Effects of Adrenoceptor Compounds on Larval Metamorphosis of the Mussel Mytilus Coruscus

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Effects of adrenoceptor compounds on larval metamorphosis of the mussel Mytilus coruscus

ARTICLE in AQUACULTURE · APRIL 2014 Impact Factor: 1.83 · DOI: 10.1016/j.aquaculture.2014.02.019

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Jin-Long Yang Yi-Feng Li Shanghai Ocean University Shanghai Ocean University

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Available from: Jin-Long Yang Retrieved on: 11 September 2015 This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights Author's personal copy

Aquaculture 426–427 (2014) 282–287

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Effects of adrenoceptor compounds on larval metamorphosis of the mussel Mytilus coruscus

Jin-Long Yang a,b,c,d,⁎, Wu-Shuang Li a, Xiao Liang a, Yi-Feng Li a,Yu-RuChena,Wei-YangBaoe,Jia-LeLia a College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China b Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai 201306, China c Shanghai Engineering Research Center of Aquaculture, Shanghai 201306, China d Shanghai University Knowledge Service Platform, Shanghai Ocean University Aquatic Animal Breeding Center (ZF1206), Shanghai 201306, China e Institute of Marine Science and Technology, Yangzhou University, Yangzhou 225009, China article info abstract

Article history: The metamorphosis responses of mussel (Mytilus coruscus) larvae to adrenoceptor compounds were investigated Received 3 October 2013 through a series of bioassays. The adrenergic agonist, epinephrine, as a positive control, exhibited signiﬁcant Received in revised form 1 February 2014 inducing activity. Phenylephrine and clonidine induced larval metamorphosis at 10−6 to 10−4 M concentrations Accepted 11 February 2014 − in both 24-h and continuous exposure assays. Dobutamine induced larval metamorphosis at 10 5 Minthe24-h Available online 26 February 2014 exposure assays, and the percentage of larval metamorphosis was very low (b5%). Methoxyphenamine exhibited inducing activity at 10−4 M in the continuous exposure assays, and the percentage of larval metamorphosis was Keywords: ﬁ Mytilus coruscus 8%. No larval mortality was observed for these ve agonists at all concentrations tested. Among these antagonists, α Larval metamorphosis 1-adrenergic receptor antagonist chlorpromazine and amitriptyline showed by far the most promising Adrenoceptor inhibiting effects, indicating that G-protein-coupled α1-adrenoceptors may be involved in the process of larval

Agonists metamorphosis in M. coruscus.Theα2-adrenergic receptor antagonist idazoxan also inhibited the larval Antagonists metamorphosis of M. coruscus, indicating that larval metamorphosis may also be mediated by G-protein- Chemical cue coupled α2-adrenoceptors. Atenolol and butoxamine also inhibited larval metamorphosis induction by epinephrine. Thus, these adrenergic agonists can be used as non-toxic and promising inducers of larval metamorphosis in this species, and to improve M. coruscus larval production for aquaculture. The present study provides a novel insight into the mechanism modulating the metamorphosis of larvae of the mussel M. coruscus. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Paul, 2001; Tebben et al., 2011). To date, fully characterized chemical cues have been conducted in only a few cases (Dreanno et al., 2006; Many marine invertebrates possess a planktonic larval phase pre- Pawlik, 1986; Tebben et al., 2011; Yvin et al., 1985). Therefore, some ceding a benthic adult phase (Crisp, 1974; Thorson, 1950). Larvae of scientists switched their focus to commercially available chemicals these marine invertebrates must recognize exogenous cues prior to including some pharmaceutical compounds that have been proposed their settlement and metamorphosis (Crisp, 1974; Hadfield, 2011; to be potentially useful inducers or inhibitors (Alfaro et al., 2011; Hadfield and Paul, 2001; Morse, 1990). Chemical cues have been Grant et al., 2013; Rittschof et al., 2003; Yang et al., 2008, 2011; Young known to play an important role in the process of larval settlement et al., 2011). and metamorphosis (Hadfield and Paul, 2001; Hay, 2009; Paul et al., Commercial pharmaceutical compounds are often well-characterized, 2011; Pawlik, 1992). target-specific substances with known pharmacological profiles in In the marine environment, natural chemical cues originate from vertebrates, clear chemical-synthesis pathways, and an alternative various resources such as biofilms (Bao et al., 2007; Ganesan et al., supply source (Rittschof et al., 2003). The use of G-protein-coupled 2010; Hadfield, 2011; Wang et al., 2012; Yang et al., 2013a), macroalgae receptor (GPCR) agonists and antagonists to elucidate a signaling (Huggett et al., 2005; Walters et al., 1996; Yang et al., 2007), conspecifics pathway can be a powerful tool for determining the signaling interac- (Clare, 2011; Dreanno et al., 2006). Despite the importance of chemical tions upon treatment with the compound of interest (Clare et al., cues for larval settlement and metamorphosis, the complete chemical 1995; Dahlström et al., 2000; Qian et al., 2013; Tran and Hadfield, identity of these natural inducers is poorly understood (Hadfield and 2012; Yamamoto et al., 1996; Yang et al., 2011). GPCR agonists and antagonists have been tested for their inductive and inhibitive activities against larval settlement and metamorphosis of many marine in- ⁎ Corresponding author at: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China. Tel.: +86 21 61900440; fax: +86 21 61900405. vertebrates, e.g. Balanus amphitrite (Dahms et al., 2004; Yamamoto E-mail address: [email protected] (J.-L. Yang). et al., 1996, 1998), Balanus improvisus (Dahlström et al., 2000, 2005),

