GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

86,424-432

(1992)

The Occurrence and in Vitro Effects of Molecules Potentially Active in the Control of Growth in the Marine Mussel Mytilus edulis L. JEAN-YVES *Laboratoire

TOULLEC,

*,l IAN ROBBINS,?~

AND MICHEL

MATHIEU?

de Biologie Marine, Coll&e de France, Concarneau, France; and fLaboratoire Universite’ de Caen, Caen, France

de Zoologie,

Accepted October 11, 1991 A molecule with a molecular weight, estimated by gel filtration, of =22 kDa and immunoreactive to anti-human hypophysial growth hormone (hGH) has been identified by radioimmunoassay in the digestive gland and hemolymph of the mussel Mytilus edulis L. The dilution curve of this molecule was parallel to that of hGH, suggesting that the antigenic site of the Mytilus molecule is similar to that of hGH. Immunoreactive fractions resulting from gel filtration failed to stimulate protein synthesis in dispersed mantle-edge cells in vitro. No hGH-immunoreactive material was detected in the cerebral ganglia. It is thus clear that a small protein-synthesis-stimulating factor (PSSF), identified in the cerebral ganglia and hemolymph by its action in vitro on dispersed mantle-edge cells, is not analogous to the Mytilus hGH-immunoreactive molecule. Likewise, a somatostatin-immunoreactive molecule present in the hemolymph of Myths did not coelute with PSSF. Evidence is presented that PSSF is a hydrophilic peptide that stimulates DNA, RNA, and protein synthesis and that is 0 1992 not tissue specific. These characteristics suggest that PSSF is a growth hormone. Academic

Press, Inc.

Many studies have indicated growth of molluscs to be controlled by neuroendocrine cells of the central nervous system (CNS). Earlier papers described the inhibitory effects of ablation or cauterization of parts of the CNS on growth and growthassociated phenomena, (Lubet, 1971; Geraerts, 1976a,b; Wijdenes and Runham, 1977; Dogterom and Jentjens, 1980; Le Gall, 1980; Dogterom and Van der Schors, 1980). These studies concerned gastropod molluscs for which specific growthpromoting cells have been localized: the light green cells (LGC) in Lymnaea stagnalis (Geraerts, 1976a,b) and the neurosecretory medial cells in Agriolimax reticula&s (Wijdenes and Runham, 1977). The peptidic

nature of the neurosecretory granules of Lymnaea LGC was demonstrated by Wendelaar Bonga (1970) and these cells show an immunopositive reaction to anti-vertebrate somatostatin (Schot et al., 1981). The possibility that a somatostatin-like molecule is responsible for the growth promoting activity of these cells is further supported by the experimental results of Grimm-Jorgensen (1983a,b) and Marchand et al. (1989). Recently Smit et al. (1988, 1990) demonstrated a series of genes coding for insulin-like peptides (MIP = molluscan insulin-like peptide) in Lymnaea LGC, opening up the possibility that insulin-like peptides are involved in the control of growth. Compared with the considerable data on gastropod molluscs, very little is known about the commercially important bivalve molluscs. Homologous extracts of the cerebral ganglia have been demonstrated to stimulate protein synthesis by dispersed mantle-edge cells both of Mytilus edulis L. (Toullec et al., 1988) and indeed of other

’ To whom all correspondence should be addressed at present address: Laboratoire de Biochimie et Physiologie du Developpement, E.N.S., CNRS-URA686, 46 rue d’Ulm, 75230 Paris cedex 05, France. * Present address: Station de Biologie Marine, Quai de la Daurade, 34200 S&e, France. 424 0016-6480/92

