THE JOURNAL OF EXPERIMENTAL ZOOLOGY 275:326-330 (1996) Characterization of Serotonin Receptor Mediating Parturition in Fingernail Clams Sphuerium (Musculium:)spp. From Eastern North America F'ETER P. FONG, SCOTT WADE, AND MARK ROSTAFIN Cettysburg College, Gettysburg, Pennsylvania 17325 ABSTRACT Serotonin (5-Hydroxytryptamine, 5-HT) induces parturition in fingernail clams (Sphaerium spp.). We characterized pharmacologically the serotonin receptor mediating parturition in Sphaerium transuersurn and S. striatinum by exposing clams to a variety of serotonergic ligands. As in previous experiments, 5-HT was a potent inducer of parturition in both species. In addition, the selective vertebrate 5-HTz agonist alpha-methyl-5-HT significantly induced parturition in both species. Other agents including the serotonin agonists TFMPP (a 5-HT1 agonist), 1-l-naphthylpiperazine (5-HT1),8-OH-DPAT (5-HTlA),oxymetazoline (5-HT1*, lB, ID), and l-m-chlorophenylbiguanide(5-HT31, as well as dopamine, acetylcholine, and fluoxetine (a 5-HT releaser) were ineffective inducers. A 2-hour pretreatment with cyproheptadine, a vertebrate 5-HTz receptor ant,agonist, blocked 5-HT-induced parturition in both species. However, neither propranolol (5-HT1) nor mianserin (5-HTz)blocked 5-HT-induced parturition, but pretreatment with mianserin delayed parturition. These results, coupled with previous published experiments that showed significant inhibition of 5-HT-induced parturition by methiothepin (5-HT1),suggest that the 5-HT receptor mediating parturition displays a mixed 5-HT1/5-HTz pharmacological profile unlike any described for vertebrates, but support recent data on 5-HT receptors regulating reproductive processes in other bivalves. o 1996 Wiley-Liss, Inc. Many reproductive processes in bivalve molluscs are regulated by endogenous biogenic monoamines. In both marine and freshwater bivalves, serotonin (5-Hydroxytryptamine; 5-HT) acts as a neurohormone regulating various processes including meiosis reinitiation and germinal vesicle breakdown (Hirai et al., '88; Krantic et al., '93; Fong et al., ,941, spawning (Braley, '85; Matsutani and Nomura, '82; Hirai et al., '88; Matsutani, '90; Fong et al., '93; Ram et al., '931, and parturition (Fong and Warner, '95). Most freshwater bivalves do not freely spawn gametes, but have internal fertilization, and either release parasitic glochidia larvae or brood and release well-developed juveniles (Mackie, '84). Fingernail clams (Sphaeridae) are small freshwater bivalves, thought to be self-fertilizing, ovoviviparous hermaphrodites which typically brood their young in specialized pouches around the gills (Mackie, '78, '84;Pennak, '89). Parturition in Sphaerium transuersum can be induced by external application of serotonin, and serotonin-induced parturition can be blocked by pre-treatment with a vertebrate 5-HT1 antagonist, methiothepin (Fong and Warner, '95). 5-HT receptor pharmacology in freshwater bivalves is known only for 0 1996 WILEY-LISS, INC. the free spawning zebra mussel Dreissena polymorpha (Ram et al., '93; Fong et al., '93). In the present study, we characterized pharmacologically the 5-HT receptor mediating parturition in two species of Sphaerium: S. transuersum and S. striatinum. The receptor pharmacology was similar in both species, and did not resemble any corresponding to vertebrates, but showed a mixed 5HT1/5HTzprofile. MATERIALS AND METHODS Specimens of Sphaerium transuersum were collected by hand in September 1995 from Conewago Creek at Dick's Dam, New Oxford, Adams Co., PA. Specimens of S. striatinum were collected in May and September 1995 from Marsh Creek, Gettysburg, Adams Co., PA All animals ranged from 5-9 mm shell length. Animals