THE JOURNAL OF EXPERIMENTAL ZOOLOGY 275339-345 (1996) Serotonin Involvement in Larval Settlement of the Barnacle, Balanus amphitrite HISASHI YAMAMOTO, AKIKO TACHIBANA, SATORU KAWAII, KIYOTAKA MATSUMURA, AND NOBUHIRO FUSETANI Fusetani Biofouling Project, ERATO, JRDC,Niigata Engineering Co. Ltd., Isogo, Yokohama 235, Japan ABSTRACT Following our previous report (Yamamoto et al. r19951 Zool. Sci., 12:391-396) in which we demonstrated that protein kinase C (PKC) plays an important role in larval metamorphosis of the barnacle Balanus amphitrite, we examined the effects of a range of neurotransmitters, including serotonin (5-hydro~ytryptamine)~ epinephrine, and norepinephrine on cyprid settlement and metamorphosis. Serotonin induced pronounced larval settlement. Furthermore, settlement was inhibited by neurotransmitter uptake blockers, such as amitriptyline, imipramine, and desipramine, in a concentration-dependent manner. Cyprids exposed t o these uptake blockers metamorphosed without settling on substrata. These results suggest that serotonin is involved in larval settlement. 0 1996 Wiley-Liss, Inc. Settlement competent larvae of sessile marine invertebrates, such as barnacles, mussels, and hydroids, are believed to initiate settlement behavior upon reception of chemical cues which are often derived from adult conspecifics or from prey organisms (Morse, '90). Cyprid larvae of barnacles have long been known to respond to protein found i n conspecific adults, known as arthropodin (Larman et al., '82). When exposed to adult extracts, cyprids usually begin to explore potential settlement substrata and eventually settle by secreting cement substance, after which metamorphosis to the juvenile barnacles takes place. It is probable that signal reception followed by signal transduction is a n integral part of this sequence. However, very little is known about the receptors or signal transduction systems involved. A receptor coupled with G-protein has, however, been suggested as the signal reception system in veliger larvae of the red abalone Haliotis rufescens (Baxter and Morse, '92). A number of neurotransmitters including dopamine, epinephrine, and norepinephrine have further been reported as inducers of larval settlement and metamorphosis in a range of marine invertebrates (Jensen and Morse, '90; Pawlik, '90; Pires and Hadfield, '91). However, it is unclear whether these neurotransmitters bind to receptors to induce metamorphosis, and no receptor molecules have as yet been isolated from marine invertebrate larvae (Morse, '90). Larvae of the hydroid Hydractinia echinata, by contrast, have been reported to metamorphose when exposed to phorbol esters, suggesting the 0 1996 WILEY-LISS, INC. involvement of a protein kinase C (PKC) signal transduction system in the metamorphosis of these larvae (Leitz and Klingmann, '90; Leitz and Muller, '87; Muller, '85; Schneider and Leitz, '94). We have already demonstrated that a PKC signal transduction system plays a n important role in larval metamorphosis of the barnacle Balanus amphitrite (Yamamoto et al., '95). Furthermore, recent evidence has demonstrated the involvement of cyclic AMP in the settlement of this species (Clare et al., '95). In order to clarify the operation of signal transduction systems in cyprid larvae, we further examined the effects of a range of neurotransmitters on settlement and metamorphosis. MATERIALS AND METHODS Materials Artificial seawater (ASW) was prepared according to the Van't Hoff formula as follows: 460 mM NaC1, 10.1 mM KCl, 9.2 mM CaCl,, 35.9 mM MgC12, 17.5 mM MgS04, and 10 mM Tris-HC1 (pH 8.2). Serotonin, epinephrine, norepinephrine, and quinacrine were purchased from Sigma Chemical Co. (St. Louis, MO). GABA (y-4-amino-n-butyric acid), L-DOPA (L-p-3,4-dihydroxyphenylalanine), dopamine, amitriptyline, imipramine, and desipramine were purchased from Wako Pure- Received August 29, 1995; revision accepted March 12, 1996. Address reprint requests to Hisashi Yamamoto, Fusetani Biofouling Project, ERATO, JRDC, c/o Niigata Engineering Co. Ltd., Isogo, Yokohama 235, Japan. 