval settlement behavior, but has little inductive effect on larval settlement and it is toxic to larvae at high concentrations. The significant inductive effect of NH4+on larval settlement behavior was observed in 0.1 mmol L−1for 36 h and 60 h, and 1 mmol L−1for 84 h (Fig.1C). The significant inhibitory effect on larval settlement behavior was observed at the concentrations of ≥ 10 mmol L−1. The significant inductive effect on larval settlement was observed in 0.1 mmol L−1of NH4+after 108 h (Fig.2C). No significant toxic effect was observed with NH4Cl at the concentrations of ≤ 1 mmol L−1. However, larval mortality was significantly affected by high concentration of NH4+, reaching 100% at the concentrations of ≥ 10 mmol L−1after 36 h (Fig.3C).

There was no significant effect of Mg2+on larval settlement behavior or settlement (Fig.1D and Fig.2D), and larval mortality was not affected by excess MgCl2(Fig.3D).

Fig.1 The percentage of larval settlement behavior of Apostichopus japonicus in response to various concentrations of different ions after 12, 24, 36, 60, 84, and 108 h. (A) KCl, (B) CaCl2, (C) NH4Cl, (D) MgCl2. The results are shown as mean ± S.D. of three different experiments. Asterisks and dots indicate significantly higher and lower than the control (0), respectively (P < 0.05). One molar stock solution of KCl, CaCl2, NH4Cl, and MgCl2was prepared by dissolving the chemicals in double-distilled water. Test concentrations were 10, 20, 30, 50 and 70 mmol L−1K+; 1, 10, 30, 50 and 70 mmol L−1Ca2+; 0.1, 1, 10, 30, and 50 mmol L−1NH4+; 10, 20, 30, 50 and 70 mmol L−1Mg2+.

Fig.2 The percentage of larval settlement of A. japonicus in response to various concentrations of different ions after 12, 24, 36, 60, 84, and 108 h. (A) KCl, (B) CaCl2, (C) NH4Cl, (D) MgCl2. The results are shown as mean ± S.D. of three different experiments. Asterisks indicate significant differences from the control (0) (P < 0.05).

Fig.3 The percentage of larval mortality of A. japonicus in response to various concentrations of different ions after 12, 24, 36, 60, 84, and 108 h. (A) KCl, (B) CaCl2, (C) NH4Cl, (D) MgCl2. The results are shown as mean ± S.D. of three different experiments. Asterisks indicate significant differences from the control (0) (P < 0.05).

3.2 Larval rResponse to Neuroactive Compounds

GABA has strong inductive effects on larval settlement behavior, but has weak inductive effects on larval settlement. GABA induced significant larval settlement behavior at all concentrations except 10−4mol L−1(Fig.4A). In the treatments of 10−4mol L−1and 10−2mol L−1, the percentages of larval settlement were 25% (Fig.5A) and 23% after 84 h (Fig.5A), respectively. Longer exposure to GABA at 10−3mol L−1after 108 h, larvae showed the highest percentage of settlement (30%) among all treatments (Fig.5A). Larval mortality was significantly affected by excess GABA at 10−2mol L−1after 36 h and 60 h (Fig.6A).

Choline chloride has significant inductive effects on larval settlement behavior but no effect on larval settlement. Although choline chloride induced significantly higher larval settlement behavior at 10−6mol L−1and 10−5mol L−1after 84 h (Fig.4B), no significant effect on larval settlement was observed (Fig.5B). Larval mortality was not affected by choline chloride at all the tested concentrations except for 10−4mol L−1(Fig.6B).

Acetylcholine chloride has little inductive effect on larval settlement behavior, but has moderate inductive effects on larval settlement. No significant effect of acetylcholine chloride at all concentrations on larvae settlement behavior was observed except in the treatment of 10−2mol L−1(Fig.4C). There was significantly higher percentage of larval settlement in 10−5mol L−1after 84 h and 108 h than that of control (Fig.5C). There was no significant toxic effect of acetylcholine chloride on larvae at all concentrations (Fig.6C).

Fig.4 The percentage of larval settlement behavior of A. japonicus in response to various concentrations of different neuroactive compounds after 12, 24, 36, 60, 84, and 108 h. (A) GABA, (B) choline, (C) acetylcholine, (D) serotonin, (E) L-DOPA, (F) dopamine, (G) epinephrine, (H) norepinephrine. The results are shown as mean ± S.D. of three different experiments. Asterisks and dots indicate significantly higher and lower than the control (0), respectively (P < 0.05). Stock solutions of neuroactive compounds were prepared by dissolving the chemicals in 0.45 µm FSW immediately before the bioassay, except for epinephrine and norepinephrine, which were dissolved by 0.005 mol L−1HCl and diluted in FSW to achieve the desired final experimental concentration.

