Induction of metamorphosis of two species of sea urchin from Sea of Japan

The induction of metamorphosis in mature larvae by selected chemical compounds and natural substrata was investigated in two species of sea urchins from the Sea of Japan, Strongylocentrotus intermedius and Scaphechinus mirabilis. Glutamine in crystalline form was added directly to water containing mature larvae, and this compound, at a final concentration of 10 g ml^-1, was an inducer of metamorphosis in S. intermedius (100% activity) and S. mirabilis (50% activity). Gutamine, or its natural mimetic molecules, may be an active component of the exogenous cue that induces metamorphosis of S. intermedius larvae. This exogenous cue was produced by the epiphytic calcareous algae, Melobesia spp. that colonized the older sea grass Zostera marina. Glutamic acid was also used as an inducer of metamorphosis for S. intermedius and S. mirabilis larvae (50 to 60% activity), but it was toxic to the larvae.     

Isolation of novel metal-binding proteins distinct from metallothionein from spotted seatrout (Cynoscion nebulosus) and Atlantic croaker (Micropogonias undulatus) ovaries

Metal-binding proteins were isolated from ovaries of the spotted seatroutCynoscion nebulosus and the Atlantic croakerMicropogonias undulatus collected in 1988 near Port Aransas, Texas, USA. Gel-filtration analysis of spotted seatrout trout ovarian cytosolic fraction on Sephadex G-75 revealed the presence of three zincbinding protein fractions. A major zinc/calcium-binding protein fraction had a low molecular weight (M _r)(6 000 to 10 000), similar to mammalian hepatic metallothionein (MT). All the metals were displaced from this fraction following saturation with exogenous cadmium. After exposure of Atlantic croaker to 2 mg cadmium l^–1 seawater for 2 mo, the majority of the cadmium in the ovarian cytosolic fraction was associated with a similar low molecular weight protein fraction. These proteins were further purified by heat treatment and sequential acetone precipitation. Three isoforms were isolated by reversephase high-performance liquid chromatography. All the isoforms were found to be distinct from mammalian MT, based on amino acid composition. The major isoform contained low amounts of cysteine (approximately 5 residues per molecule) and aromatic amino acids, compared to high amounts of cysteine (typically 17 to 20 residues/molecule) and a lack of aromatic amino acids for mammalian MT. All the ovarian protein isoforms contained more glutamate than mammalian MT. The spotted seatrout and Atlantic croaker ovarian isoforms showed a high degree of homology with metal-binding proteins isolated from mammalian gonadal tissues. The results suggest a physiological role for these metal-binding proteins in developing vertebrate ovaries as well as an involvement in the sequestration of cadmium following environmental exposure.     

Distribution,habitat preferences and breeding periods in the crustaceans Sesarma cinereum and S. reticulatum (Brachyura: Decapoda: Grapsidae)

The distribution and habitat preferences of Sesarma cinereum and S. reticulatum were studied in the region of Beaufort, North Carolina, USA. S. cinereum inhabits the supralittoral zones of marshes characterized by high salinities (mean=27.9%) and sandy substrates, while s. reticulatum were found to prefer eulittoral marshes with brackish salinities (mean=16.2%) and silty substrates. Major differences have also been found in the cycling of egg production between these two species. Populations of S. cinereum produce approximately 4 to 6 egg batches in close synchrony with the lunar phase, whereas populations of S. reticulatum produce only from 2 to 3 batches of young per breeding season. It appears that female S. cinereum invest less time in carrying their eggs (approximately 1 lunar month), while female S. reticulartum invest a greater amount of time (eggs carried for approximately 45 days). These and other specific differences in reproductive strategies suggest that populations of S. cinereum are responding to density-independent selective pressures, while populations of S. reticulatum exhibit characteristics of density-dependent selective effects.     