were immediately transported to the laboratory where they were maintained in aquaria supplied with either artificial pond water (modified from Dietz et al., '82) com- Received December 18, 1995; revision accepted April 1,1996. Address reprint requests to Peter I? Fong, Department of Biology, Gettysburg College, Gettysburg, PA 17325. SEROTONIN MEDIATES PARTURITION IN CLAMS 327 RESULTS posed of 0.5 mM NaC1, 0.4 mM CaC12, 0.2 mM NaHC03, 0.05 mM KC1, and 2 mM Hepes, pH 7.8, As in previous experiments (Fong and Warner, or spring water at room temperature (23°C). All '95), serotonin proved to be a n effective inducer animals were tested within 3 days of collection. of parturition in Sphaerium spp. Individuals of both S. transuersum and S. striatinum gave Parturition assay birth in response to 5-HT (Figs. 1, 2, Table 1). 5-HT significantly induced parturition comMost of the procedures were identical to those reported in Fong and Warner ('95). Serotonin was found previously to be a potent inducer of paturition i n S. transversum. This was confirmed in s. striatinum. We exposed animals to a variety of serotonergic ligands, as well as two other neurotransmitters (dopamine and acetylcholine), and fluoxetine (a 5-HT releaser), and either spring water or artificial pond water (Fong and Warner, '95) as controls. Clams were monitored over a 4-hour period for juvenile release. A positive parturition response was scored if a n indi5-HT spring water vidual clam released at least one extra-marsupial 1 mM larva (Mackie, '78). Initially, all adults were gently wiped of adhering material and placed into vials (I clam per vial) containing 4.5 ml of either spring water or artificial pond water and allowed to acclimate for 20-30 minutes. Thereafter, 0.5 ml of either lo-' M, lov3M, or lo-* M of desired chemical was added to the appropriate vials, Thus, the final concentrations were 10-fold less than the added concentrations. The pH of all solutions was adjusted to a final pH of 7.8, however slightly acidic (pH 6.8) artificial pond water alone did not 5-HT alpha-m-5-HT spring water induce parturition. 1 mM 1 mM Inhibition of parturition was tested by pre-treating individuals of both species with the selective c c 5-HT antagonists cyproheptadine, mianserin, and 12 10 propranolol. Cyproheptadine and mianserin are both vertebrate 5-HT2receptor antagonists known to inhibit 5-HT-induced spawning in the freshwater bivalves (Fong et al., '93). Propranolol is a selective 5-HT1 antagonist. Clams were treated as above except that following acclimation, 0.5 ml of antagonist (lo3 M) was added to the vials. After a 2-hour exposure, clams received 5-HT to achieve the final concentrations. S.W. 10-5 lo4 10-3 After all experiments, clams which did not re[Alpha-methyl 5-HT] lease juveniles were dissected to determine if juveniles were present i n order to assess reproFig. 1. Percent parturition of Sphaerium transversurn ductive maturity. For all experiments, initial stimulated by 1 mM 5-HT. Numbers of clams tested are given samples sizes ranged from n = 10-15 clams in above bars. *P < 0.05. Fig. 2. Percent parturition of Sphaerium each group tested and results were analyzed striatinum stimulated by 1 mM 5-HT and 1 mM alpha-mestatistically using Fisher's Exact Test (Sokal thyl-5-HT. Numbers of clams tested are given above bars. *P < 0.05. Fig. 3. Percent parturition of Sphaerium transversum and Rohlf, '811, and null hypotheses were re- in different concentrations of alpha-methyl-5-HT. Numbers jected were P < 0.05. of clams tested are given above bars. *P< 0.05. P.P. FONG ET AL. 328 TABLE 1. Parturition induced b y various neurochemicals in Sphaerium transversum and S. straiatinum' Chemical Serotonin (5-HT) 1mM 0.1 mM 0.01 mM Alpha-methyl 5-HT 1mM 0.1 mM 0.01 mM Dopamine 1mM Acetylcholine 1mM TFMPP 1mM 8-OH-DPAT 1mM 0.1 mM Oxymetazoline 1mM 1-M-CP 1mM 0.1 mM 1-NP 1mM Fluoxetine 1mM Receptor selectivity S. transversum (no. of parturitions/total) S. straitinum (No. of parturitionsltotal) 54/73 6/g2 0/S2 24/43 9/10 11/12 2/10 219 1/12 0115 419 Endoengous 5-HT2 Endogenous Endogenous 5-HT1 EI-HT~A ~-HTIA,IB,I~ 5-HT3 0110 0115' 0/1l2 on0 3/11 018 017 018 014 5-HT1 1/15 0111 'TFMPP: m-trifluoromethylphenylpiperazine,8-OH-DPAT: 8-hydroxydipropylaminotetralin hydrobomide; 1-M-CP: l-methyl-chlorophenylbiguanide; 1-NP: 1-(1-naphthy1)piperazine. 'Data from Fong and Warner, 1995. pared t o the spring water control (Fisher's Exact Test, P < 0.0001 for both species). Furthermore, the vertebrate 5-HT2 receptor agonist, alpha-methyl-Ei-HT, was also a potent parturition inducer. In S. striatinum, parturition was induced by M alpha-methyl-5-HT (Fisher's Exact Test, P < 0.04; Fig. 2). In S. transversum, parturition was induced by both and M alpha-methyl-5-HT (Fisher's Exact Test, P < 0.001 for both concentrations; Fig. 3). However, other agonists and neurotransmitters were either marginally effective or completely ineffective at inducing parturition (Table 1).Particularly notable by t heir lack of effectiveness were TFMPP and 1-1-naphthylpiperazine, both 5-HT1 agonists, which are potent inducers of spawning in zebra mussels (Fong et al., '93). 5-HT-induced parturition was blocked by the 5-HTz antagonist cyp roheptadine in both species. A 2-hour pre-treatment with cyproheptadine ( lo4 M) significantly inhibited 5-HT(10-3 M)-induced parturition i n S. transversum (Fisher's Exact Test, lD< 0.006; Fig. 4), and 5 HT( M)-induced parturition in S. striatinum (Fisher's Exact Test, P c 0.007; Fig. 5 ) . Two other antagonists, propranolol (5-HT1) and mianserin (5-HT2)did not block 5-HT-induced parturition in S. traitsuersum, but mianserin M) did delay parturition (Fig. 6). DISCUSSION In previous experiments, Fong and Warner (1995) showed that 5-HT was a potent inducer of parturition in Sphaerium transversum. However, the specific 5-HTlAagonist 8-OH-DPAThad no effect on parturition. Conversely, methiothepin, an antagonist with a mixed 5-HT1/5-HT2selectivity completely inhibited parturition. In the present study, the pharmacological profile of the 5-HT receptor mediating parturition in two species of Sphaerium has been elucidated. The most potent parturition inducer in S. transversum was alpha-methyl-!j-HT, a 5-HT2receptor agonist. This agonist induced parturition in over 90% of the clams tested at both lo4 and lo3 M. For S. striatinum, 5-HT was the most potent inducer, and alpha-methyl-5-HT the second most effective. No other neurotransmitter or 5-HT ligand tested was a n effective parturition inducer in either species. Cyproheptadine M), a 5-HTz receptor antagonist, blocked 5-HT-induced parturition in both species. The only difference between species was t h a t in S. striatinum, cyproheptadine blocked an equimolar concentration of 5-HT, whereas in S. transversum, cyproheptadine blocked parturition i n a 10-fold higher concentration of 5-HT. In terms of reproductive biology, the major SEROTONIN MEDIATES PARTURITION IN CLAMS loo] S. transverswn 15 * " 5-HT 1mM 23 cyproheptadine qproheptadiae 0.1 mM 0.1 mM+ 5-HT 1 mM spring water S. striatinurn " 5-HT 0.1 mM 104 0 cyproheptadine cyproheptadine spring water 0.1 mM 0.1 m M + 5-HT 0.1 mM i 3 Time of experiment (hours) 1 2 4 Fig. 4. Effect of a 2-hour pretreatment with 0.1 mM cyproheptadine on parturition of Sphaerium transversum. Numbers of clams tested are given above bars. *P < 0.06. Fig. 5. Effect of a 2-hour pretreatment with 0.1 mM