$4.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

CONTROL

OF GROWTH

lamellibranchs; the active factor (proteinsynthesis-stimulating factor, PSSF) was shown to have a molecular weight of around 1 kDa (Toullec et al., 1988, 1989). In addition, immunoreactivity both to antivertebrate somatostatin (Toullec et al., to anti-Lymnaea MIP C-chain (Mathieu, unpublished results) has been detected in Mytilus cerebral ganglia. Hemolymph somatostatin immunoreactivity was also shown to be inversely proportional to body size in Pecten maximus, i.e., small, fast-growing individuals had high concentrations of circulating somatostatinimmunoreactive material (Toullec et al., s clear that neurosecretory cells of the lay a major role in the control of molluscan growth, and that the active factors are of low molecular weight, whether they are somatostatin-like or insulin-like. The CNS is the only clearly established endocrine center in the mussel. It remains possible, however, that larger molecules related to vertebrate hypophysial growth hormones (GH) and secreted by non-CNS tissues are also involved in growth regulation of molluscs. GH molecules compose a polymorphic family that also encompasses the prolactins and placental lactogens. The members of this family have probably evolved from a common parent molecule (Nicoll and Licht, 1971) that may find its ancestry amongst the invertebrates. Some evidence is available to support this hypothesis. Indeed, it has been demonstrated that the administration of vertebrate GH or prolactin promotes the growth and developlluscan larvae (Morse, 1984; 986, 1988; Paynter et al., 1990) -like molecules have been pufrom the gastropod Haliotis dissicus yana et al., 1989). e present investigation concerns the sence of a large molecular weight, verrate-like GH in Mytilus and the possible actions of the low molecular weight PSSF.

IN Mytilets edulis L.

Mussels. Adult M. e&is (4-6 cm length), obtained from a commercial mussel farm (Normandy, France), were cleared of fouling organisms and mainlined. in sea water for a maximum of 3 days prior to study. Antibiotics (penicillin 500,000 IU . liter-’ and aureomycin 30 mg liter-‘) were also added to the sea wa ter 48 hr before study. Radioimmunoassays. haolecules ~mmu~oreact~ve towards antibodies against human growth hormone (hGH) and vertebrate synthetic somatostatin-14 were estimated with commercial radioimm~n~assay lcits (CIS ORIS Industrie, France). Each extract or fraction was tested in duplicate. Dispersed

ceEi suspensions.

Tissues

were

hkissoci-

ated as previously described (Toullec et al., 1988) with pronase (0.1%) and collagenase (0.05%) in Ma&s culture medium adapted for marine invertebrates ( noir and Mathieu, 1986). Bioassays. The dispersed celi suspensions were diluted with fresh medium to give a final concentration of ca. lQ-15 X 106 cells . ml-‘. The exact cell concentration was estimated by hemocytometry (Thoma). Aliqoots of 500 p,I were incubated in IO-ml sterile tubes with 100 pl radiotracer and lyophilized hormonal extract dissolved, at appropriate dose, in 50 )~l of culture medium. The following radiotracers w mpioyed: (i) L-[3,4,5-3H]1eucine (sp act = 61 Ci ’ 1, 2 pm tube) to estimate protein synthesis, (ii) [6-“IIJthymidine (sp act = 93 Ci . m&z-‘, 1 &i/tube) to estimate DNA synthesis, (iii) [2-‘“Cluridine (sp act = 57.8 mCi . mk-‘. 0.6 @.X/tube) to estimate RNA. synthesis. The tubes were incubated in darkness at 1Y and under continuous gentle agitation. Incubation was stopped by adding 500 ~1 of ice-cold 10% (w/v) trichloroacetic acid (TCA) to each tube. The tubes were left for at least 2 hr at 4°C. A 200~p+l aliquot of each precipitate was deposited on a Whatman GFlC filter and washed with 25 ml of 5% TCA followed by 10 ml of ethanol and 2 ml of ether. The fillers were air dried and the radioactivity was determined with a scintillation counter using 5 ml PicoFluor 15 sci~t~I~a~i~~ fluid. Counts were corrected for quenching using an external standard. Hormanal extracts. Cerebral ganglia were homogenized in 10 mA4 ammonium acetate (hotter-~~ve~~rn). The homogenate was left at 4” for 16 hr and then centrifuged at 9,ODOgfor 4 min (4”). The supernatant was lyophilized. Hemolymph was collected from the posterior adductor muscle with a hypodermic syringe and centrifuged at 4,OOOgfor 10 min (4”) to separate ~ern~~~~~s from the serum. The hemocytes were homogenized in 10 mM ammonium acetate and extracted by the same procedure as for the ganglia.