Egg protein insolubility inLymantria dispar versus other forest Lepidoptera

Summary A standard buffer (5 mM phosphate at pH 7) which is used to extract protein from insect eggs provided complete protein solubility for eggs from three of four tree-feeding lepidopteran species: obliquebanded leaf roller (Choristoneura rosaceana), forest tent caterpillar (Malacosoma disstria), and the eastern tent caterpillar (Malacosoma americanum). Under the same extraction protocol, egg proteins from the gypsy moth (Lymantria dispar), remained nearly insoluble. An array of methods typically used to solubilize insect egg proteins were tried and all but the most denaturing (2% SDS) were ineffective. Extraction buffers with typically high pH values were then evaluated. The results indicated that 1) solubility of gypsy moth egg proteins was pH dependent, and full solubility of most egg proteins required the extraction buffer to have a pH of 12 or more prior to the addition of eggs. We also determined that 2) the gypsy moth egg has a buffering capacity which must be surpassed for complete protein extraction, 3) low salt/high pH buffers gave slightly higher total protein values than did high salt/high pH buffers, 4) parental nutritional history (host species utilized) and egg developmental state (pre-embryonatedvs postembryonated/pre-hatch) were unrelated to the requirements for complete egg protein solubilization, and 5) the presence of soluble phenolics, compounds that have the potential to bind to protein and cause insolubility, was confirmed for the gypsy moth egg with 2-D paper chromatography and several other tests. Based on these results, we present a hypothesis about the cause of egg protein insolubility in the gypsy moth.     

Subitaneous, diapause, and delayed-hatching eggs of planktonic copepods from the northern Gulf of Mexico: morphology and hatching success

Experiments were conducted to examine the morphology and hatching success of eggs, either spawned by freshly caught planktonic copepods or recovered from bottom sediments in the northern Gulf of Mexico. Collections were made between August 1992 and September 1995. Eggs of nine species were described and these differed in their diameter, color and surface attributes. Three types of eggs were distinguished: subitaneous, diapause, and delayed-hatching. Three species, Labidocera aestiva Wheeler, Acartia tonsa Dana, and Centropages velificatus (Oliveira) produced only subitaneous eggs. Hatching success varied greatly among these species. Two species, Labidocera mirabilis Fleminger and Centropages hamatus (Lilljeborg) produced diapause eggs and subitaneous eggs. The length of the refractory phase of the diapause eggs differed greatly both within and between these two species. A third type of dormant egg, delayed-hatching, was recognized in Labidocera scotti Giesbrecht and Pontella meadi Wheeler. The existence of delayed-hatching eggs may be an adaptive response of subtropical species to less seasonal fluctuation. Based upon morphological characteristics of the eggs and rearing of nauplii to an identifiable stage, benthic dormant eggs of eight species of calanoid copepods were also identified. Received: 9 September 1996 / Accepted: 1 October 1996     

Isolation and characterization of naturally and artificially induced hepatic metallothioneins from two species of flatfish: Limanda limanda and Microstomus kitt

The separation of hepatic metallothioneins of Limanda limanda and Microstomus kitt by gel-filtration chromatography, anion-exchange high-performance liquid chromatography (HPLC) and reversed-phase HPLC is described. Two isoforms of metal-binding proteins were isolated by DEAE HPLC chromatography from fish caught in the field and fish injected intraperitoneally with Cd. The amino acid composition of these proteins further purified by reversed-phase HPLC chromatography revealed the existence of metallothioneins. The route of contamination (natural or artificial) of the fish has no effect on the nature of these proteins and one of the two protein isoforms from each species has similar ionic properties.     

The cladocerans of the Aegean Sea: Occurrence and seasonal variation

The little-known cladoceran population of the Aegean Sea was studied. One hundred and seventy-six planktonic samples were collected during 8 cruises in the Aegean Sea (4 in the northern part, 4 in the southern part). From these samples, the following 4 species of Cladocera were determined: Evadne spinifera Muller, Evadne tergestina Claus, Penilia avirostris Dana, Podon intermedius Lilljeborg. All species were found both in the north and in the south Aegean Sea, with the exception of P. avirostris, which was found only in the northern Aegean Sea. Its absence from the southern part is attributed to the higher salinities and the pelagic character of the area. With regard to abundance, E. spinifera is by far the commonest in this area; P. avirostris and E. tergestina could be characterised as frequent forms; P. intermedius is scarce. P. avirostris, E. spinifera and E. tergestina are thermophilic; their occurrence is more or less restricted to the warm season; P. intermedius is psychrophilic and appears only in winter.     