340 H. Y-OTO chemicals Co. (Osaka, fJapan). Octopamine was obtained from Aldrich Chemical Co. (Milwaukee, WI). Larval settlement assays Competent cyprid larvae were obtained from laboratory cultured adu It Balanus amphitrite using methods described previously (Yamamoto et al., '95). Settlement assays were performed using six-well polystyrene plates (Corning Cell Wells, Corning, NY).Six milliliters of ASW (see Materials) was added to each well, plus 10 2 2 cyprid larvae. The plates were then placed on a n orbital shaker at 22°C. Test solutions were prepared by the addition of 6 ~1 of aqueous or diluted HCl solution to 6 ml of ASW in each well. Epinephrine and L-DOPA were dissolved in dilute HC1, but HCl alone had no effect on cyprids at concentrations of up t o 1 x 10" M. Plates were observed daily for 5 or 6 days under a binocular dissection microscope. Daily counts were made of the numbers of settled and metamorphosed larvae in each well. Significant effects on l a r d settlement were tested using analysis of variance (ANOVA), following which Tukey-Kramer multiple comparison tests (at P < 0.05) were used t,o identify specific effects. Microinjection of cyprid larva Larval movement was restrained by overnight storage at 4"C, following;which cyprids were fixed loosely between two glass coverslips. Microinjection was performed as Tollows: a 7 pm diameter glass micropipette containing test solution was held by a micromanipulator allowing peritoneal (intraantennular) injection of cyprids. Injected cyprids were immediately transferred to six-well polystyrene plates (one cyprid/well) containing 6 ml of ASW, and plates were then placed on a n orbital shaker at 22°C. The volume of test solution microinjected into each cyprid was determined by the fluorescence intensity of quinacrine (Ex/Em: 380/540). When quinacrine solution at a concentration of 10 mM was injected in a cyprid larva, the final concentration of quinacrine within the cyprid was calculated as approximately 20 pM (500 x dilution). Each dlrug treatment was replicated at least five times. ET AL. A 100 z 0o +serotonin Q - norepinephrine --X--epinephrine - r T r 0 0.1 1 10 100 conc. [ pM ] B l o80 o c - Q- - L-DOPA Fig. 1. A: Effects of 0.1-100 FM serotonin, epinephrine, and norepinephrine on B. amphitrite cyprid larval settlement. B: Effects of 0.1-100 FM GABA, L-DOPA, dopamine, and octopamine on B. amphitrite cyprid larval settlement. Data presented are means f S.D. With the exception of epinephrine (F = 1.3430, ns), all drugs tested were found to have a highly significant effect on the settlement of B. amphitrite cyprids over the range of concentrations tested (Table 1).Statistical significance was as follows (ANOVA): serotonin (F = 23.4958, P < O.OOOl), norepinephrine (F = 4.5896, P < O.OOl), GABA (F = 13.4492, P < O.OOOl), L-DOPA (F = 105.5102, P < O.OOOl), dopamine (F = 90.6277, P < O.OOOl), octopamine (F = 219.0287, P < 0.0001). However, serotonin promoted cyprid settlement at concentrations of 100 pM, while other neurotransmitters had no settlement-promoting effects. The time RESULTS course of cyprid settlement promoted by serotoLarval settlement assays nin is presented in Figure 2. Statistical signifiThe effects of various neurotransmitters, includ- cance at each concentration of serotonin over 5 ing serotonin, epinephrine, and norepinephrine, days was observed as follows (ANOVA): day 1 (F on the larval settlement and metamorphosis of = 12.1598, P c O.OOOl), day 2 (F = 32.1722, P < Balanus amphitrite are presented in Figure 1. O.OOOl), day 3 (F = 27.5201, P c O.OOOl), day 5 (F 341 SEROTONINAND BARNACLE SETTLEMENT TABLE 1. Pairwise comparison for all neurotransmitters tested' 0 0.1 Serotonin Norepinephrine Epinephrine GABA L-DOPA Dopamine Octopamine 0 1 0 10 0 0 100 0.1 1 0.1 10 0.1 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 10 100 10 100 [pMl 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 'Statistical significancewas assessed at P = 0.05 (Tukey-Kramermultiple comparison test). 