426

TOULLEC,

ROBBINS,

Digestive glands (~10 g) were homogenized in 10 vol of 10 n&f ammonium acetate and extracted by the same procedure as for the ganglia. Gel chromatography. A lyophilized extract of 90 ml hemolymph serum was dissolved in 5 ml of elution buffer (ammonium acetate, 10 mM) and applied to a 2.6 x 58-cm column of Sephadex G-50 superfine (Pharmacia) equilibrated with the same buffer. The column was eluted at a flow rate of 6 ml . hr-’ and 3-ml fractions were collected. The absorbance of the fractions was measured at A = 280 nm. The pooled active fractions from this column were applied to a 1.7 x 45-cm column of Biogel P-4 (Biorad) equilibrated and eluted with the same buffer as above. Fractions of 3.2 ml were collected and their absorbance at X = 280 nm was measured. Similarly, a lyophilized extract of 1000 cerebral ganglia was dissolved in elution buffer and applied to this column. In another series of experiments to compare the elution volumes of PSSF with somatostatin-like immunoreactivity, extracts of 1000 cerebral ganglia were applied to a 0.6 X 140-cm column of Sephadex G-50 superfine and eluted with 10 mM ammonium acetate. Trypin digestion. The cerebral and hemolymph extracts were incubated at room temperature and under continual orbital shaking for 30 min with 250 IU of TPCK-treated trypsin attached to DITC glass beads (Sigma). Incubations were terminated by centrifuging the glass beads at 200s for 2 min (4”). The supernatants were collected and lyophilized. In control experiments, extracts were incubated as above, but the trypsin was added immediately prior to centrifugation. C,, reverse-phase extraction. The extracts of hemolymph and cerebral ganglia were passed through a Sep-Pak Cis cartridge (Waters) according to manufacturer’s instructions. The cartridge was washed with 10 ml water (aqueous phase) and retained material eluted with 5 ml methanol and evaporated under a stream of nitrogen (methanol phase). Chemicals. Radiotracers were supplied by CEA Saclay (France). PicoFluor 15 scintillation fluid was from Packard. Pronase, collagenase, and Hanks’ 199 powdered medium were supplied by Boehringer and antibiotics by Sigma. All other chemicals were of analytical grade. Double glass distilled water was used throughout.

AND -MATHIEU

bra1 ganglia, even at high concentrations of the latter (64 ganglia equivalent). The dilution curve of the hGH positive material of Mytilus hemolymph was parallel to that of hGH (Fig. 1). When Mytilus hemolymph was fractionated on a column of Sephadex G-50, an hGH-like immunoreactive peak was observed with an apparent molecular mass of about 22 kDa (Fig. 2). Two peaks of somatostatin-like immunoreactivity were detected by radioimmunoassay of the fractions obtained by gel chromatography of Mytilus cerebral ganglia on Sephadex G-50 (Fig. 3). The first peak was associated with the void volume and is possibly nonspecitic, though it could indicate that a large molecular weight somatostatinimmunoreactive protein occurs. The second peak eluted with an apparent molecular mass of -2.5 kDa. Protein synthesis stimulating factor (PSSF). A significant, dose-dependent in-

crease in the incorporation of [3H]leucine by dispersed mantle-edge cells was induced by hemolymph-serum extracts from a dose as low as 15 pg serum-protein equivalents (Fig. 4). The stimulatory activity of hemolymph-serum and cerebral ganglia extracts was significantly reduced by the action of trypsin (Fig. 5a). The active factor

l/2,

Dilutions 3!4

\

RESULTS Radioimmunoassays. hGH-immunoreac-

tive molecules were detected in the digestive gland (0.80 +- 0.30 ng * liter-‘, n = 4) and hemolymph serum (0.77 -+ 0.25 ng - liter-l; n = 6) of M. edulis. No hGH positive reaction was found with the cere-

-2100

1000

1GQCQ

pgitube FIG. 1. Dilution curves of human GH (filled squares) and Mytilus hemolymph serum (open circles) for measured GH concentrations in an hGH radioimmunoassay. The results are expressed in picogram equivalent hGH.

