THE JOURNAL OF EXPERIMENTAL ZOOLOGY 274:365-372 (1996)

Monoarnines and the Release of Gametes by the Scallop Argopecten purpuratus

GLORIA MARTINEZ, FERNANDO SALEH, LIVIA METTIFOGO, ELISE0 CAMPOS, AND NIBALDO INESTROSA Departamento de Biologia Marina, Facultad de Ciencius del Mar, Universidad Catdlica del Norte, Coquimbo, Chile (G.M., KS. , L.M.), and Laboratorio de Neurobiologiu Molecular,' Pontificia Uniuersidad Catdica de Chile, Santiago, Chile (E.C., N.I.)

ABSTRACT Levels of dopamine (DA), noradrenaline (NA) and serotonin (5-HT) were mea- sured fluorometrically in the ganglia, gonads and adductor muscle of the functionally hermaphro- ditic scallop Argopectea purpurutus at different times during the induced release of gametes. The levels of these amines were analyzed in samples before stimulation, when animals looked stimu- lated, during the release of sperm, during the release of oocytes, and 24 hours after the end of spawning. Levels in muscles of the three monoamines changed during spawning and the greatest change was detected in the level of NA. In nervous tissue, the level of 5-HT decreased, whereas those of DA and NA increased significantly with the release of sperm. In both the female and male portions of gonad, levels of DA and NA increased during spawning while those of 5-HT decreased. However, levels of 5-HT in the female portion of gonads did not show any changes in a group of scallops that did not release sperm before releasing oocytes. Separate analysis of levels of monoa- mines in the visceral ganglion from those in the cerebropedal ganglion, showed that, in spawning animals, levels of DA and NA did not change in cerebropedal ganglia but decreased in the visceral ganglia, whereas levels of 5-HT decreased only in the cerebropedal complex. The results are con- sistent with the existence of two different mechanisms for the control of the release of sperm and oocytes by hermaphroditic pectinids. @ 1996 Wiley-Liss, Inc.

The role of a variety of external factors, such as temperature, food and photoperiod, in the re- lease of gametes by bivalve molluscs has been ex- tensively investigated (Giese and Kanatani, '87). Less attention has been paid to the endogenous factors that regulate this process. The first sug- gestions of a role for neurosecretions from the ce- rebral ganglia in the release of gametes by bivalves came from observations that the removal of the ce- rebral ganglia led to precocious spawning by Mytilus edulis (Lubet, '56) and by Crassostrea uirginica (Nagabhushanam, '62).

Most of the knowledge about factors that con- trol spawning in bivalves has come from experi- mental assays that involve artificial induction of this process by injections of homogenates of gan- glia (Matsutani and Nomura, '82) and of some neurotransmitters, mostly serotonin (Matsutani and Nomura, '82; Gibbons et al., '83; Gibbons and Castagna, '84; Braley, '85; Hirai et al., '88; Velez et al., '90, Ram et al., '93).

Osada et al. ('87) reported a decrease in levels of gonadal dopamine (DA) after an induced spawn- ing in male and female Patinopecten yessoensis. 0 1996 WILEY-LISS, INC.

Matsutani ('90) found an increased level of sero- tonin (5-hydroxytryptamine, 5-HT) in the testis and ganglia during spawning of Chlamys farreri nipponensis, at the same time as levels of this amine and of DA decreased in the ovary. Martinez and Rivera ('94) reported a decrease in levels of DA and 5-HT in gonads of Argopecten purpuratus during the first hours after a spontaneous spawn- ing. Smith ('87) showed that algae-induced spawn- ing in Myti lus cali fornianus required intact connections between visceral and pedal ganglia and was associated with an increase in the level of dopamine in the visceral ganglia and a decrease in the pedal ganglia.

The aim of the present study was to analyze the levels of the amines 5-HT, DA and noradrena- line (NA) in different tissues of the functionally hermaphroditic scallop A. purpuratus, during an induced release of gametes.

Received June 28, 1995; revision accepted February 2, 1996. Address repr in t requests to Gloria Martinez, PO Box 117,

Coquimbo, Chile.

