Mussel Hemolymph Research Articles

Oxidative effects of inorganic and organic contaminants on haemolymph of mussels

We applied a newly-established method in haemolymph of mussels, Mytilus galloprovincialis, exposed to different concentrations of heavy metals, such as zinc and cadmium and organic pollutants, such as PAHs and lindane, for the detection of total antioxidant capacity (TAC). The susceptibility of exposed mussels was increased in relation to oxidative stress induced by contaminants tested. Oxidative modifications of proteins were estimated by measuring protein carbonyl content (PCC) and malondialdehyde levels (MDA). For PCC measurement, a highly sensitive and accurate ELISA method, which requires only 5 µg of protein, was used. The significant increase of PCC and MDA in haemolymph of exposed mussels reinforces its role as biomarkers of oxidative stress. Significant correlation of TAC assay, PCC and MDA was conducted in order to evaluate the utility of PCC and TAC assay, used in the present study, as tools for determining oxidative effects of pollutants in mussels. The results reinforce the application of PCC method as useful tool for the determination of PCC alterations in haemolymph of mussels exposed to different levels of contaminants. In addition, the TAC method gives encouraging results, concerning its ability to predict antioxidant efficiency in haemolymph of mussels exposed to inorganic and organic contaminants.

Influence of β-glucans on the immune responses of carpet shell clam ( Ruditapes decussatus) and Mediterranean mussel ( Mytilus galloprovincialis)

The effects of β-glucans on several immune functions of carpet shell clam ( Ruditapes decussatus) and Mediterranean mussel ( Mytilus galloprovincialis) hemocytes were determined. Nitric oxide (NO) production increased significantly in β-glucan treated mussels and clams. In mussels, β-glucans increased by themselves the release of free oxygen radicals and also were able to enhance the phorbol 12-myristate 13-acetate (PMA) mediated effect on this hemocyte activity. However, high doses of β-glucans when combined with zymosan decreased this respiratory burst. In clams, hemolymph treated with several doses of β-glucans limited the growth of the three bacteria, Vibrio algynolyticus (strain TA15), Vibrio splendidus (strain TA2) and Escherichia coli (strain ATCC 13706). This modulation on the antibacterial activity, however, was not observed when mussel hemolymph was incubated with β-glucans. These results suggest that the immune responses of these animals can be up and down modulated by external stimuli and, although clams and mussels are both relatively closely related species, their behaviour concerning immune responses can be different.

High sequence variability of myticin transcripts in hemocytes of immune-stimulated mussels suggests ancient host–pathogen interactions

Small cationic antimicrobial peptides (AMPs) are host defense molecules detected in virtually all groups of organisms. To investigate the immune response mechanisms of Mytilus galloprovincialis, primary and suppression subtractive hybridization libraries were prepared from hemolymph of mussels injected with heat-inactivated bacteria or poly I:C, the latter mimicking viral infection. After DNA sequencing, sequence processing and similarity searching, a remarkable abundance of AMP mRNAs were identified. In detail, 25.9% and 32.4% AMP sequences from mussels infected with bacteria and 43.4% and 40.6% from mussels stimulated with poly I:C were detected by selective amplification of 180 differentially expressed genes and random sequencing of 967 cDNA clones, respectively. The 232 ESTs matching with myticin A and B ( Mytilus spp.) displayed considerable sequence variability and revealed a third cluster proposed here as myticin C. Phenetic analysis of the translated myticin ESTs yielded 74 and 25 variants of the precursor and active peptide, respectively, and confirmed the high polymorphism of the new form. Myticin C shows typical features of the CS αβ AMP family (eight-cysteine array and secretory signal peptide) as well as amino acid variation, mainly in the anionic C-terminal region. The sequencing of one intronic region from genomic DNA, allowed us to detect 13 variants in 9 individual mussels referring them to one gene only. In addition to hemolymph, myticin C transcripts were detected in various mussel tissues, oocytes and early larval stages. The striking sequence variability and expression levels of myticins in mussels confirm the fundamental role of these natural antibiotics in the ancient host–pathogen interplay of mutual inhibition, evasion and adaptation strategies.

