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C.M. 1985/F:61

Mariculture Committee/

A RAPID METHOD TO INDUCE SPA\~ING IN A NUMBER OF BIVALVE BY INJECTION OF SEROTONIN SOLUTION

l l 2 M. Castagna • M. C. Gibbons and J. Goodsell

lVirginia Institute of Marine Science, School of Marine Science, College cf William and Mary Eastern Shore Laboratory. Wachapreague, VA 23480.

2Rutgers Research Laboratory. P.O. Box 587, Port Norris, NJ 08349.

ABSTRACT

Injection of serotonin (5-hydroxytryptamine, creatinine sulfate complex) • solution was used to induce spawning in a number of bivalve species. Studies at the Virginia Institute of Marine Science demonstrated that intergonadal

injection induced spawning in , virginica,

Mercenaria mercenaria and Spisula solidissima. Intermuscular injection of

serotonin solution induced spawning of , cancellata,

Cyrtopleura costata, Geukensia demissa, Hiatella arctica, Mercenaria

mercenaria, Mesodesma arctatum, arenaria, Mya truncata, Mytilus edulis

and Rangia cuneata. \"'., ...

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Patinopecten yessoensis, Patinopecten caurinus, Hinnites multirugosus,

Argopecten circularis, Pinctada mazatlantica, Protothaca asperrima,

sp., Tridacna gigas, Tridacna derasa, Tridacna crocea, Hippopus hippopus

have also been induced to spawn by injection of serotonin (Hatsutani and

Nomura, 1982; personal communication - Neil Boume, Pacific Biological

Station, Nanaimo, B.C., Canada; John W. Ewart, University of Delaware,

School of Marine Science, Lewes, Delaware, USA; and Richard D. Braley, 4It School of Zoology, University of South Wales, P.O. Box 1, Kensington, New South Wales 2033, Australia).

Concentration of serotonin was 0.2 to 2.0 mM solution with 2.0 ~1

most eommonly used. Dosages were adjusted to size of the specimen with

0.4 ml used for average sized 4 to 8 cm in length or elams.

Symptomatic behavior was observed almost immediately following injection

of the solution. Spawning usually began within 15 minutes.

I NTRODUCTI ON

Many methods of indueing bivalve mollusks to spawn have been described. • Temperature cyeling and the addition of sperm or ova are the most eommonly used methods (Loosanoff and Davis, 1963).

The chemical serotonin, 5-hydroxytryptamine (5-BT), has been shown to be an effeetive inducer of spawning for many bivalves (Matsutani and Nomura,

1982; Gibbons et al., in press; Gibbons and Castagna, 1984). Injection of

serotonin into the gonad or adductor museie of ripe bivalves will often

induee spawning without additional stimuli. ~1ale bivalves respond and

spawn in greater number than females treated with serotonin, and unripe individuals who cannot spawn will display the usual behavioral response

to thc serotonin. ..L...... ,.,.'.:.•,,·,4 .~

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Serotonin is a neurotransmitter naturally occurring in the nervous

system of mollusks (I'lelsh and Moorhead, 1960). It stimulates heart

beating and ciliary beating of gills in bivalves (Leake and Walker, 1980).

However, the role of serotonin in spawning of bivalves is unknown.

Studies were carried out to test the effectiveness of serotonin on

different species.

MATERIALS AND METHODS • Serotonin (S-hydroxytryptamine, ereatinine sulfate eomplex) was dissolved in 1 pm fi1tered seawater to the desired eoneentration before

use. Onee a solution is mixed it is kept refrigerated since it deteriorates

at room temperature or in light. Matsutani and Nomura (1982) found the

optimal eoncentration for Patinopecten yessoensis was 2 Illi-l so this was the·

concentration common1y used.

When using this chemica1, the biva1ves to be spawned are either placed

in individual dishes or in a common spawning trough filled with filtered

seawater. The serotonin was injected either into the gonad or into the • adductor museie or on occasion into the foot. A new needle was used for each bivalve to prevent transference of gonadal products, blood, or other

tissue which may act as a stimuli. A small nick was made into the edge of

the shell to allow insertion of the needle in species which close tightly

such as oysters. This was accomp1ished with a sma11 stee1 file. Most of

the bivalves were injected with 0.4 ml of 2 mr-t solution. I'lhen smaller sized .species were used such as Mulinia lateralis, a smaller amount was

injected. All controls were injected with the same amount of 1 pm

filtered seawater as the test .

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Testsofindependence using the g-statistic and Williams correction for

a 2 x 2 contingency table were conducted to determine whether or not

spawning and injection with serotonin were independent (SokaI and Rohlf,

1981). The level of statistical reference was chosen to be 0(. = 0.01.