Evaluation of the significance of metal-binding proteins in the gastropod Littorina littorea

Measurements of metals in Littorina littorea from clean and contaminated sites in Great Britain confirmed that body concentrations of Ag, Cd and Hg vary according to environmental contamination, while the essential elements Cu and Zn are regulated. Investigation of the cytosolic distribution of metals in L. littorea, using Sephadex G-75 gel-permeation chromatography, revealed important distinctions in the partitioning of elements. In contrast to Ag and Hg, which were mainly associated with high molecular weight ligands, Cd was bound predominantly to an intermediate molecular weight, soluble protein (CdBP-I, apparent mol. wt=20 000). However, although the main function of CdBP-I, both in clean as well as contaminated individuals, was sequestration of Cd, significant quantities of other non-essential metals (Ag, Hg) were similarly bound. Laboratory experiments confirmed the role of CdBP-I in complexing cadmium, and also revealed the induction of a second cadmium-binding protein (CdBP-II) in response to high cadmium levels. The apparent molecular weight (10 000), absorbance characteristics and high (inducible)-SH content of CdBP-II suggest similarities with metallothionein. A dose-related increase in the-SH content of very low molecular weight (>3 000) fractions was also observed in cadmium-exposed L. littorea, although no cadmium was associated with these ligands. A detoxifying role is tentatively proposed for the metal-binding proteins CdBP-I and II in L. littorea. However, some spillover of cadmium to the high molecular weight protein pool was observed in individuals exposed to cadmium in the field and laboratory. The use of L. littorea in biochemical assays of environmental contamination is discussed.     

Subcellular localisation and identification of95mTc- and241Am-binding ligands in the hepatopancreas of the lobsterHomarus gammarus

Following uptake of technetium-95m and americium-241 by lobsters (Homarus gammarus) from food, the subcellular distribution and binding in the major organ of accumulation, the digestive gland, were analysed.^95mTc was predominantly bound to soluble proteins; approximately 40% was bound to two proteins displaying the same elution characteristics as Cdthioneins as indicated by gel permeation and ion-exchange chromatography, indicating reduction and sequestration by metallothionein in vivo. The identity of a third anionic low molecular weight Tc-binding component was not established. No evidence was found for binding of Tc to haemocyanin, ferritin or transferrin. Particulate Tc [probably TcO(OH)₂] was bound to plasma membranes and was also present in lysosomes, indicating reduction of pertechnetate and pinocytosis in the digestive gland. In contrast,²⁴¹Am was mainly sequestered by the lysosomal/vacuolar system, indicative of uptake by pinocytosis. Approximately 2 to 3% of the Am was bound to ferritin and 20% was bound to unidentified ligands of 10 and 20 kdaltons mol. wt. There was no evidence for sequestration of Am by metallothionein. These studies prove that waste radionuclides discharged by the nuclear industry are metabolically active and enter the normal metal metabolism and detoxication pathways of a marine shellfish.     

Hemolysins from the mucus of Spirographis spallanzani (Polychaeta: Sabellidae)

Specimens of Spirographis spallanzani (Polychaeta: Sabellidae) were collected in the Gulf of Taranto, Italy, in 1991, and their mucous secretion was examined. The mucus possesses thermolabile biologically active proteins which produce hemolysis in vertebrate erythrocytes. The molecules are ineffective against both unfertilized and fertilized echinoid eggs and spermatozoa. The lytic reaction against rabbit erythrocytes varies as a function of cations, pH and temperature, and displays a fast time-course. No immunological cross-reaction could be demonstrated between hemolysins of S. spallanzani and Eisenia foetida andrei. SDS-PAGE analysis of mucus proteins reacting with sheep erythrocytes revealed that at least three major (16, 22 and 40 kDa) and two minor (29 and 34 kDa) components interact with membrane targets.     

Chemical aspects of mass spawning in corals. I. Sperm-attractant molecules in the eggs of the scleractinian coral Montipora digitata

This paper provides the first evidence for sperm chemotaxis in the Scleractinia. Montipora digitata Dana, 1845 (Scleractinia: Coelenterata) is a hermaphroditic coral which reproduces bi-annually, releasing egg-sperm bundles during the mass spawning at Magnetic Island (19°10S; 146°52E) in late spring-early summer, and autumn each year. The buoyant egg-sperm bundles float to the surface where they break apart, releasing eggs and sperm into the ocean. Fertilisation occurs after 30 min. Unfertilized eggs were collected, washed free of sperm, and freeze-dried. The eggs were extracted with dichloromethane, fractionated by chromatography on silica gel, and the fractions assayed for their ability to attract M. digitata sperm. The active fraction was further fractionated by high-performance liquid chromatography, resulting in the isolation of three highly unsaturated fatty alcohols: (1) dodeca-2,4-diynol; (2) tetradec-13-ene-2,4-diynol; (3) (14Z)-heptadeca-14,16-diene-2,4-diynol. Of these three compounds, only Compound 1 attracted sperm of M. digitata. Synthetic Compound 1, produced from simple precursors by known reactions, possessed sperm-attracting activity comparable to the naturally derived attractant. Preliminary experiments suggest that the natural mixture of Compounds 1, 2 and 3 in the ratio 1:4:9 is more effective in attracting sperm from M. digitata than sperm from other Montipora species. Sperm attractants may act to reduce the incidence of hybridisation between different species of Montipora.     