Serotonin hydrochloride has weak inductive effects on larval settlement behavior, and has no inductive effect on larval settlement. There was no significant inductive effect of serotonin hydrochloride at all concentrations on larval settlement behavior except for 10−4mol L−1after 84 h and 10−3mol L−1after 36 h (Fig.4D). Serotonin did not induce a significant larval settlement at all tested concentrations (Fig.5D). The significant negative effects on larval survival were observed after 108 h in all treatments, and the mortality was significantly increased, reaching 100% in 10−2mol L−1after 12 h and 10−3mol L−1after 60 h (Fig.6D).

L-DOPA has strong inductive effects on larval settlement behavior and larval settlement, and it is identified as one of the most efficient inducers of larval settlement of A. japonicus. L-DOPA induced significantly higher larvae settlement behavior at 10−6mol L−1and 10−5mol L−1after 12 h, 60 h, and 84 h, 10−7mol L−1after 60 h and 84 h (Fig.4E). In the 10−5mol L−1treatment, L-DOPA induced significantly higher percentages of larval settlement after 36 h, 84 h and 108 h, with the highest value (33%) among all the treatments at 84 h (Fig.5E). Meantime, larval mortality was not significantly affected at these concentrations compared to controls (Fig.6E). However, the mortality reached 100% after 12 h at higher concentrations of L-DOPA (10−4mol L−1, 10−3mol L−1, and 10−2mol L−1).

Fig.5 The percentage of larval settlement of A. japonicus in response to various concentrations of different neuroactive compounds after 12, 24, 36, 60, 84, and 108 h. (A) GABA, (B) choline, (C) acetylcholine, (D) serotonin, (E) L-DOPA, (F) dopamine, (G) epinephrine, (H) norepinephrine. The results are shown as mean ± S.D. of three different experiments. Asterisks and dots indicate significantly higher and lower than the control (0), respectively (P < 0.05).

Dopamine also has strong inductive effects on larval settlement behavior and larval settlement, and it induced the highest percentage of larval settlement. The significant inductive effects of dopamine on larval settlement behavior were observed at 10−5mol L−1after 24 h and 10−7mol L−1after 60 h and 84 h (Fig.4F). Dopamine induced significantly higher percentages of larval settlement than controls at 10−7mol L−1after 108 h, 10−5mol L−1after 36 h, 60 h, and 84 h (Fig.5F). In the treatment of 10−5mol L−1, dopamine induced the highest percentage of larval settlement (40%) at 36 h and 60 h, and no significant negative effect on larval survival was observed. However, dopamine was toxic and the mortality reached 100% at higher concentrations (10−4mol L−1, 10−3mol L−1, and 10−2mol L−1) after 12 h (Fig.6F).

Fig.6 The percentage of larval mortality of A. japonicus in response to various concentrations of different neuroactive compounds after 12, 24, 36, 60, 84, and 108 h. (A) GABA, (B) choline, (C) acetylcholine, (D) serotonin, (E) L-DOPA, (F) dopamine, (G) epinephrine, (H) norepinephrine. The results are shown as mean ± S.D. of three different experiments. Asterisks indicate significant differences from the control (0) (P < 0.05).

Epinephrine has little inductive effect on larval settlement behavior, and no effect on larval settlement. There were significant inductive effects of epinephrine on larval settlement behavior in the concentrations of ≥ 10−5mol L−1at 12 h than controls (Fig.4G), while no significant inductive effect on larval settlement was observed (Fig.5G). Long exposure of larvae to all tested concentrations of epinephrine increased the mortality after 108 h (Fig.6G). In the treatment of 10−2mol L−1, the mortality reached 100% after 24 h.

Norepinephrine has little inductive effect on larval settlement behavior and larval settlement, and it is toxic to the settled larvae. The significant inductive effects of norepinephrine on larval settlement behavior were observed in the concentrations of ≥ 10−4mol L−1at 12 h, but not in the concentrations of ≤ 10−5mol L−1(Fig.4H). The percentage of larval settlement in 10−5mol L−1of norepinephrine was significantly higher than that of controls at 36 h but decreased after 60 h of exposure or more (Fig.5H). Larval mortality in 10−5mol L−1of norepinephrine increased significantly after 84 h and 108 h (Fig.6H). There were significant negative effects of norepinephrine on larval survival after long exposure to all the concentrations except for 10−6mol L−1(Fig.6H).