366 G. MARTINEZ ET AL.

MATERIALS AND METHODS

Mature specimens ofA. purpuratus (80-100 mm in shell length) were collected from hanging cul- tures in La Herradura Bay, Coquimbo, Chile (30"s). They were maintained in aquaria with re- circulating seawater until use. Spawning was in- duced by an increase in water temperature and addition of a high concentration of microalgae. Samples of tissues were obtained from groups of four scallops which were killed at the following times: (1) before exposure to the stimulus; (2) about 60 minutes later, when animals appeared clearly to have been stimulated (with enlarged, white gonoducts); (3) about 15 minutes after the release of sperm had begun; (4) about 15 minutes after release of oocytes had begun; and (5) 24 hours after completion of spawning. After killing of the animals, the adductor muscle, nervous sys- tem and gonads were quickly removed. The male and female portions of the gonads were dissected separately. The tissues were homogenized with an ultrasonic homogenizer in cold 0.4 N perchloric acid to which sodium metabisulfite had been added (0.1 gA). After centrifugation at 20,000 g for 10 min, the supernatant fraction was kept at -20°C until the extraction and analysis of the amines as previously described by Martinez and Rivera ('94). Briefly, NA and DA were separated by adsorption on alumina at pH 8.5 and poste- rior elution with HCl .025 N (Anton and Sayre, '62; modified by Shellenberger and Gordon, '71; Campuzano et al., '75). After oxidation with io- dine solution, NA and DA were assayed with a Shimadzu RF-540 spectrofluorometer. 5-HT was extracted by alkaline butanol from the liquid remnant after adsorption on alumina and, af- ter purification with heptane, was detected by the fluorescence of the products of its reaction with ninhidrin (York and Twarog, '73). Stan- dardization was done with internal standards. All chemicals were the highest grade reagent commercially available. Since some of the scal- lops among those that were stimulated did not release sperm before releasing oocytes, samples of gonads of these animals were analyzed sepa- rately (the release of gametes by A. purpuratus is usually, but not always, protandrous; Disalvo e t al., '84).

With the aim of defining more clearly the role of the nervous system as a possible regulator of the spawning process, we placed another group of mature animals in individual aquaria and sub- jected them to the stimulus (as described above)

for the release of gametes. Under these conditions, not all the animals responded positively to the stimulus. Four of those that responded by spawn- ing and four of those that did not, were killed and the visceral and cerebropedal ganglia were dis- sected separately for the analysis of the amines.

One-way ANOVA was used to examine differ- ences in the levels of monoamines within each tis- sue analyzed. Homogeneity of variances was tested by Barlett's test. Tukey's test was used to evaluate the significance of possible differences and, in the figures, groups which do not share at least one letter are significantly different (P < 0.05). In some cases, as indicated, other statisti- cal tests were applied.

RESULTS Analysis of the adductor muscle

An analysis of variance showed that the levels of the three monoamines studied changed signifi- cantly (P < 0.05) in the muscle during the release of gametes by A. purpuratus. However, the pat- terns of changes differed among the amines (Fig. 1). The most striking feature was a considerable increase in levels of NA which was detected upon the release of sperm and was followed by a quick return to prestimulation values. Levels of DA and 5-HT changed less abruptly, increasing signifi- cantly after the release of oocytes and remaining elevated for the following 24 hours.

Analysis of the nervous system The levels of DA and NA in the ganglia of scal-

lops increased significantly (P < 0.05) after appli- cation of the stimulus for spawning and then they fell to the prestimulus values (Fig. 2). Unlike cat- echolamines, the level of 5-HT decreased signifi- cantly after the release of sperm. Regression analysis showed that this decrease was progres- sive (r = -0.74, P < 0.05) from just before the stimulus until the release of sperm.

Analysis of the gonad Female portion

The levels of the three amines in the female portion of the gonad changed significantly with the release of gametes (ANOVA, P < 0.05), but the changes in levels of the two catecholamines (DA and NA) differed from those in levels of 5- HT. While levels of DA and NA increased sig- nificantly (P < 0.05) with the release of oocytes, levels of 5-HT decreased after the release of sperm (Fig. 3A). Twenty-four hours after spawn-

MONOAMINES AND SPAWNING OF A. PURPURATUS

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Fig. 1. Levels of endogenous monoamines in the adduc- tor muscle of A. purpurutus at different times during the in- duced release of gametes. Each value is a mean k S.E. (n = 4). Means with same letter (for each monoamine) are not sig- nificantly different (Tukey’s test, P < 0.05).

ing had ended, the levels of the three monoam- ines revealed a tendency to return to the val- ues prior t o stimulation. In this portion of the gonad, those scallops that did not release sperm before releasing oocytes, showed similar in- creases in the levels of DA and NA with spawn-

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Fig. 2. Levels of endogenous monoamines in the nervous system ofA. purpurutus at different times during the induced release of gametes. Each value is a mean 2 S.E. (n = 4). Means with same letter (for each monoamine) are not significantly different (Tukey’s test, P < 0.05).

ing, although the levels attained by these amines were significantly lower (Student’s t- test, P < 0.05) than those attained in the scal- lops that released sperm. However, the most striking difference between the two groups of animals was that the scallops that did not re-

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G. MART~NEZ ET AL.