CONTROL

OF GROWTH

IN Mytilus edulis L.

0 0 100

200

300

400

Elution volume (ml) FIG. 2. Gel chromatography of an extract of Mytilus kemolymph serum (90~ml equivalent) on a 2.6 x 58-cm column of Sephadex 650 superfine. Elution buffer, 10 mM ammonium acetate. The absorption of the col‘ected fractions was measured at X = 280 nm and the concentration of hGH-like molecules estimated by radi&mmunoassay. The results are expressed in picogram equivalents of hGH. The column was calibrated with (I) bacitracin (1.45 kDa), (2) adrenocorticotropic hormone (4.57 kDa), (3) cytochrome C (12.3 kDa), (4) myoglobin (18.9 kDa), and (5) chymotrypsinogen (24.5 kDa).

was not retained on a Sep-Pak C,, reverse phase cartridge (Fig. 5b). wo peaks of PSSF activity were observed following the gel chromatography of ~y~i~~~ cerebra1 ganglia extracts (Fig. 6)

50

and hemolymph-serum extracts ( Sephadex G-50, one associated void volume, the other with an molecular weight of l-2 low molecular weight lap. The pooled fractions constituti hemolymph low molecular weight peak were applied to a column of the void volume, with an apparent molecular weigh

serum (Fig. 9). The low molecular wei P-4 fractionation of cerebral ganglia bo 10

20

30

40

50

60

Elution voIume (ml) Fro. 3. Gel chromatography of an extract of 1000 Mytilus cerebral ganglia on a 0.6 X 140~cm column of Sephadex GSO superfine. Elution buffer, 10 mM ammonium acetate. The absorption of the collected fractions was measured at A = 280 nm and the concentration of somatostatin-like molecules estimated by radioimmunoassay. The results are expressed in picogram equivalents of vertebrate somatostatin-14. The column was calibrated with (1) crustacean red pigmentconcentrating hormone (1.00 kDa), (2) bacitracin (1.45 kDa), (3) glucagon (3.55 kDa), (4) insulin (5.7 kDa), and (5) chymotrypsinogen (24.5 kDa).

100

150

~emoIym~h ( p. g of pro FIG. 4. Dose-dependent activation of incorporation (PSSF activity) by disperse mantle-edge cells provoked by Mytilus bemo~ymph serum extracts. The results are expressed as % activation with respect to control C SD (n = 4).

428

TOULLEC,

ROBBINS,

AND MATHIEU

60 40 20 0

Ganglia

Serum

Ganglia

Serum

FIG. 5. (a) The effects of trypsinization of cerebral ganglia (four ganglia equivalents) and hemolymph serum (0.4-ml equivalent) extracts on their capacity to stimulate [3H]leucine incorporation (PSSF activity) by dispersed M~&u mantle-edge cells. The results are expressed as % activation with respect to control + SD (n = 6). *Significantly different to control (Student’s t test; P < 0.05). (b) The effects of the aqueous and methanol phases resulting from the Sep Pak C,, reverse phase separation of cerebral ganglia (four ganglia equivalents) and hemolymph serum (0.4-ml equivalent) extracts on the stimulation of [3H]leucine incorporation (PSSF activity) by dispersed Mytilus mantle-edge cells. The results are expressed as % activation with respect to control f SD (n = 6). *Significantly different to control (Student’s t test; P < 0.05).