A methodology for screening haemolymph of intertidal mussels, Mytilus edulis, using FT-IR spectroscopy as a tool for environmental assesment

Developing effective, rapid and inexpensive methods for monitoring and conserving aquatic resources is an important issue for environmental managers. This study focuses on Mytilus edulis, a keystone species of many coastal marine communities, which is frequently used as a biomonitor for a range of pollutants. Recent advances in post-genomic technologies have provided new methods of biochemical screening, and Fourier transform-infrared spectroscopy (FT-IR) is one such method that could enable bioindicator species to be used for environmental assessment. This paper develops a methodology to apply the FT-IR approach to marine intertidal M. edulis and addresses three methodological issues: First, the optimum physical location for biofluid sampling is examined (i.e. laboratory versus field). Secondly, the effects of transportation of frozen biofluid sampling from either the field-site or laboratory to the analytical facility are considered. Finally, the effect of repeated FT-IR measurements on collected M. edulis haemolymph samples is examined. From these results we suggest sampling haemolymph from M. edulis at the top of the shore prior to immediate snap-freezing in liquid nitrogen. Sample transportation can occur on ice for up to eight hours before storage at −80 °C. FT-IR measurements should occur within three months of collection and samples should not be used or thawed more than twice. We show how this method can be used to differentiate successfully between four different estuarine environments. Ultimately, through addressing these methodological questions, we provide a protocol to allow efficient sampling and FT-IR measurement of M. edulis as collected from the intertidal areas of rocky and muddy shores. We conclude that due to current monitoring needs presented by the European Water Framework Directive such an approach could prove to be an invaluable future tool for assessing coastal water quality.

Linker chains of the gigantic hemoglobin of the earthworm Lumbricus terrestris: Primary structures of linkers L2, L3, and L4 and analysis of the connectivity of the disulfide bonds in linker L1

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major kinds of globin chains: a, b, c, and d, present in equimolar proportions, and additional non-heme, non-globin scaffolding chains called linkers that are required for the calcium-dependent assembly of the full-sized molecule. The amino acid sequences of all four of the globin chains and one of the linkers (L1) have previously been determined. The amino acid sequences via cDNA of each of the three remaining linkers, L2, L3, and L4, have been determined so that the sequences of all constituent polypeptides of the hemoglobin are now known. Each linker has a highly conserved cysteine-rich segment of approximately 40 residues that is homologous with the seven ligand-binding repeats of the human low-density lipoprotein receptor (LDLR). Analysis of linker L1 shows that the connectivity of the three disulfide bonds is exactly the same as in the LDLR ligand-binding repeats. The presence of a calcium-binding site comprising one glutamyl and three aspartyl residues in both the LDLR repeats and in the linkers supports the suggestion that calcium is required for the folding and disulfide connectivity of the linkers as in the LDLR repeats. Linker L2 is markedly heterogeneous and contains unusual glycine-rich sequences near the NH2-terminus and a polar zipper-like sequence with imperfect repeats of Asp-Asp-His at the carboxyl terminus. Similar Asp-Asp-His repeats have been found in a protein homologous to superoxide dismutase in the hemolymph of certain mussels. These repeats may function as metal-binding sites.

Evaluation of methods for improved detection of Cryptosporidium spp. in mussels ( Mytilus californianus)

Bivalve molluscs concentrate Cryptosporidium oocysts from fecal-contaminated aquatic environments and are therefore useful in monitoring water quality. A real-time TaqMan polymerase chain reaction (PCR) system was developed to allow for large scale quantitative detection of Cryptosporidium spp. in mussels ( Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) assays, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a 1 log 10 improvement when samples were first processed by IMS. For tank exposed mussels, TaqMan and conventional PCR methods detected C. parvum in < 5% of hemolymph samples. No gill washings from these same mussels tested positive by TaqMan PCR or DFA analysis even with IMS concentration. All methods detected the highest prevalence of C. parvum-positive samples in digestive gland tissues of exposed mussels. In conclusion, the most sensitive method for the detection of C. parvum in oocyst-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive. TaqMan PCR was comparable to conventional PCR for detection of C. parvum oocysts in mussels and additionally allowed for automated testing, high throughput, and semi-quantitative results.

Encystment in vitro of the Cercariae Himasthla elongata (Trematoda: Echinostomatidae)

Trigger and toxic effects of Mytilus edulis (Bivalvia) hemolymph on encystment of cercariae Himasthla elongata obtained from infestated Littorina littorea (Prosobranchia) was evaluated as a result of 24-h experiments in vitro. The contact of H. elongata larvae with the whole hemolymph or mussel acellular plasma led to an intensive transformation of cercariae into metacercariae. In both tested media, the cercariae had to complete the encystment phase as fast as for 2 h, otherwise the risk of the larvae injury by humoral and cellular components of the mussel hemolymph would increase dramatically. The cercaria mortality after 24 h in the whole hemolymph was twice higher than in plasma (40% and 20%, respectively) and much higher than in the control medium (sea water). Both toxic and trigger effects of plasma was revealed to depend on its concentration, with the maximal larva mortality in the undiluted medium and with the highest number of successful transformations in the medium diluted more than 4 times. There is shown both the strong individual variability of toxicity of the individual mussel hemolymph for cercariae and the variability of the resistance to the toxic factors of the cercariae obtained from various L. littorea individuals. These experiments not only offer a method of the massive encystment of H. elongata cercariae, but also propose a perspective model for the study of the systemic defensive response of Bivalvia to invasion of multicellular parasite.