RE SU LTS

In these studies, serotonin was effective in inducing spawning in 83% 4It of bay Argopecten irradians, 70% of oysters Crassostrea virginica, 60% of surf Spisula solidissima, 45% of ribbed musseIs Geukensia

demissa, 45% of hard clams Mercenaria mercenaria and 27% of quahogs

Arctica islandica. In these bivalves males were much more sensitive to serotonin since they predominated the spawners. For the hermaphroditic bay

scallops, 97% released sperm while for dioecious bivalves between 67 and

100% of the spawners in each test lot were males. This data was not

extracted from tests with other species due to insufficent numbers of

spawners.

Reaction of ripe male bivalves to serotonin is almost immediate. • Oysters gaped and spawned as quickly as 2-5 minutes after injection and most of them had spawned within 30 minutes. Ribbed musseIs spawned

within 15 minutes. The behavior of biva1ves injected with serotonin was

to extend the foot and probe the bottom, gape and usually pump more

vigorous1y.

All broodstock survived the stress of injection with serotonin solution

or the filtered seawater used for the controls. Often the notches in the

shell showed new growth within one week. ..

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The authors successfully demonstrated that intergonadal or inter­

muscular injection of serotonin solution induced spawning in 14 species

(Table I).' It was not successful in two species of blood arks, Noetia

ponderosa and Anadara ovalis. 80th species released quantities of blood

cells but no gonadal products were observed.

Other investigators have reported that injection of serotonin solution

successfully induced spawning in 11 additional species (Table 11).

Spawning induced by serotonin has the advantages of ease of application,

does not require temperature cycling, and speed and synchronization of

induction. Serotonin may also be used to successfully induce spawning in

other bivalves which cannot be spawned using traditional spawning stimuli .

• . .

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LITERATURE CITED

Boume, Nei1. Personal communication.

Bra1ey, Richard D. Personal communication.

Ewart, John W. Personal communication.

Gibbons, M.C., J.G. Goodse11, M. Castagna, and R.A. Lutz. Chemica1 induction

of spawning by serotonin in the ocean quahog Arctica is1andica. J. of

She11fish Research. In press.

Gibbons, M.C. and M.. Castagna. 1984. Serotonin as an inducer of spawning

in six biva1ve species. Aquacu1ture 40: 189-191.

Leake, L.D. and R.J. Walker. 1980. Invertebrate Neuropharmaco1ogy. John

Wi1ey, New York, pp. 102-143.

Loosanoff, V.L. and H.C. Davis. 1963. Rearing of biva1ve mo11usks. Adv.

Mar. Bio1. 1: 1-136.

Matsutani, T. and T. Nomura. 1982. Induction of spawning by serotonin in

the sca110p Patinopecten yessoensis (Jay). Mar. Bio1. Lett. 3: 353-358.

Soka1, R.R. and F.J. Roh1f. 1981. Biometry. W.H. Freeman, San Francisco,

CA, 2nd edition, 859 pp . • We1sh, J.H. and M. Moorhead. 1960. The quantitative distribution of 5­ hydroxytryptamine in the invertebrates, especia11y in their nervous

systems. J. Neurochem. 6: 146-169. · .

Table I. Species of bivalves induced to spawn by injection with serotonin.

Common Name Scientific Name

ocean quahog Arctica islandica

bay Argopecten irradians

cross-barred Chione cancellata

American Crassostrea virginica

angel wing Cyrtopleura costata

ribbed musseI Geukensia demissa

arctic rock borer Hiatella arctica

hard Mercenaria mercenaria

arctic wedge clam Mesodesma arctatum

soft-shell clam Mya arenaria

truncate soft-shell clam Mya truncata

blue musseI MytHus edulis

common rangia Rangia cuneata

surf clam Spisula solidissima · .

Tab1e 11. Additional species of biva1ves induced to spawn by injection

with serotonin.

Species Reported by

Patinopecten yessoensis Matsutani and Nomura. 1982

Patinopecten caurinus Nei1 Bourne. personal communication

Hinnites mu1tirugosus Nei1 Boume, personal communication

Argopecten circu1aris John W. Ewart, personal communication

Pinctada mazat1antica John W. Ewart. personal communication

Protothaca asperrima John W. Ewart, personal communication

Ensis sp. John W. Ewart, personal communication

Tridacna gigas Richard D. Bra1ey, personal communication

Tridacna derasa Richard D. Bra1ey, personal communication

Tridacna crocea Richard D. Bra1ey. personal communication

Hippopus hippopus Richard D. Braley, personal communication