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purpuratus at different times during the induced release of gametes. A: Levels of amines in scallops that released sperm and oocytes. B: Levels of amines in scallops that did not re- lease sperm. Each value is a mean f S.E. (n = 4). Means with same letter (for each monoam- ine) are not significantly different (“ukey’s test, P c 0.05).

lease sperm, did not show any changes in lev- els of 5-HT in the female portion of the gonad upon spawning (Fig. 3B).

Male portion The levels of DA and 5-HT in the male por-

tion of the gonad changed similarly t o those in the female portion upon the induced release of gametes. However, levels of NA showed a sus- tained increase from the time when the stimu- lus was applied until the end of the spawning process (regression analysis, r = 0.96, P < 0.05; Fig. 4A).

As in the female portion of the gonad, the lev- els of DA and NA, after the release of oocytes,

were significantly lower (Student’s t-test, P < 0.05) in the scallops that did not release sperm than in those that did. However, unlike the female por- tion, the male portions of gonads from this group of animals showed the same decrease in levels of 5-HT after the release of oocytes as those from the group that released sperm (Fig. 4B).

Analysis of separate ganglia The analysis of levels of monoamine in sepa-

rate ganglia of A. purpuratus revealed that levels of both catecholamines were higher in the visceral ganglion (which innervates mainly the female part of the gonad) than in the cerebropedal complex (which innervates mainly the male portion) and,

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MONOAMINES AND SPAWNING OF A. PURPURATUS

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Fig. 4, Levels of endogenous monoamines in the male portion of the gonad of A. purpuratus at different times during the induced release of gametes. A Levels of amines in scallops that released sperm and oocytes. B: Levels of amines in scallops that did not re- lease sperm. Each value is a mean 2 S.E. (n = 4). Means with same letter (for each monoam- ine) are not signiflcantly different (Tukey's t.est, P < 0.05).

conversely, the level of serotonin was higher in the cerebropedal ganglion (Fig. 5).

When scallops were individually induced to spawn, only some of them responded to the stimu- lus. In these animals, levels of DA and NA de- creased only in the visceral ganglion, while that of 5-HT decreased only in the cerebropedal com- plex. No such changes were detected in those scal- lops that did not spawn (Fig. 5).

DISCUSSION The present study showed that levels of DA, NA

and 5-HT change with the release of gametes by the scallop Argopecten purpuratus. These changes, detected in nervous, muscular and gonadal tis-

sues, support the view that these monoamines are involved in the physiological process of spawning of bivalves.

In adductor muscle, the monoamines might function as neurotransmitters at the neuromus- cular junction. During the entire process of ga- mete release, the adductor muscle shows clear muscular activity. When a stimulus for spawning is applied, scallops clap their shells repetitively. When the release of gametes begins, this activity declines but each expulsion of gametes is accom- panied by a clapping of shells. We ignore the spe- cific neurotransmitter at the n e u r ~ m u ~ c u l a r junction of adductor muscle of scallops but the striking increase in NA levels during sperm re-

370 G. MART~NEZ ET a.

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Fig. 5. Levels of endogenous monoamines in, visceral and cerebropedal ganglia of A. purpuratus that had been indi- vidually induced to spawn. Values for animals that responded positively to the stimulus and for those that did not are rep- resented as means * S.E. (n = 4). Means with same letter (for each monoamine) are not significantly different (Tukey's test, P < 0.05).

lease might mean that this catecholamine is in- volved in the muscular response associated with this release. An association of monoamines with the spawning process has been demonstrated in nervous tissue of pectinids (Osada et al., '87;

Matsutani, '90). A decrease in the level of DA and no change in that of NA were detected during spawning in the ganglia of I? yessoensis (Osada et al., '87). In A. purpuratus (present results), lev- els of DA and NA in the nervous tissue increased as the animals were stimulated and, when oocytes were ejected, levels returned to prestimulus val- ues. By contrast, Matsutani ('90) did not detect any significant changes in levels of either DA or NA in ganglia of C. farrerri nipponensis during spawning. These differences might be due to the different kinds of stimulus applied for spawning. Therefore, in induced spawning, the changes in monoamine levels detected in nervous tissue might well represent the response to the applied stimulus, which, in the present study, was an in- crease of temperature and addition of an excess of microalgae. In the study of I? yessoensis (Osada et al., '87), spawning was induced by U.V. irra- diation of seawater and Matsutani ('90) induced the spawning of C. farreri nipponensis by raising the temperature and the pH of seawater. Martinez and Rivera ('94) detected no changes in levels of monoamines in ganglia during the first hours af- ter spontaneous spawning of A. purpuratus.