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Bugula neritina (Dahms et al., 2004; Yu et al., 2007), Hydroides elegans clonidine, dobutamine, methoxyphenamine, chlorphromazine, amitrip- (Dahms et al., 2004), and Halocordyle disticha (Edwards et al., 1987). tyline, idazoxan, atenolol and butoxamine are shown in Table 1. Stock For molluscs, larvae of the oyster Crassostrea gigas were induced to solutions of epinephrine, atenolol and butoxamine were prepared by metamorphose by cirazoline, epinephrine and phenylephrine (Coon dissolving this chemical in autoclaved filtered seawater (AFSW) with and Bonar, 1987). Epinephrine has also been demonstrated to induce 0.1 ml of 1 M HCl. Except for epinephrine, atenolol and butoxamine the larval metamorphosis of the clams Venerupis pullastra and Ruditapes stock, solutions of other chemicals tested were prepared by directly philippinarum (García-Lavandeira et al., 2005). On the other hand, the dissolving this substance in AFSW. Test solutions assayed were pre- larval metamorphosis of C. gigas could be inhibited by the GPCR agonist pared by diluting stock solutions in AFSW to desired concentrations chlorpromazine, prazosin and phentolamine (Coon and Bonar, 1987). (Table 1). All stock solutions and test solutions were prepared on the As with other marine invertebrates, the larval settlement and meta- same day of the assay. Tested concentrations used in the present inves- morphosis of the mussels can be also induced or inhibited by various tigation were chosen based on the previous bioassays on other mussel GPCR agonists (Alfaro et al., 2011; Dobretsov and Qian, 2003; species and an oyster (Alfaro et al., 2011; Coon and Bonar, 1987; Sánchez-Lazo et al., 2012; Yang et al., 2008, 2013b; Young et al., 2011) Satuito et al., 1999; Yang et al., 2008, 2011). and antagonists (Satuito et al., 1999; Yamamoto et al., 1998; Yang et al., 2011). Although a variety of commercially available compounds 2.3. Larval metamorphosis bioassays were identified in above-mentioned researches, molecular mechanisms of how these pharmacological compounds act have not been fully The inductive effects of different adrenergic agonists on larval meta- described (Qian et al., 2013; Sánchez-Lazo et al., 2012). morphosis were investigated following the methods of previous reports The mussel, Mytilus coruscus Gould, 1860, is a common species (Coon and Bonar, 1987; Satuito et al., 1999; Yang et al., 2008, 2013b). inhabiting the temperate zone along the coastal waters of East Asia Twenty pediveliger larvae were released in each glass Petri dish (Chang, 2007). In China, this species is one of the most heavily commer- (Ø 60 mm × 19 mm height) containing 20 ml of the chemical compound cially exploited marine bivalves, particularly within the Zhejiang Prov- solution in AFSW. Larvae were subjected to the test solution either in a ince (Chang and Wu, 2007; Zhang and Zhao, 2003). Consequently, 24-h exposure bath (Coon and Bonar, 1987; Satuito et al., 1999; Yang hatchery development may be an effective means of restocking the et al., 2008, 2013b) or in continuous exposure (Yang et al., 2008) wild population to meet market demands. However, progress in the throughout the experimental period. Larvae that were given the 24-h mussel culture industry is still hampered by inefficient and unstable exposure bath were rinsed three times with AFSW and then finally larval settlement and metamorphosis (Chang and Wu, 2007; Yang transferred to Petri dishes containing 20 ml of AFSW. Two groups of et al., 2013b; Young et al., 2011). pediveligers were, respectively, exposed only to epinephrine in the In the present study, the authors investigated the effects of G- 24-h and continuous exposure assays, as positive controls. protein-coupled adrenoceptor agonists and antagonists on the larval The inhibitive