that of the human antigen, suggesting that the antigenic sites of the vertebrate and molluscan molecules are similar. This is not of course definitive confirmation of an hGH-like peptide in Mytilus, but the molecular weight close to that of the 22 kDa hGH suggests that Mytilus may possess a form of 3-

such a peptide. Further evidence for molluscan GH-like peptides has been provided by Moriyana et al. (1989) who have demonstrated two peptides of 30 and 22 kDa in Haliotis and Sellos (personal communication) who has demonstrated a peptide of 32 kDA in Pecten maximus. GH immunoreac-

I

1 z2 c1

P \ -

40 50 Elution volume (ml) FIG. 6. Gel chromatography of an extract of 1000 Myths cerebral ganglia on a 0.6 x 140-cm column of Sephadex GSO superfine. Elution buffer, 10 mA4 ammonium acetate. The absorption of the collected fractions was measured at h = 280 nm (dashed line). The fractions were tested for their ability to stimulate protein synthesis in dispersed mantle-edge cell suspensions (PSSF activity). Results are expressed as % activation with respect to control (solid line). The column was calibrated with (1) crustacean red pigmentconcentrating hormone (1 .OOkDa), (2) bacitracin (1.45 kDa), (3) glucagon (3.55 kDa), (4) insulin (5.70 kDa), and (5) chymotrypsinogen (24.5 kDa).

0

100

200

300

400

0

2

)

Elution volume (ml) FIG. 7. Gel chromatography of an extract of Mytihs hemolymph serum (90~ml equivalent) on a 2.6 x 58-cm column of Sephadex G50 superfine. Elution buffer, 10 rnM ammonium acetate. The absorption of the collected fractions was measured at A = 280 nm (dashed line). The fractions were tested for their ability to stimulate protein synthesis in dispersed mantle-edge cell suspensions (PSSF activity). Results are expressed as % activation with respect to control (solid line). The column was calibrated with (1) bacitracin (1.45 kDa), (2) adrenocorticotropic hormone (4.57 kDa), (3) cytochrome C (12.30 kDa), (4) myoglobin (18.90 kDa), and (5) chymotrypsinogen (24.50 kDa).

CONTROL

OF GROWTH

E 05 c “w N

0.4

2

Q,3

d 4

0.2 I 0s *” “2” 0

50

1W

150

Elution volume (ml) Fro. 8. Gel chromatography, on a 1.7 x 45cm column of Biorad P-4, of the pooled active fractions, collected during the Sephadex GSO fractionation of Myrilus hemolymph serum. Elution buffer, 10 mM ammonium acetate. The absorption of the collected fractions was measured at h = 280 nm (dashed line). The fractions were tested for their ability to stimulate protein synthesis in dispersed mantle-edge cell suspensions (PSSF activity). Results are expressed as % activation with respect to control (solid line). The column was calibrated with (1) actinomycin C (1.28 kDa), (2) bacitracin (1.45 kDa), and (3) insulin B chain (3.495 kDa).

tivity has also been thropods (Patton and et al., 1985; Verhaert and Van Wormhoudt, Broeck, 1990). These

shown in several arKuo, 1977; Veenstra et al., 1986; Toullec 1987; Van den results are supportive

Fi 0 2

2

I 2

1 / ” 0

50

100

150

Elution volume (ml) FIG. 9. Gel chromatography of an extract of 1000 cerebral ganglia on a 1.7 x 45cm column of Biorad P-4. Elution buffer, 10 rnM ammonium acetate. The absorption of the collected fractions was measured at X = 280 nm (dashed line). The fractions were tested for their ability to stimulate protein synthesis in dispersed mantle-edge cell suspensions (PSSF activity). Results are expressed as % activation with respect to control (solid line). The column was calibrated with (1) actinomycin C (1.28 kDa), (2) bacitracin (1.45 kDa), and (3) insulin B chain (3.495 kDa).

IN Mytihs

edulis

L.