The breeding cycles of two species of grapsid crabs (Crustacea: Decapoda) from the North Coast of Kyushu,Japan

The incidence of ovigerous females in populations of two grapsid crabs, Hemigrapsus penicillatus (de Haan, 1835) and Sesarma (Parasesarma) pictum (de Haan, 1835) were followed from August 1975 to November 1976. H. penicillatus, which inhabits the lower intertidal region near the mouth of Tatara-Umi Estuary, breeds from March to November. S. pictum, which inhibits crevices and abandoned holes of other species and is abundant at and above the upper intertidal region of the estuary, breeds from May to September. H. penicillatus matures when the female reaches 6 to 7 mm carapace width, whereas S. pictum becomes mature when the carapace width is 12 to 13 mm, although the maximum size attained by females of both species is almost the same. H. penicillatus produces 5 to 6 broods, S. pictum 2 to 3 broods, during a breeding season. The size of a newly laid egg of H. penicillatus is smaller than that of S. pictum. The number of eggs produced by a female H. penicillatus is greater than that of S. pictum during a breeding season. In both species the peak of the breeding season is during summer, at which time the ovarian activity is also apparently accelerated. The major environmental factor which controls the breeding in these crabs appears to be temperature. H. penicillatus is submerged at every high tide, and is relatively inactive from late November to February when the ambient water and air temperatures are rather low. S. pictum is dormant in crevices or understones of the splash zone from November to March. The length of the breeding season of these crabs appears to be inversely proportional to the period of their winter dormancy.     

Diets of the demersal fishes on the shelf off Iwate,northern Japan

Diets of demersal fishes were determined on the shelf (ca. 130 m deep) off Iwate, Japan. Samples were taken in three different types of habitat, an artificial reef (AR) site, a natural reef (NR) site, and sandymud bottom (SB) site, from May 1987 to September 1991, mostly every two months. A total of 67 prey items were recognized in the stomachs of 45 predator fish species. The most important preys were the pelagic fishes Sardinops melanostictus and Engraulis japonicus, which comprised 37% wet wt of the overall stomach contents. The percentage of pelagic fishes was highest at AR site, where fish density was highest. The dominant ten species could be divided into five feeding types. The pelagic fish feeders Physiculus maximowiczi and Gadus macrocephalus fed mainly on S. melanostictus. The dietary breadth of P. maximowiczi was wide, while that of Gadus macrocephalus was narrow. The pelagic crustacean feeder Theragra chalcogramma mostly consumed Themisto japonica and euphausiids and showed the least dietary overlap with other fishes. Benthic fish feeders were Hemitripterus villosus and Liparis tanakai. The benthic crustacean feeders Alcichthys alcicornis and Hexagrammos otakii consumed benthic crustaceans as well as pelagic and benthic fishes and showed the largest dietary breadth. The benthic invertebrate feeders Gymnocanthus intermedius, Dexistes rikuzenius and Tanakius kitaharai fed mainly on polychaetes and benthic crustaceans. But Gymnocanthus intermedius consumed a significant proportion of pelagic fishes. Ontogenetic dietary shift was recognized for these fishes. Pelagic fishes were consumed more intensively by larger individuals, especially true of A. alcicornis, Theragra chalcogramma and Gadus macrocephalus. Predominancy of the two most adundant species, P. maximowiczi and A. alcicornis, may be supported by their wide dietary breadth and the significant proportion of pelagic fish in their diets. Interspecific dietary overlap was low in most cases suggesting that food resources were well partitioned, although some high overlap was observed among the pelagic fish feeders, A. alcicornis, and Gymnocanthus intermedius, and among the benthic invertebrate feeders. Interspecific competition seemed more likely in the benthic invertebrate feeders than in the pelagic fish feeders partly because of superabundance of the pelagic prey S. melanostictus.     