effects of adrenergic antagonists on larval metamor- metamorphosis of M. coruscus. The purpose was to gain the insight phosis were investigated following the methods of previous studies into the mechanisms of the larval metamorphosis of M. coruscus, and (Coon and Bonar, 1987; Satuito et al., 1999; Yang et al., 2011). Twenty identify potential inducers for application in hatchery production in pediveligers were exposed to test solutions of different concentrations this species. of these antagonists for 15 min (Table 1). Epinephrine was then added to the test solutions described above to a final concentration of 10 2. Materials and methods −4 M. After 3 h exposure to solutions containing the antagonists and 10−4 M epinephrine, pediveligers were rinsed three times with AFSW 2.1. Spawning and larval culture and then transferred to Petri dishes each containing 20 ml of AFSW. A group of pediveligers was also exposed only to 10−4 M epinephrine Adult M. coruscus were collected from populations growing about for 3 h, as a positive control. In addition, pediveligers were exposed to 2 miles northeast from the coast of Shengsi, Zhoushan (122°44′E; five adrenergic antagonists without the addition of 10−4 M epinephrine 30°73′N), China. Spawning in the laboratory was induced following for the purpose of checking for any effect, including toxicity, of these the modified method of Yang et al. (2008, 2013b). Mussels were cleaned compounds during 24-h exposure time. by brushing off material attached on the shell surfaces and rinsing In all assays, pediveliger larvae were checked after 48 h and 72 h in seawater (salinity: 30 ppt), packed in ice overnight and then from the commencement of assays and evaluated for metamorphosis transferred to a 10 l polycarbonate tank with filtered seawater (ace- by verification of post-larval shell growth. Dead pediveligers in each tate-fiber filter: 1.2 μm pore size, FSW) at ca. 21 °C. The final water Petri dish were also recorded. Petri dishes, each containing 20 temperature was ca. 18 °C. Mussels that started spawning were trans- pediveliger larvae and 20 ml of AFSW were always set as negative ferred to individual 2 l glass beakers. Eggs were collected using a glass pipette and were transferred to a beaker containing FSW. Fertilization was achieved by gently mixing with a sperm suspension in FSW. The Table 1 suspension was then left undisturbed for 20 min. Fertilized eggs were Chemical compounds used in the present study and their respective concentrations of filtered onto a nylon plankton net (mesh size: 20 μm) to remove excess stock and tested concentrations. sperm, washed thoroughly with FSW and left undisturbed for 2 days in Chemical compound Concentration (M) an incubator maintained at 18 °C. After two days, swimming straight- Stock solution Tested concentrations hinge veliger larvae were collected, washed gently with FSW and −3 −6 −5 −4 −1 Phenylephrine 10 10 10 10 cultured in 2 l glass beakers at an initial density of 5 larvae ml .Larvae − − − − Clonidine 10 3 10 6 10 5 10 4 4 −1 −1 were fed a diet of Chaetoceros gracilis at 5 × 10 cells ml day .The Isoproterenol 10−3 10−6 10−5 10−4 culture water was changed every other day and the temperature Dobutamine 10−3 10−6 10−5 10−4 − − − − maintained at 18 °C. Larvae were cultured to the pediveliger stage of Methoxyphenamine 10 3 10 6 10 5 10 4 −3 −6 −5 −4 growth and were used in the metamorphosis bioassays. Chlorpromazine 10 10 10 10 Amitriptyline 10−3 10−6 10−5 10−4 Rauwolscine 10−3 10−6 10−5 10−4 2.2. Chemical compounds Idazoxan 10−3 10−6 10−5 10−4 Atenolol 10−3 10−6 10−5 10−4 −3 −6 −5 −4 Concentrations of stock and test solutions of adrenoceptor compounds Butoxamine 10 10 10 10 Epinephrine 10−3 10−6 10−5 10−4 tested in the present investigation, viz. epinephrine, phenylephrine, Author's personal copy