4

of the apparent growth promoti vertebrate GH and larvae (Kawauchi, 1 many more data are require whether the molluscan peptides p antigenicity to vertebrate GH a are indeed members of the 61-11fa whether they play a physiolo growth m The cerebral PSSF is not Indeed, GH-like immunoreactivit~ observed in the cerebral ganglia GH-immunoreactive fractio chromatography of h~molyrn~~ not stimulate protein synthesis in mantle-edge cells. A low molecular weight PSS ilar molecular weight to that

although

it is not kno

from hemolymph and ganglia do not e overlap in the Sephadex G-50 se~a~a~~~~~ must be considere cases, as were the molecular wei ers used to calibrate the colum sible that wall effects in the n umn influenced the migration grated with identical apparent weight on the P-4 column. L not be concluded that these factors are growth hormones merely because t stimulate protein synthesis in a single rowth hormone shoul of all organs withou proportional weight. This s strates, however, that the chromato~~a fractions exhibiting PSS stimulate other metabolic acteristic of growth factors, viz. DNA synthesis. Pn additions bot and hemolymph PSSF-active stimulate DNA an persed gill cells, indicating tissue specific. These data support t

430

TOULLEC,

n

ROBBINS,

AND MATHIEU

3 H leucine 70 60

0

Ganglia

Serum

Ganglia

Serum

10. (a) The effects of cerebral ganglia (four ganglia equivalents) and hemolymph serum (0.4-ml equivalent) extracts on the incorporation of 13H]leucine, [3H]thymidine, and [i4C]uridine by dispersed Mytilus mantle-edge cells. The results are expressed as % activation with respect to control + SD (n = 6). Treatments were in all cases significantly different to controls (Student’s t test; P < 0.05). (b) The effects of cerebral ganglia (four ganglia equivalents) and hemolymph serum (0.4-ml equivalent) extracts on the incorporation of [3H]leucine and [3H]thymidine by dispersed Mytilus gill cells. The results are expressed as % activation with respect to control f SD (n = 4). Treatments were in all cases significantly different to controls (Student’s t test; P < 0.05). FIG.

pothesis that PSSF is a growth factor similar to that described for the gastropods. L. stagnaZis (see Joosse, 1988) and A. reticulatus (Wijdenes and Runham, 1977), though it will be necessary to further purify PSSF to determine whether a single factor stimulates protein, RNA, and DNA synthesis. The PSSF activity associated with the void volume during gel chromatography could be linked to nonspecific effects provoked by the high protein content of these fractions or, more probably, to molecular associations between low molecular weight PSSF molecules or between PSSF and other proteins. The fact that the low molecular weight fractions from G-Xl gel chromatography form two peaks (one eluting in the void volume), when rechromatographed on P-4 supports the latter hypothesis. The molecular weight of = 1 kDa, the partial sensitivity to trypsin, and the fact that it is not retained on a Sep-Pak C,, RP cartridge suggest that PSSF is a small hydrophilic peptide, as described for the cerebral growth hormone of L. stagnalis (Ebberink and Joosse, 1985; Geraerts et al., 1988). The fact that the sensitivity to trypsin was partial could be due to one of many rea-

sons: (i) digestion time was insufficient, (ii) the truncated peptide has diminished activity, (iii) more than one factor is involved. With such a low apparent molecular weight, and in spite of the fact that other factors may influence the migration of peptides in gel chromatography, it would appear unlikely that PSSF represents an insulin-like molecule. In addition, the insulinlike molecules of Lymnaea LGC possess hydrophobic properties not observed for PSSF (they do not elute with the void volume during Cl8 reversed phase chromatography; Ebberink et al., 1987). Likewise, PSSF would not appear to be a somatostatin-like molecule as it does not comigrate with the anti-somatostatin-immunoreactive peak during gel chromatography. ACKNOWLEDGMENTS This study was supported by the GCS-BBA (Groupe de Cooperation Scientifique-Bases Biologiques de l’Aquaculture), the “Fond Regional de Recherche et de Developpement” (Region Basse Normandie) and by an IFREMER grant to J-Y. Toullec. The authors thank Mme. Jeanne Mathieu for her technical assistance.