0, significantly different. = 20.3872, P < 0.0001). During day 1, with the exception of 0 and 0.1 pM, 0 and 1 yM, and 0.1 and 1 pM, all other painvise comparisons for serotonin were found t o be statistically significant. On the second day, except for 0 and 10 pM and 0.1 and 1 @ all!I comparisons , were significant. On the third and fifth days, except for 0 and 1 pM, 0 and 10 yM, and 1 and 10 pM,all comparisons were significant. Within 5 days, all settled larvae metamorphosed fully into juveniles. Induction of larval settlement by serotonin at low concentrations prompted examination of the effects of serotonin uptake blockers on cyprids as follows: amitriptyline (Hyttel et al., '80; Kriiger et al., '86), imipramine (Barbaccia et al., '83; Barondes, '94; Javaid et al., '79; Kruger et al., '86), and desipramine (Checkley et al., '81; Javaid et al., '79; Lee et al., '83). All these well-known serotonergic uptake blockers inhibited larval settlement at 10100 pM but promoted metamorphosis at 1-10 pM (Fig. 3). Each serotonin uptake blocker treatment was found to have a highly significant effect on larval settlement (Fig. 3). Results of ANOVA were 100 unsettled juvenile s loo 5 Y .- .-bP .E CI s - I c 60 - 40 Q e 0.1 ASW 20 80 100 60 80 100 60 80 100 40 60 juvenile [YO] - 100 1 h' 3 10 a, .-c E .-E 1 0.1 E iz 2. a, .- 0 20 0 20 40 juvenile [Oh] loo 10 C E l .G 0.1 ! 40 a, ASW al u, 1 0 Y 80 10 a, E c r 20 0 Fig. 2. Effects of 0.1-100 pM serotonin on the time course of B. amphitrite cyprid larval settlement. Data presented are means -c S.D. 40 juvenile [Oh] Fig. 3. Effects of 0.1-100 pM serotonin uptake blockers on B. amphitrite cyprid larval settlement and metamorphosis. Data presented are means f S.D. 342 H. YAMAMOTO ET AL. as follows: amitriptyline (F = 336.1766, P < O.OOOl), imipramine (F = 171.1640, P < O.OOOl), desipramine (F = 154.3397, P < 0.0001). All three treatments demonstrated a relatively high proportion of unsettled individuals at high concentrations of uptake blocker. This was less marked in the case of desipramine, with which no unsettled juveniles were observed below a concentration of 1 pM. Cyprids normally settle by attachment to surface substrata by the secretion of cement substance, followed by metamorphosis to the juvenile barnacle. However, although metamorphosis proceeded as observed in natural settlement, attachment to surface substrata did not take place when cyprids were exposed to amitriptyline, imipramine, and desipramine (Fig. 4). Finally, we examined the effects of amitriptyline, imipramine, and desipramine on the activity of serotonin. These uptake blockers all inhibited the activity of serotonin. A combination of 1-100 pM serotonin and 1 pM serotonin uptake blockers produced larvae which were unable to undergo settlement and attachment but were still capable of full metamorphosis into juvenile barnacles (Fig. 5 ) . While! combinations of serotonin and imipramine (F =I 1.4795, ns) and seroto- nin and desipramine (F = 2.6429, ns) had no significant effects on settlement at the concentrations employed, serotonin plus amitriptyline had a highly significant effect (F = 50.5666, P < 0.0001) on the number of larvae which settled and fully metamorphosed into juveniles. Serotonin plus amitriptyline also had the most pronounced effect on the production of unsettled juveniles (I? = 19.8128, P < O.OOOl), and production of the latter was highest at 1 pM amitriptyline plus 100 pM serotonin (Tukey-Kramer, P < 0.05). A similar pattern was observed for imipramine (F = 22.5714, P < 0.0001); however, no significant effect was recorded for serotonin plus desipramine (F = 3.7377, P = 0.0482), although the numers of unsettled larvae did differ significantly by pairwise comparison between 1:land 1:lOO (P < 0.05). These larvae had the same appearance as those described in Figure 4. For amitriptyline and imipramine, the percentage of settled juveniles increased with increasing serotonin concentration. The agents tested were nontoxic to cyprid larvae at concentrations of up to 10 pM. Most cyprids which failed to settle and metamorphose did not die but did eventually stop swimming, usually after 5 days. Some cyprids died at concentrations of 100 pM; however, this was a limited effect, and the majority of cyprids were still alive during assays. Microinjection of cyprid larva The cyprid microinjection process is presented in Figure 6, and the results of settlement assays I settled juvenile unsettled juvenile I ami+ 1 ami+ 10 ami+100 imi+ 1 h i + 10 imi+100 des+ 1 des+ 10 des+100 Fig. 4. B. amphitrite cyprid.s in ASW alone and following treatment with 1 p M amitriptyline. a: Cyprid larva. b Normal settled juvenile. c: Unsettled juveniles resting