The changes in levels of monoamines detected in gonads of A. purpuratus differ from those re- ported by Osada et al. ('87) and by Matsutani ('90). In male and female R yessoensis, a decrease in gonadal levels of DA was detected during spawn- ing but no change was observed in levels of NA (Osada et al., '87). Similar changes in DA values were detected by Matsutani ('90) in the ovary of C. farreri nipponensis. By contrast, an increase in levels of these two catecholamines was found in both male and female portions of gonads of A. purpuratus. An increase in 5-HT content of the testis and a tendency for a decrease in the ovary of C. farreri nipponensis were detected during spawning (Matsutani, '90). In A. purpuratus, both portions of the gonads showed a decrease in 5-HT content during the release of sperm and of oocytes. Differences between the present results and those of Osada et al. ('87) and Matsutani ('90) may be explained by differences in reproductive biology; A. purpuratus is a functional hermaphrodite and P. yessoensis and C. farreri nipponensis are gonochoristic.

The apparent discrepancy between some of the present results (gonadal DA levels) and those pre- viously reported (Martinez and Rivera, '94) is not real because the experiments are not comparable. In the case of spontaneous spawning (previous study) we don't know the values previous to the

MONOAMINES AND SPAWNING OF A. PURPURATUS 371

release of oocytes so it is not possible to ensure that an increase, between stimulus, sperm release, and spawning did not occur. Matsutani and Nomura ('82) induced the release of gametes by males and female F! yessoensis by intragonadal injections of 5-HT, and effective doses of the amine were higher for females than for males. Similar results were obtained with C. farreri nipponensis (Matsutani, '90). However, 5-HT did not induce the release of oocytes but only of sperm by the her- maphroditic scallops Argopecten irradians (Gibbons and Castagna, '84) and Pecten ziczac (V6lez et al., '90). NA and DA induced spawning only in male C. farreri nipponensis but the effective doses were higher than those of 5-HT (Matsutani, '90).

Matsutani ('90) suggested that the different changes in levels of 5-HT in male and female gonads, together with the different responses to inductive injections of 5-HT, might reflect a difference in the physiological process of the re- lease of gametes. However, in A. purpuratus, lev- els of 5-HT, NA and DA changed similarly both in male and female parts of the gonad. Nonetheless, when scallops did not release sperm but only oo- cytes (this study), no change was detected in lev- els of 5-HT in the female portion upon spawning while the levels decreased in the male portion dur- ing the release of oocytes.

Further evidence for differential control of the release of male and female gametes comes from the analysis of separate ganglia of A. purpuratus. In the visceral ganglia, only levels of catecholamines changed with spawning, while in cerebropedal gan- glia, only 5-HT showed a quantitative change. We don't have exact knowledge about the innervation from each ganglion to the different parts of the go- nad of this hermaphrodite scallop, but connectives from the visceral ganglion arrive rather directly at the female portion and the male portion seems nearer the cerebropedal ganglion. In the hermaph- rodite scallop Pecten maximus, Paulet et al. ('93) found that serotonin neurons were mainly distrib- uted in the cortices of the pedal and cerebral gan- glia, whereas in the gonads they are distributed mainly in the male acini. All above findings sup- port the hypothesis that external stimulus for spawning is detected by specific receptors and the signals are communicated, through some monoam- inergic mechanism, from the nervous system to the gonads, where the signal induces the action of some other compound or a physiological event that finally results in the release of gametes. The results are also consistent with two different ways for the control of the release of sperm and of oo-

cytes by the gonads. We suggest that serotoner- gic ways would conduct information for the re- lease of male gametes while catecholamines would be involved in the release of oocytes.

ACKNOWLEDGMENTS The authors are very grateful to Raul Vera for

his technical assistance. This work was supported by project FONDECYT 194-1125.

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