Flexible reproductive strategies in tropical and temperate Sepioteuthis squids

A major difficulty confronting the determination of cephalopod reproductive life history is assessing over what portion of the life span an individual is reproductively mature and actively depositing eggs. This paper assesses the potential of the tropical Sepioteuthis lessoniana and two genetic types of the temperate Sepioteuthis australis, to spawn multiple batches of eggs at discrete times throughout the adult life span. This is achieved by histological examination of the ovarian gametogenic cycle and detailed morphological assessments of the reproductive system, in conjunction with other biological information. The genetic type of S. australis found at the northern limits of its Australian distribution showed evidence of a high correlation between body size and quantity of mature eggs, suggesting that eggs may be accumulating to be laid in a single batch. Although maturation was also a size-related process in S. lessoniana and Tasmanian S. australis, oviduct size was not correlated with body weight in mature females, which is indicative of multiple spawning. Further supporting evidence includes relatively low gonadosomatic indices, the heavier weight of the ovary relative to the oviduct, and the feeding activity of mature animals. Mature S. lessoniana and S. australis individuals were present at each location over very wide age and size ranges. In Tasmanian waters, there were distinct seasonal differences in the reproductive biology of S. australis. Summer-caught individuals had much higher gonadosomatic indices and may have been laying larger batches of eggs compared with winter-caught individuals. Summer-caught females also showed a negative correlation between egg size and egg number within the oviduct, suggesting that some individuals were producing fewer, larger eggs and others many smaller eggs. Evidence suggests that considerable flexibility is inherent in the reproductive strategy of both S. lessoniana and S. australis. Received: 11 May 2000 / Accepted: 12 September 2000     

Purification of metallothionein from thymus and spleen,induced by zinc administration

Zinc is an essential trace element to maintain human and generally mammal homeostasis; its biological signification could be explained through the participation or as structural element of more than 200 protein molecules in mammals.

The synthesis of proteins in spleen and thymus of rats is induced by zinc, a fact which moved us to purify and isolate such proteins, before proceeding to its identification or characterization. Rats are treated with zinc during 4 weeks and then sacrificed; blood is extracted under light ether anesthesia. The extracts of thymus and spleen were prepared by homogenization with Tris‐HC1 buffer 1.0 × 10^‐2M pH 7.4 and submitted to Sephadex G‐75 column (40 × 1.6 cm) after centrifugation and filtration through 0.45 μ filters. The elution profile of proteins were established at 253 and 280 nm, and zinc concentration was measured by atomic absorption spectrophotometry. The peak containing zinc was submitted to a second column of Sephadex G‐25 (40×1.6 cm) obtaining a peak of purified protein with physico‐chemical characteristics similar to metallothionein.     

Distribution and mortality of diapause eggs from calanoid copepods in relation to sedimentation regimes

Distribution, abundance and age of diapause eggs from three species of calanoid copepods (in particular from Acartia spp. most likely Acartia tonsa, and Centropages hamatus and less numerous from Temora longicornis) were recorded in sediment profiles by enumerating hatched nauplii from incubated sediment samples. Phytoplankton pigments and ²¹⁰Pb and ¹³⁷Cs analyses indicated that the sedimentation regimes were different between two southern and two northern stations of the island Funen, Denmark. Significant variations in vertical distribution, abundance and mortality of diapause eggs were found between the stations. Dating of the sediment cores suggested a ~70-year maximum age of viable eggs on the northernmost stations, and ~28 year at the southernmost stations. The eggs exhibited a significantly higher mortality rate at the southernmost stations compared with the northernmost, 0.35–0.53 year⁻¹ vs. 0.07–0.08 year⁻¹ with no systematic pattern among species. The differences in abundance, mortality and age of the diapause eggs are suggested to be due to the sediment characteristics in which they are buried.     