284 J.-L. Yang et al. / Aquaculture 426–427 (2014) 282–287 controls during assays. All assays were conducted at 18 °C in a dark en- after 72 h, while no inducing activity was observed in methoxyphena- vironment. All assays were conducted with six replicates per treatment. mine (p N 0.05) during the 72-h assay period (Fig. 1b). No mortality of larvae was observed in the 24-h exposure assays of five adrenergic 2.4. Data analysis agonists. Percentages of post-larvae exposed to the five adrenergic agonists The metamorphosis inducing activities of the chemical compounds after 48 h and 72 h in the continuous exposure assays are as shown in were expressed as percentages of post-larvae. Prior to statistical analy- Fig. 2. Larval metamorphosis was observed in epinephrine (Kruskal– sis, all data expressed in percentages were arcsine-transformed and Wallis test: p b 0.01), phenylephrine (p b 0.01) and clonidine tested for their normality and homogeneity. Normality of distribution (p b 0.01) after 48 h (Fig. 2a). After 72 h, methoxyphenamine was assessed using Shapiro–Wilk test. Homogeneity of variance (p b 0.01) also induced larvae to metamorphose (Fig. 2b), and the was verified using O'Brien test. Data for amitriptyline, atenolol and inducing activity was 8%. In contrast, dobutamine (p N 0.05) showed butoxamine were analyzed using the one-way analysis of variance no inducing activity even after 72 h exposure. In the case of continuous (ANOVA)followedbyTukey–Kramer honestly significant difference exposure assays, no larvae mortality was observed during the 72-h test. Data for epinephrine, phenylephrine, clonidine, dobutamine, exposure period. methoxyphenamine, chlorphromazine and idazoxan were analyzed – using the Kruskal Wallis test followed by Steel with a control test 3.2. Effects of adrenergic antagonists on larval metamorphosis because these above data did not meet parametric assumption even after transformation. All statistical computations were performed The effects of adrenergic antagonists on larval metamorphosis in the ™ using JMP software (version 10.0). Differences were considered presence of 10−4 M epinephrine are as shown in Fig. 3. Chlorpromazine fi b signi cant at p 0.05. inhibited larval metamorphosis at 10−6 M(Kruskal–Wallis test: p b 0.01) and the percentages of post-larvae were 6 ± 2% and 10 ± 1% 3. Results after 48 h and 72 h, respectively. Amitriptyline also showed inhibition at 10−5 M(ANOVA:p b 0.05) after 48 h and at 10−6 M(ANOVA:p b 0.05) Throughout all experiments, the percentage of metamorphosis to after 72 h. In the vertebrate alpha2 adrenergic antagonist idazoxan, post-larvae of M. coruscus in negative controls was always 0%. larval metamorphosis was inhibited at 10−4 M (Kruskal–Wallis test: p b 0.01) after 48 h and 10−6 M (Kruskal–Wallis test: p b 0.05) after 3.1. Effects of adrenergic agonists on larval metamorphosis 72 h. In the vertebrate beta1 adrenergic antagonist atenolol, the inhibition was observed at 10−4 M(ANOVA:p b 0.05) after 48 h and all fi Percentages of post-larvae exposed to the ve adrenergic agonists the concentrations (ANOVA: p b 0.05) tested after 72 h. Similarly, after 48 h and 72 h in the 24-h exposure assays are as shown in Fig. 1. butoxamine also showed inhibitive activity at 10−4 M (ANOVA: – b After 48 h, only epinephrine (Kruskal Wallis test: p 0.01), phenyleph- p b 0.05) after 48 h and all tested concentrations (ANOVA: p b 0.05) b b rine (p 0.05) and clonidine (p 0.01) exhibited inducing activity when after 72 h. When pediveligers exposed to different concentrations of comparing with the negative control (Fig. 1a). After 72 h, the percent- the five adrenergic antagonists in the absence of epinephrine, no larval ages of post-larvae in these three agonists further increased (Fig. 1b). metamorphosis was observed even after the 72-h assay period. In Dobutamine (p b 0.05) also induced the post-larval metamorphosis