on the bottom of experimental wells. Bar..300 pm. I 0 20 tM I 40 60 juvenile [%] I I 80 100 Fig, 5. Inhibition of 1-100 ,LLMserotonin activity by 1yM serotonin uptake blockers in B. amphitrite cyprid larvae. Data presented are means c S.D. ami, 1 pM amitriptyline; imi, 1 pM imipramine; des, 1 pM desipramine; 1, 10, 100, serotonin concentration (pM). SEROTONINAND BARNACLE SETTLEMENT 343 conducted using injected cyprids are presented in Table 2. Only serotonin induced larval settlement, whereas the other neurotransmitters demonstrated no such effect, with the exception of a single cyprid which metamorphosed following microinjection with dopamine. When using the microinjection assay, the results of drug treatments were the same as those already obtained using the routine plate assays. Following microinjection with ASW alone, 2/8 cyprids settled and metamorphosed. Similarly, a single cyprid (1/9>settled and metamorphosed following injection with dopamine. Moreover, as in previous trials using external reagents, serotonergic uptake blockers induced nonsettled, nonattached, but fully metamorphosed juveniles. Quinacrine and the other neurotransmitters tested had no effect on cyprids. Following microinjection, most cyprids were either still actively swimming, resting on the bottom surface of the experimental wells, or floating at the water surface and therefore appeared to have been unharmed by the microinjection process. DISCUSSION It is widely believed that the process of larval settlement and metamorphosis in marine invertebrates can be described by the following process: exogenous chemical cues bind to chemosensory receptors on the larva, resulting in signal transduction by neurotransmitter to the interior cells, which leads t o cement secretion. Hitherto, no receptor molecules have been isolated from marine invertebrate larvae (Morse, '90). In barnacles, competent cyprid larvae initially swim and then crawl in search of appropriate TABLE 2. Results of trials in which B. amphitrite cyprids were microinjected with a variety of neurotransmitters and serotonin uvtake blockers Cn 2 6j1 Settled Unsettled juvenile juvenile 5 rnM (10 pM, n = 6) 50 mM (100 pM, n = 6) 100 mM (200 pM, n = 9) Epinephrine 100 mM (200 pM, n = 6) Norepinephrine 100 mM (200 pM,n = 6) GABA 100 mM (200 pM, n = 9) L-DOPA 100 mM (200pM, n = 9) Dopamine 100 mM (200 pM, n = 9) Octopamine 100 mM (200 pM, n = 6) Amitriptyline 5 mM (10 pM, n = 9) Imipramine 5 mM (10 pM, n = 12) Desipramine 5 mM (10 pM, n = 6) ASW in = 8) Serotonin Fig. 6. Microinjection of a B. amphitrite cyprid. a: Microinjection of cyprid larvae was performed by the insertion of a 7 pm diameter glass micropipette containing test solution. b: Cyprid following microinjection with test solution. Cyprids appeared unharmed by the microinjection process. Bar, 300 pm. 2 4 6 0 0 0 0 1 0 0 0 0 2 0 0 0 0 0 0 0 0 0 8 9 4 0 '(pM, n), (approximate internal agent concentration, sample size) 344 H. YAMAMOTO ET AL. settlement substrata arid finally settle on a suitable settlement site where they then attach and metamorphose (Crisp, '84;Walker et al., '87). Larval settlement and metamorphosis are often influenced by a range of environmental and synthetic factors which have been reported as follows: settlement surface color (Yule and Walker, '841,water movement (Crisp, '55), conspecific adult-derived proteins (Larman et al., '821, synthetic peptide analogs of barnacles settlement pheromone (Tegtmeyer and Rittschof, '861, and bacterial films (Maki et al., '90). In addition t o these exogenous factors, the involvement of signal tra nsduction systems including PKC (Yamamoto et al., '95) and CAMP(Clare et al., '95) have also been reported. More than this, however, little is known about the signal transduction systems involved. Various neurotransmjtters, including epinephrine, norepinephrine, and dopamine, have been reported as settlement and metamorphosis inducers (Jensen and Morse, '90; Pawlik, '90; Pires and Hadfield, '91). However, it is suggested that such agents act directly on receptors of the larval nervous system of the red abalone (Huliotis rufescens (Morse, '90). In the present study, we have demonstrated that serotonin induced cyprid larval settlement (Figs. 1, 2), with 0.1 pM serotonin