Induction of larval settlement and metamorphosis of the sea urchin Strongylocentrotus intermedius by glycoglycerolipids from the green alga Ulvella lens

In aquaculture centers of the northern region of Japan, "Nami-ita" (waved polycarbonate plates), on which the green alga Ulvella lens Crouan frat. (Chaetophoraceae: Chaetophorales) was cultured, are used to promote larval settlement and metamorphosis of the sea urchin species Strongylocentrotus intermedius (A. Agassiz) and S. nudus (A. Agassiz). We investigated chemical inducer(s) for larval settlement and metamorphosis of these sea urchins with extracts of U. lens. Bioassay-guided separation of the methanol extract using a combination of column and thin-layer chromatography led to the isolation of several active compounds, the chemical structures of which were determined by spectral and chemical methods. These active compounds were identified as glycoglycerolipids, all comprising several molecular species: sulfoquinovosyl monoacylglycerols (SQMGs), sulfoquinovosyl diacylglycerols (SQDGs), monogalactosyl monoacylglycerols (MGMGs), monogalactosyl diacylglycerols (MGDGs), digalactosyl monoacylglycerols (DGMGs) and digalactosyl diacylglycerols (DGDGs). Among these glycolipids, SQMGs, MGMGs, MGDGs and DGMGs induced larval metamorphosis of the sea urchin S. intermedius. SQMGs and MGDGs induced larval metamorphosis at a concentration of 5 µg ml^-1, whereas SQDGs and DGDGs only induced larval settlement. These glycoglycerolipids are new congeners of chemical inducers to settlement and metamorphosis of planktonic larvae of sea urchins. The findings would provide a better understanding of larval settlement and metamorphosis in sea urchins.     

Changes in biochemical and fatty acid composition of the razor clam <Emphasis Type="Italic">Solen marginatus</Emphasis> (Solenidae: Bivalvia) during larval development

Aquaculture studies have revealed that polyunsaturated fatty acids are critical for maintaining substantial growth, survival and reproductive rates, and high food conversion efficiencies for a wide variety of marine and freshwater organisms. The aim of this study was to investigate the gross biochemical and fatty acid composition of both neutral and polar lipid compartments of the razor clam Solen marginatus throughout embryonic and larval development. High levels of stored reserves in S. marginatus eggs allow a short larval development, lasting only 8 days. The energy required for embryogenesis was obtained from stored proteins. During larval development from D-shaped veliger until settlement, protein, lipid, and carbohydrate reserves were indistinctly stored for metamorphosis. Although total lipids increased, fatty acids in both neutral and polar lipids decreased during embryonic development. The depots allow a short larval development in which settlement is reached with lower amounts of stored neutral and polar lipids than the contents found in the oocytes. Non-methylene-interrupted dienoic fatty acid levels were similar to those of some polyunsaturated fatty acids, with increasing percentages at the onset of metamorphosis. This study indicates that S. marginatus exhibits a different pattern in the use of gross biochemical and fatty acid reserves during larval development compared to other razor clam and bivalve species, mainly due to the large size of its eggs and the short larval development stage reported in this species.     

Effects of low oxygen concentrations on the hatching and viability of eggs of marine calanoid copepods

Hatching of the eggs of four species of copepods, Acartia tonsa, Labidocera aestiva, Tortanus discaudatus, and Centropages hamatus was suppressed at oxygen concentrations less than 0.02 ml O₂/l. When such eggs were subsequently exposed to normal oxygen concentrations, hatching varied among the species, indicating variability in the capacity of eggs to survive exposure to low oxygen. Incubation of eggs at low oxygen concentrations resulted in an increase in the duration of embryonic development. Experiments were conducted in closed systems and oxygen concentration was determined by thermal conductivity using gas chromatography.     

Metallothionein induction inMytilus edulis exposed to cadmium

The exposure of mussels,Mytilus edulis, collected from Whitsand Bay, southwest England, in August 1988, to sublethal concentrations of cadmium (400µg l^–1) for 65 d resulted in the induction of metallothionein (MT) synthesis in the soft tissues. In cadmium-exposed mussels, metallothionein concentrations, measured by differential pulse polarography, increased by a factor of three, from 2 to 3 mg g^–1 to a maximum of 9 mg g^–1 after 30 d. No significant changes could be detected in controls. Cadmium accumulated in the soft tissues of mussels correlated significantly with metallothionein concentrations and can be described by the relationship: MT (mg g^–1)=0.045 Cd (µg g^–1)+3.03 (r=0.803,P<0.001). Gel chromatography of heat-treated cytosolic extracts showed that the accumulated cadmium is bound principally to the newly formed metallothioneins. Copper and zinc were also analysed in the whole soft-tissues and in subcellular fractions of cadmium-exposed mussels. Although copper concentrations were not affected by cadmium-exposure, zinc levels were significantly reduced. The results demonstrate that the induction of metallothioneins inM. edulis is a quantifiable biological response to sublethal levels of cadmium exposure.