Fig. 1. Percentages of M. coruscus post-larvae on ﬁve adrenergic receptor agonists after 48 Fig. 2. Percentages of M. coruscus post-larvae on ﬁve adrenergic receptor agonists after 48 h (a) and 72 (b) at various concentrations in 24-h exposure assays. Data are means (±SE) h (a) and 72 (b) at various concentrations in continuous exposure assays. Data are means of 6 replicates. * = p b 0.05. (±SE) of 6 replicates. * = p b 0.05. Author's personal copy

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Fig. 3. Percentages of M. coruscus post-larvae after 48 h (open boxes) and 72 h (shaded boxes) obtained by exposure to different concentrations of the ﬁve adrenergic receptor antagonists in the presence of 10−4 M epinephrine. Data are means (±SE) of 6 replicates. * = p b 0.05.

addition, no larval mortality was observed when exposed to these ﬁve metamorphosis. Although β-adrenergic receptor agonist dobutamine adrenergic antagonists in the presence and absence of epinephrine. and methoxyphenamine also exhibited inducing activities, the percentages of larvae metamorphosis were very low (b10%). In addition, mere- 4. Discussion and conclusions ly exposing larvae to dobutamine may have not been sufﬁcient to induce metamorphosis, but transferring treated larvae to AFSW could It is well established that a variety of pharmacological compounds have physiologically shocked larvae and thus triggered the metamor- are effective inducers or inhibitors of larval settlement and metamorphosis. Further research needs to be conducted. Phenylephrine, an α1- phosis of many marine invertebrates. The purpose of these studies adrenoreceptor agonist, also exhibited inducing activity in C. gigas was to explain the mechanism of larval settlement and metamorphosis (Coon and Bonar, 1987)andMytilus galloprovincialis (Yang et al.,

(Clare et al., 1995; Coon and Bonar, 1987; Dahlström et al., 2005; Morse, 2011), suggesting that α1-adrenoreceptor is involved in larval meta- 1990; Pawlik, 1992; Tran and Hadﬁeld, 2012; Yamamoto et al., 1996), morphosis. Clonidine, an α2-adrenoreceptor agonist, inhibited the set- and to provide valuable information that has application in ﬁelds of tlement of cyprid larvae of B. improvisus (Dahlström et al., 2000), and aquaculture and biofouling (Alfaro et al., 2011; Dahlström et al., 2000; exhibited no inducing effects on C. gigas, while it induced larval meta- Dahms et al., 2004; Dobretsov and Qian, 2003; Sánchez-Lazo et al., morphosis in the present species. It indicates that clonidine may inter-

2012; Yang et al., 2011, 2013b; Zhou et al., 2009). In the present study, act with α2-adrenoreceptor and participate in the larval the authors have demonstrated that G-protein-coupled adrenoceptor metamorphosis in M. coruscus. agonists such as phenylephrine and clonidine induce the larval meta- The present investigation demonstrates that adrenergic receptor morphosis of M. coruscus. Moreover, no larval mortality was observed antagonists tested exhibited inhibiting properties of the larval meta- for these two agonists at all concentrations tested, indicating that morphosis in M. coruscus. Among adrenergic receptor antagonists, these agonists can be used as non-toxic and active inducers of larval chlorpromazine and amitriptyline showed by far the most promising re- metamorphosis in this species. sults. Chlorpromazine, an α1-adrenergic receptor antagonist, inhibited Although a number of neurotransmitters have been known to in- the larval metamorphosis of M. coruscus in the present investigation. duce the larval metamorphosis of marine invertebrates, knowledge on This is consistent with previous studies on C. gigas (Coon and Bonar, whether these neurotransmitters bind to receptors to induce metamor- 1987)andM. galloprovincialis (Yang et al., 2011). In contrast, chlor- phosis is limited (Morse, 1990; Tran and Hadﬁeld, 2012; Yamamoto promazine was reported to be a stimulus to the larval metamorphosis et al., 1996). Yamamoto et al. (1996) demonstrated that serotonin of Crepidula fornicata in a 24 h exposure assay although it blocked could induce B. amphitrite larval settlement, which could be inhibited the stimulatory action of excess potassium ions on the metamorphosis by serotonin uptake blockers, indicating that serotonergic neurons (Pechenik et al., 2002). Alternatively, chlorpromazine may instead play an important role in the process of settlement. In the present be stimulating metamorphosis by elevating concentrations of study, the α-adrenergic receptor agonist phenylephrine and clonidine calmodulin-dependent cyclic nucleotide (Pechenik et al., 2002). In addi- successfully induced the larval metamorphosis of M. coruscus,in- tion, chlorpromazine is known to have several other pharmacological dicating that the α-adrenoreceptor may participate in the process of effects in other model systems (Pechenik et al., 2002). For example, Author's personal copy

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Amador-Cano, G., Carpizo-Ituarte, E., Cristino-Jorge, D., 2006. Role of protein kinase C, G- chlorpromazine can act as a histamine H1 antagonist in some systems protein coupled receptors, and calcium flux during metamorphosis of the sea urchin (Martinez and Coleman, 1990; Oken, 1995). Strongylocentrotus purpuratus. Biol. Bull. 210, 121–131. Idazoxan, an α2-adrenergic receptor antagonist, has been reported Bao, W.Y., Yang, J.L., Satuito, C.G., Kitamura, H., 2007. Larval metamorphosis of the mussel to inhibit larvae settlement of some species of marine invertebrates, Mytilus galloprovincialis in response to Alteromonas sp. 1: evidence for two chemical cues? Mar. Biol. 152, 657–666. such as B. amphitrite (Dahms et al., 2004), B. neritina (Dahms et al., Chang, Y.Q., 2007. Stock Enhancement and Culture in Mollusks (in Chinese). China 2004), H. elegans (Dahms et al., 2004). 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