4 Discussion

Settlement is one of the most critical stages in the lifehistory of many marine invertebrates. For sea cucumber, because the loss of cilia and vitelline reserves was gradual, larvae slowly lost buoyancy and explored the substrate, as their few remaining cilia and most probably the prevailing water flow helped them move about, indicating presettlement behavior of the larvae as described by Hamel and Mercier (1996) for Cucumaria frondosa. Larvae were considered to have settled in this study when they reached late pentactula stage with the loss of capability to swim, fully extension of buccal podia and the development of tube feet according to previous descriptions (Hamel and Mercier, 1996; Mercier et al., 2000; Morgan, 2008; Hu et al., 2010).

Increased external K+may activate metamorphosis by depolarizing externally accessible cells in an inductive pathway according to Yool et al. (1986), and it has proven to be an effective inducer in a variety of invertebrates (Wendt and Woollacott, 1995; Zhao et al., 2003; Yu et al., 2008). Conversely, the effectiveness of potassium does not imply that K+is necessarily involved in transducing natural signals for metamorphosis (Yool et al., 1986). In this study, the optimal concentration of KCl for settlement was 10 mmol L−1, consistent with the previous study in which K+induced the highest percentage of settlement of pearl oyster (Pinctada maxima) with a concentration between 10 and 30 mmol L−1(Zhao et al., 2003). However, no inductive effect of KCl on larval settlement of A. japonicus was observed in the study of Matsuura et al. (2009). High concentrations of K+were toxic to P. maxima and P. fucata martensii, but there was no statistically significant toxic effect on the survival of A. japonicus larvae.

The effects of higher concentration of Ca2+in medium suggest a role of calcium in signal transduction in marine invertebrate larvae (Yool et al., 1986). Although Zhao et al. (2003) and Yu et al. (2008) demonstrated that Ca2+promoted larval settlement in pearl oyster, there were no inductive effect on green mussel Perna viridis, abalone Haliotis rufescens (Baloun and Morse, 1984; Ke et al., 1998). There were significant inductive effects of Ca2+with 30, 50, and 70 mmol L−1on settlement behavior of A. japonicus larvae, but no obvious effect on settlement was observed in this study.

The little inductive effect of NH4Cl on larval settlement in this study indicated that NH4Cl is not an effective inducer on larval settlement of A. japonicus. Similarly, the weak inductive effect of NH4Cl on larval settlement was found in P. maxima (Zhao et al., 2003). However, NH4Cl had significant inductive effects on larval settlement of C. gigas and P. fucata martensii (Coon et al., 1990; Yu et al., 2008). The different effects of NH4Cl on larval settlement among species may be owing to the concentrations of NH3rather than NH4+in experimental seawater (Bower and Bidwell, 1978). Further experiments are needed to determine the role of NH3and NH4+in the induction of larval settlement of A. japonicus.

In many marine invertebrate species, magnesium ion appeared to have no effect on competent larvae (Baloun and Morse, 1984; Yool et al., 1986; Yu et al., 2008). In this study, there was also no significant inductive or inhibitory effect of MgCl2on larval settlement of A. japonicus. In contrast, in the presence of metamorphically active microbial films, excess magnesium inhibits the process of sea urchin metamorphosis by competing with calcium for binding molecules involved in neurotransmitter release at neuromuscular junctions (Cameron et al., 1989). The inhibitory effects of Mg2+on larval settlement are not found in A. japonicus probably because of the absence of metamorphically active microbial films or compounds.

GABA, as an inhibitory neurotransmitter in both vertebrate and invertebrate systems, produces depolarization of cells capable of activating metamorphosis (Baloun and Morse, 1984). To date, it has been reported that GABA can induce larval settlement in many mollusc species, including mussel Mytilus galloprovincialis, clam Venerupis pullastra and Ruditapes philippinarum, oyster Ostera edulis, pearl oyster P. maxima and P. fucata martensii (Zhao et al., 2003; Garcia-Lavandeira et al., 2005; Yu et al., 2008), black chiton Katharina tunicate (Rumrill and Cameron, 1983), and abalone H. rufescens and H. diversicolor supertexta (Baloun and Morse, 1984; Li et al., 2006). In echinodermata, GABA triggered the metamorphosis of four genetically divergent sea urchins of the genus Echinometra at concentration of 10−3mol L−1and 10−2mol L−1(Rahmani and Ueharai, 2001). In our study, the effective concentration for settlement of A. japonicus larvae was 10−3mol L−1, similar to that for the settlement of sea urchin Echinometra, but much higher than that for abalone larvae (10−7–10−5mol L−1, Morse et al., 1980). However, the inductive effect of GABA on A. japonicus larvae was not observed in the study of Matsuura et al. (2009). Although there was no evidence of deleterious effect or toxicity even at higher concentrations of GABA in Echinometra, larval mortality was significantly affected by excess GABA at 10−2mol L−1in A. japonicus, suggesting deleterious effects at higher concentrations of GABA in this species.