Mercury- and copper-induced lysosomal membrane destabilisation depends on [Ca 2+] i dependent phospholipase A2 activation

Heavy metals are environmental pollutants able to produce different cellular effects, such as an alteration of Ca 2+ homeostasis and lysosomal membrane destabilisation. The latter is one of the most used stress indices in biomonitoring programs. Recently, it has been demonstrated that cytosolic calcium increase can modulate lysosomal membrane destabilisation via activation of Ca 2+-dependent phospholipase A2 (cPLA2). The aim of this work was to investigate the possible involvement of Ca 2+-activated PLA2 in lysosomal membrane destabilisation induced by heavy metals in mussel haemolymph cells. We have studied the effects of Hg 2+ and Cu 2+ on free cytosolic calcium using Fura2/AM-loaded cells and lysosomal membrane destabilisation using neutral red (NR) staining. Hg 2+ induced a [Ca 2+] i rise from 100 to 780 nM in 30 min, and a lysosome destaining of 70% after 60 min that indicates destabilisation of lysosomal membranes. Both effects were reduced in a Ca 2+-free medium, suggesting a cause-effect relationship. Exposure to Cu 2+ produced the same effects, but with an intensity of about 50% respect to Hg 2+. Metal-induced lysosomal destabilisation was also reduced in cells pre-exposed to a specific Ca 2+-dependent cPLA2 inhibitor (AACOCF3). Conversely, haemocyte pretreatment with a Ca 2+-independent PLA2 inhibitor (bromoenol-lactone (BEL)) did not prevent the destabilizing effect of heavy metals on lysosomes. Exposure to heavy metals also produced an increase in lysosomal volume of 1.8–2-folds, that was prevented by pre-incubation with AACOCF3 but not with BEL. These data indicate an involvement of cPLA2 in lysosomal membrane destabilisation induced by heavy metals.

Role for mannose-sensitive hemagglutinin in promoting interactions between Vibrio cholerae El Tor and mussel hemolymph.

The role of mannose-sensitive hemagglutinin (MSHA) in Vibrio cholerae O1 El Tor interactions with hemolymph of the mussel Mytilus galloprovincialis was studied. Bacterial adherence to and association with hemocytes were evaluated at 4 and 18 degrees C, respectively. In hemolymph serum, the wild-type strain N16961 adhered to and associated with hemocytes about twofold more efficiently than its mutant lacking MSHA. In artificial seawater (ASW), no significant differences between the two strains were observed. N16961 was also more sensitive to hemocyte bactericidal activity than its MSHA mutant; in fact, the percentages of killed bacteria after 120 min of incubation were 60 and 34%, respectively. The addition of D-mannose abolished the serum-mediated increase in adherence, association, and sensitivity to killing of the wild-type strain without affecting the interactions of the mutant. A similar increase in N16961 adherence to hemocytes was observed when serum was adsorbed with MSHA-deficient bacteria. In contrast, serum adsorbed with either wild-type V. cholerae El Tor or wild-type Escherichia coli carrying type 1 fimbriae was no longer able to increase adherence of N16961 to hemocytes. The results indicate that hemolymph-soluble factors are involved in interactions between hemocytes and mannose-sensitive adhesins.

Biomonitoring: Integration of biological end-points into chemical monitoring

Biomonitoring is currently performed at two levels, assessing exposure to pollutants and effects monitoring by bioassays. As an example for the first approach, vitellogenin (VTG) in male fish of Abramis brama as an endpoint for estrogen exposure is discussed. However, similar changes of VTG or VTG-like proteins in the hemolymph of mussels could not be detected. Enzyme-linked receptor assays for monitoring estrogenic effects at the molecular level serve as an example for the second category. Applications of the enzyme-linked receptor assay (ELRA) developed in our laboratory are presented. Detection limits of 0.02 μg/l 17β-estradiol were recently achieved with the chemiluminescent format. Although effect monitoring provides information in terms of toxicity equivalents, it is not possible to relate the signals to specific pollutants and their concentrations. For this purpose, chemical analysis is required. New approaches are reported for the direct coupling of bioassays and chemical analysis. This concept is defined as bioresponse-linked instrumental analysis. It combines biomolecular recognition, initiating a biological effect, and chemical analysis. In addition to the classical bioanalytical approaches, new strategies in genomics and proteomics have been developed. This may lead to multimarker approaches opening this area to environmental analytics.

Vitellogenin levels in mussel hemolymph—a suitable biomarker for the exposure to estrogens?