CONTROL

OF GROWTH

EFERENCES Dogterom, A. A., and Jentjens, T. (1980). The effect of the growth hormone of the pond snail Lymnaea stagnalis on periostracum formation. Comp. Biothem. Physiol. A M(4), 687-690. Dogterom, A. A., and Van der Schors, R. C. (1980). The effect of the growth hormone of Lymnaea stagnalis on (bi)carbonate movements, especially with regard to shell formation. Gen. Comp. Endocrinol. 41(3), 334-334. Ebberink, R. H. M., Van Loenhout, H., Van Beek, J., de Wilde, K., and Van Minnen, J. (1987). Characterization of peptides isolated from growth-controlling neuro-endocrine cells of Lymnaea stagnalis with immunoreactivity to antiinsulin. 1n “Neurobiology: Molluscan Models” (H. H. Boer, W. P. M. Geraerts, and J. Joosse, EC&.), pp. 224-227. North-Holland, Amsterdam. Ebberink, R. H. M., and Joosse, J. (1985). Molecular properties of various snail peptides from brain and gut. Peptides 6(suppl.), 451-457. Geraerts, W. P. M. (1976a). Control of growth by the neurosecretory hormone of the light green cells in the freshwater snail Lymnaea stagnalis. Gen. Camp. Endocrinol. 29, 61-71. Geraerts. W. P. M. (1976b). The role of the lateral lobes in the control of the growth and reproduction in the hermaphrodite freshwater snail Lymnaea stagnalis. Gen. Comp. Endocrinol. 29, 97108.

Geraerts, W. P. M., Vreugdenhil, E., and Ebberink, R. H. M. (1988). Bioactive peptides in molluscs. In “Neurohormones in Invertebrates” (M. C. Thonndyke and G. J. Goldsworthy, Eds.), pp. 262-288, Cambridge University Press, Cambridge. Grimm-Jorgensen, Y. (1983a). Possible physiological roles of thyrotropin releasing hormone and a somatostatin-like peptide in gastropods. In “Proceedings of the International Minisymposium of Molluscan Endocrinology” (J. Lever and H. Boer, Eds.). Amsterdam, 1982. Grimm-Jorgensen, Y. (1983b). Immunoreactive somatostatin in two pnlmonate gastropods. Gen. Camp. Endocrinof. 49, 108-l 14. Joosse, 3. (1988). The hormones of molluscs. In “Endocrinology of Selected Invertebrate Types,” pp. 89-140. A. R. Liss, New York. Kawauchi, l-l. (1986). Biofutur 69. Kawauchi, H. (1988). Growth hormone and prolactin family in fish. h “Molecular Biology Congress.” Center of Marine Biotechnology, Baltimore, MD. Le Gall, P. (1980). Etude experimentale de l’association en chaine et de son influence sur la croissance et la sexualitt chez Ia crepidule Crepidula forni-

IN Mytilus edulis L. cata (Mollusque mesogasteropode). These de doctorat d’Etat, Universite de Caen. Lenoir, F., and Mathieu, M. (1986). ~ti~sation des cultures de cellules dissociees dans l’etude des controies exerces sur la gametogtnese chez la moule Mytilus edulis k. C.R. Acad. Sci. (Paris) 303, 523-528.

Lubet, P. (1971). Ynfluence des ganglions cCr&roides sur la croissance de Crepidula fornicata Phii. (Mollusque mtsogasteropode). C.R. Acad. Sci. (Paris) 273, 2309-23 1I. Marchand, C. R., Assaka, L., and Strosser: M. T. (1989). Variations of somatostatin-like immunoreactivity in the circumoesophageal ganglia, the hepatopancreas, the mantle edge, and the hemolymph of shell-repairing snails (Helix aspersa). Gen. Camp. Endocrinol. 73, 59-68. Moriyana, S., Atsuta, S., Kobayashi, M., and cm. H. (1989). Growth hormone-like substance of habalone. Haliotis dissicus hannai. Pn “XI”” International Symposium on Comparative EndoctiMorse, D. E. (1984). ochemical and geneticengineering for imp ed production of abalones and other valuable moliusks. Aquaculture, 39, 263-282.