cyproheptadine on parturition of Sphaerium striatinum. Numbers of clams tested are given above bars. *P < 0.07. Fig. 6 . Cumulative percent parturition of Sphaerium transversum over time stimulated by 1 mM 5-HT, with and without a 2-hour pretreatment with 0.1 mM mianserin. difference between the two species is that in S. transversum fecundity is higher, juveniles are released with a smaller body size and lighter shells, and parturition is easier to induce by 5HT ligands. Although more 5-HT analogs were tested on S. transversum than on S. striatinum, the responses of both species were generally simi- 329 lar to the same analog. Thus, we can discuss in general terms the pharmacological profiles of Sphaerium spp. The 5-HT pharmacology for both parturition induction and inhibition of parturition in Sphaerium spp. displays a mixed vertebrate 5 HT1/5-HT2profile unlike any yet described for vertebrates. However, this mixed profile is emerging as a common one for 5-HT receptors mediating reproductive processes in bivalve molluscs. For example, in the freshwater zebra mussel (Dreisena polymorpha), 5-HT1 agonists ($-OH-DPAT, TFMPP, 1-1-naphthylpiperazine, metergoline) induce spawning, but 5-HT2 (cyproheptadine, mianserin) or 5-HT1/5-HT2(methiothepin) antagonists are the most effective blockers (Fong et al., '93). Moreover, 8-OH-DPAT induces GVBD in zebra mussel oocytes (Fong et al.,'94). In the marine clam Ruditapes philippinurum, 8-OH-DPAT and TFMPP induce GVBD, and mianserin and ritanserin (both 5-HT2)inhibit it (Gobet et al., '94). In the surf clam Spisula solidissima, 8-OH-DPAT (Krantic et al., '91) and alpha-methyl 5-HT (Kadam et al., '91) induce GVBD. For antagonists, Kadam et al. ('91) found that mianserin and ketanserin (both 5-HT2)effectively blocked 5-HTinduced GVBD in Spisula, and Krantic et al. ('93) found these two antagonists as well as metaclopramide (5-HT3)the most effective blocker of 5-HT-induced GVBD. In discussing 5-HT receptors it should be mentioned that receptor sites named 5-HT1, 5 HT2, 5-HT3etc. have been identified in rat brain membranes (Peroutka, '88). The 5-HT receptors mediating the various reproductive responses in bivalves, and indeed in other invertebrates, may not resemble these mammalian 5-HT receptors. It is interesting that reproductive responses in bivalves appear to be sensitive to 5-HT1 and 5 HT2ligands. Both of these receptor sites in vertebrates are coupled to G-proteins, as is 5-HT1,, a cloned receptor in a freshwater snail, Lymnaea stagnalis (Sugamori et al., '93). Serotonin regulates a number of reproductive processes in bivalves including GVBD (Fong et al., ,941, spawning (Ram et al., '93), sperm motility (Kadam and Koide, ,901, and release of oocytes from ovarian fragments (Matsutani and Nomura, '87). Although undoubtedly important in controlling reproduction, our knowledge of 5-HT receptors in bivalves is limited to a small number of mainly economically important species (Fong et al., in press). In these studies, the 5-HT receptors mediating the above processes are probably located on either gonads or gametes. In fingernail P.P. FONtG ET AL. 330 clams which are probahly self-fertile, the embryos and juveniles develop iin specialized chambers adjacent t o the gills (Mackie, '84).Recently, Dimock and Strube ('94) used 5-HT t o induce the release of glochidia larvae from the unionid bivalves Pyganodon cataracta and Utterbackia imbecillis. The glochidia are brooded in the interlamellar spaces of marsupial gills (Ruppert and Barnes, '94). Bivalve gills are known to respond to a variety of applied neurotransmitters including 5-HT (Gardiner et al., '91; Duncan et al., '94). 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