inhibiting cyprid settlement while 100 pM serotonin promoted it. These results suggest that there may be two different kinds of serotonin binding sites, with either high oir low affinities for serotonin. On exposure to 0.1 pM serotonin, high affinity binding sites may affect the inhibition of serotonergic transduction. By contrast, when exposed t o 100 pM serotonin, low affinity binding sites may affect the promotion of serotonergic transduction. The serotonin uptake blockers, amitriptyline, imipramine, and desipramine (0.1-100 pM), were found to inhibit cyprid settlement and metamorphosis. At lower concentrations, however, these agents induced metamorphosis but not settlement (Fig. 3). These results provide further evidence that these upt,ake blockers may inhibit serotonin binding in cylprids. In order t o clarify these results, combinations of 1-100 pM serotonin and 1 pM serotonin uptake blocker were examined (Fig. 5). In the presence of 1 pM amitriptyline or imipramine and 1-100 pM serotonin, percentages of unsettled juveniles increased depending on increases in serotonin concentration. Desipramine also induced unsettled juveniles; however, the percentages decreased dependent on increases in serotonin concentration. This difference may be due to specificity of the serotonin binding block. It is known that desipramine blocks not only serotonin uptake but also norepinephrine uptake (Lee et al., '83). Our results suggested that desipramine acted as a serotonin binding blocker, but its potency was not so strong as that of amitriptyline and imipramine. Serotonin alone was found to promote settlement at 100 pM (Figs. 1, 2). Moreover, induction of settlement by serotonin was inhibited by serotonergic uptake blockers such as amitriptyline, imipramine, and desipramine (Fig. 5 ) , indicating the involvement of serotonin in signal transduction for settlement, while serotonin promoted general searching behavior. In addition, these uptake blockers produced nonsettled but fully metamorphosed juveniles. These results suggest that serotonin binding t o receptor is necessary for the natural cyprid settlement process. In order t o confirm the involvement of serotonin in cyprid settlement, serotonin and serotonergic uptake blockers were individually microinjected into cyprids (Fig. 6). Injection of 50 mM serotonin resulted in an internal concentration of approximately 100 pM in the cyprid, which was found t o promote larval settlement in a dose-dependent manner. However, 5 mM imipramine (calculated as 10 pM imipramine in the cyprid) induced nonattached, nonsettled juveniles. Microinjected 50 mM (100 pM in cyprid) serotonin uptake blockers induced unsettled juveniles. Results obtained from the microinjection experiments, although generally similar to those obtained from the routine settlement assays, were calculated to be approximately 500 times more sensitive (Table 2). Serotonin is known t o mediate signal transduction in many invertebrates species, most notably in molluscs. It has been suggested that serotonin activity is modulated by cAMP (Deterre et al., '81; Walker, '851, and Clare et al. ('95) have recently reported that cAMP was involved in the pheromonal modulation of cyprid settlement. Our results therefore provide further evidence that a relationship exists between serotonin and cAMP in the larval settlement process in B. amphitrite. In conclusion, serotonin induced cyprid settlement, which was inhibited by serotonin uptake blockers, suggesting that serotonergic neurons play an important role in the settlement of B. amphitrite cyprids. A PKC signal transduction system is already known to be involved in the larval metamorphosis of the same species (Yamamoto et al., '95). The present results provide further evidence that settlement and metamorphosis may be physiologically independent events in this species. SEROTONINAND BARNACLE SETTLEMENT ACKNOWLEDGMENTS We thank Dr. C. G. Satuito, K. Natoyama, and M. Yamazaki for their help with larval culture. LITERATURE CITED Barbaccia, M.L., 0. Grandolfi, D.M. Chuang, and E. Costa (1983) Modulation of neuronal serotonin by a putative endogenous ligand of imipramine recognition sites. Proc. Natl. Acad. Sci. U.S.A., 80.5134-5138. 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