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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). Thus, the
5-HT receptors mediating both parturition and
release of glochidia coiild be located on the gill
cell membranes. Additional studies on effects of
5-HT on bivalves and particularly, radioligand
binding and other structural studies would enhance our present limit,ed knowledge of 5-HT receptors in bivalves.
ACKNOWLiEDGMENTS
We thank two anonymous reviewers for helpful
comments. Funding was kindly provided by the
Department of Biology and the Office of the Provost of Gettysburg College, Gettysburg, PA.
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