As a precursor of acetylcholine, choline is a bound constitute of the membranes surrounding all cells (Yu et al., 2008). Zhao et al. (2003) and Yu et al. (2008) reported that there were inductive effects of both choline and acetylcholine on larval settlement in pearl oyster P. maxima and P. fucata martensii. In this study, acetylcholine induced effective larval settlement in 10−5mol L−1after 84 h and 108 h, while no significant inductive effect of choline was observed in A. japonicus larvae.

Serotonin, a derivative of tryptophan, acts as a neurotransmitter and modulator in vertebrate and invertebrate nervous systems (Pawlik, 1992). Serotonin was considered as the most effective inducer of larval settlement and metamorphosis in P. maxima and P. fucata martensii (Zhao et al., 2003; Yu et al., 2008), whereas it has not been observed to stimulate any response in larvae of the molluscs H. rufescens, H. discus hannai, Phestilla sibogae or C. gigas (Pawlik, 1992). In this study, serotonin did not induce a significant larval settlement of A. japonicus, and some deleterious effects were observed at higher concentrations.

L-DOPA and the catecholamines (dopamine, norepinephrine and epinephrine) are derivatives of tyrosine that have diverse biological functions (Darnell et al., 1986). In the present study, dopamine and L-DOPA both showed strong inductive effects on larval settlement and no toxic effect at low concentrations. The similar inductive effects on larval settlement were also found in the abalone H. discus hannai and the pearl oyster P. margaritifera larvae (Akashige et a1., 1981; Doroudi and Southgate, 2002). In common with this study, the significant inductive effects of L-DOPA, dopamine and norepinephrine on larval settlement of A. japonicus have been observed by Matsuura et al. (2009). However, the significant inductive effects of epinephrine were found in Matsuura et al.’s study (2009) but no inductive effect was detected in this study.

The studies confirm that the inductive effects of 10−5mol L−1of L-DOPA, dopamine, and norepinephrine on larval settlement of A. japonicus are effective and consistent across studies. Those discrepancies in the effects of KCl, GABA, and epinephrine between the two studies may be caused by the differences in culture systems, such as test solution volume and larval density. The toxic effects of 10−4mol L−1of L-DOPA and dopamine observed in this study suggest that sea cucumber larvae are very sensitive to concentrations of these compounds and should be treated with less than 10−4mol L−1at low culture density.

In conclusion, the significant inductive effects of K+(10 mmol L−1), NH4+(0.1 mmol L−1), GABA (10−3mol L−1), acetylcholine (10−5mol L−1), L-DOPA (10−5mol L−1), norepinephrine (10−5mol L−1), and dopamine (10−7mol L−1and 10−5mol L−1) on settlement of larvae of the sea cucumber A. japonicus were determined in the present study. Among these chemical cues, a 10−5mol L−1concentration of either L-DOPA or dopamine was proved to be the most effective in inducing A. japonicus larvae to settle. This study not only evaluate the stability and feasibility of chemical stimulus for settlement in different culture systems, extend our knowledge of potential chemical cues, but also indicate that the concentrations of chemical cues should be cautiously treated in hatchery production. However, the chemical induction might have cumulative effects on the health status of newly settled pentactula and juveniles. The short-term and long-term toxicity studies of them in A. japonicus should be performed in further studies. Furthermore, the potential synergistic or antagonistic effects of the chemical cues on larval settlement should be assessed in larger systems in presence of diatoms or microbial films.

Acknowledgements

The study was supported by the grants from National Marine Public Welfare Research Program (201205023), the Scientific and Technical Supporting Program (2011 BAD13B03), and Shandong Seed Project.

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(Edited by Qiu Yantao)

(Received August 15, 2012; revised September 30, 2012; accepted July 17, 2013)

© Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2014

* Corresponding author. Tel: 0086-532-82031622

E-mail: qili66@ouc.edu.cn