Nicoll, C. W., and Licht. P. (1971). Evolutionary biology of prolactins and somatotropins. HB. EIectrophoretic comparison of tetrapod somatotropins. Gen. Comp. Endocrinol. 17, 490-507. Patton, R. I., and Kuo, C. 61. (1977). The distribution of the STH-like protein in insects. hsecf Biothem. 7, 487-489. Paynter, K. T., Tang, Y. L., and Chen, T, T. (1990). The effect of biosynthetic trout growth harmone on oyster growth. J. Shellfish Res. &hot, L. P. C., Boer, H. II., Swaab, D. H., and Van Noorden, S. (1981). ~mmunocytocbe~~caI denonstration of peptidergic neurons in the centraI nervous system of the pond snail Lymmwa stugnabs with antisera raised to biologically active peptides of vertebrates. Cell. Tissue RDS. 216, 273-291.

Vreugdenhii, E., Ebberink, , W. P. I%, Klootwijk, J., and (1988). Growth-controlling molluscan neurons produce the precursor of an insulin-related peptide. Iva?ure 331, 535-538. Smit. A. B. (1990). The organization, neuronai expression and evolution of a family of i~s~Ii~-reIated genes in the mollusc Lymnaea stagnalis. thesis, University of Amsterdam. Toullec. J. Y., and Van Wor~houdt, A. (1987). Variations quantitatives durant Ie cycie d’intermue de peptides apparent& a I’hormone de croissance humaine chez Palaemon serrates (Crustacea Decaooda). CA. Acad. Sci. Paris 305. 265-263.

432

TOULLEC,

ROBBINS,

Toullec, J. Y., Lenoir, F., Van Wormhoudt, A., and Mathieu, M. (1988). Nonspecies-specific growth factor from cerebral ganglia of Mytilus edulis. J. Exp. Mar. Biol. Ecol. 119, 11 l-117. Toullec, J. Y., Lenoir, F., Van Wormhoudt, A., and Mathieu, M. (1989). Approche experimentale du controle de la croissance chez les bivalves. Oceanis 15, 511-517. Toullec, J. Y. (1990). “Recherche de substances hormonales actives sur la croissance d’invertebres marins d’interet aquacole.” These Sciences, Paris. Van den Broeck, J., Cardoen, J., Neyts, J., Swinnen, K., and DeLoof, A. (1990). Detection of substances recognized by antisera directed against vertebrate somatotropin, prolactin and placental lactogen, within the brain of the insect Locusta migratoria: A comparison of immunocytochemical localization patterns. Comp. Biochem. Physiol. A 97, 3540. Veenstra, J. A., Romberg-Privee, H. M., Schooneveld, H., and Polak, J. M. (1985). Immunocyto-

AND MATHIEU chemical localization of peptidergic neurons and neurosecretory cells in the neuroendocrine system of the Colorado potato beetle with antisera to vertebrate regulatory peptides. Histochemistry 82, 9-18. Verhaert, P., Huybrechts, R., Schols, D., Van den Braeck, J., De Loof, A., and Vandesande, F. (1986). Characterization of vertebrate peptide hormone-like materials in American cockroach. Different methods employed to investigate these substances and their results. In “Insect Neurochemistry and Neurophysiology” (A. B. Borkovec and B. B. Gelman, Eds.), pp. 229-233. The Humana Press, Clifton, NJ. Wendelaar Bonga, S. E. (1970). Ultrastructure and histochemistry of neurosecretory cells and neurohemal areas in the pond snail Lymnaea stagnalis L. Z. Zellforsch 108, 190-224. Wijdenes, J., and Runham, N. W. (1977). Studies on the control of growth in Agriolimax reticulatus (Mollusca, Pulmonata). Gen. Comp. Endocrinol. 31, 1X-156.