Increased vitellogenin (vtg) levels in the blood of male fish are frequently used as an indicator of estrogenic exposure. Similar responses are expected for mussels, where the concentration of vtg-like proteins has been reported to depend on estrogens. To verify the role of hemolymph during vitellogenesis of mussels, the saltwater mussel Mytilus edulis and the freshwater mussel Anodonta cygnea were exposed to 17β-estradiol (E 2) and wastewater treatment plant effluents, known for their estrogenic potential. Gel electrophoresis did not reveal any significant induction (or repression) of plasma proteins compared to control plasma. Our results do not support the hypothesis that mussel hemolymph is a carrier of estrogen-dependent major egg-yolk precursors (vtg-like proteins). However, additional information on a 35±2-kDa hemolymph protein, previously reported to bind heavy metals, was obtained by high-resolution two-dimensional electrophoresis. It was resolved in a cluster of single proteins with properties that match the characteristics of a previously reported histidine-rich glycoprotein.

Autometallographic localization of heavy metals in resin sections, cryosections and isolated cells of the mussel Mytilus galloprovincialis (L.).

In the present study, autometallography (AMG) has been performed on resin-embedded tissue sections, cryosections, as well as on isolated cells from the digestive gland, the gills and the haemolymph of mussels (Mytilus galloprovincialis) collected from the field. The AMG reaction was more intense in cryosections and isolated cells compared to resin sections. This intense AMG reaction found in cryosections and in isolated cells and the time effectiveness of the procedures enhances their use in a variety of studies, such as biomonitoring, even if small amount of metals are to be detected. In addition, the preparation of isolated cells does not require specific instruments or qualified personnel and, thus, the use of isolated cells is encouraging for AMG applications, although further laboratory investigation is required. On the other hand, the use of resin-embedded tissue sections for AMG applications, even though it is complicated and time consuming, leads to a high preservation of structural morphology and allows the exact localization of metals in discrete cellular compartments.

Toxicity of domoic acid in the marine mussel Mytilus edulis

The neurotoxic, immunotoxic and genotoxic effects of domoic acid (DA) on the blue mussel Mytilus edulis were investigated by biomarkers, acethylcholinesterase (ChE) activity in gills, DNA fragmentation in digestive glands, vitality and phagocytosis activity of haemocytes in haemolymph of mussels. After intra muscular injection of DA at the concentrations ranging from 1–500 ng/g body weight (bw), no neurotoxic effect was detected within incubation times of 48 h and 7 d. The vitality of haemocytes remained in all mussels at the level of control samples within 48 h, and increased significantly after 7 d ( P<0.05). At DA concentrations ranging from 1 to 100 ng/g bw haemocytes suggested a great phagocytosis activity, but no alteration in their number by both incubation times. By increasing DA concentration of 500 ng/g bw, the number of haemocytes doubled in 48 h without any change in phagocytosis activity. Primary DNA lesions in digestive glands of all injected mussels were determined in acute phase of poisoning within 48 h, and rapidly repaired after 7 d of incubation.

Evidence of coprostanol estrogenicity to the freshwater mussel Elliptio complanata

Coprostanol (5β(H)-cholestan-3βol) is a reduced metabolite of cholesterol produced by micro-organisms found in the intestinal tract of mammals. This substance abounds in urban effluents and is accumulated by organisms living in the vicinity of municipal effluent outfalls. In an earlier study, freshwater mussels exposed to contaminated river water for 62 days accumulated large quantities of coprostanol (Cop) in their soft tissues (16 μg/g dry wt.). Moreover, these mussels were found to have elevated levels of vitellin in their hemolymphs, suggesting estrogenic effects. Although municipal wastewaters are known to contain other estrogenic compounds capable of inducing Vn synthesis in mussels, the estrogenic potential of coprostanol was singled out for examination. To this end, mussels were first injected with concentrations of coprostanol via the abductor muscle route, and allowed to stand in aerated water for 72 h at 15°C. The levels of Vn in mussel hemolymph were assayed using the organic alkali-labile phosphate method. A competitive estradiol-binding assay was then devised to measure the ability of coprostanol to compete in the binding of fluorescein-labeled estradiol-albumin to cytosolic proteins. Coprostanol partially reversed the binding of labeled estradiol-albumin to cytosolic proteins with an EC 50 of 1 mM. In addition, injections of coprostanol and estradiol-17β led to increased levels of vitellins in the hemolymph of treated mussels. Moreover, incubation of cop in gonad homogenate extracts in the presence of NADPH led to the formation of two compounds, as determined by high-performance thin-layer chromatography. One of these compounds appears to be the C17 oxidation product of coprostanol, whose polarity is similar to that of estradiol. The results present evidence that coprostanol is estrogenic to freshwater mussels.

Surface interactions between Escherichia coli and hemocytes of the Mediterranean mussel Mytilus galloprovincialis lam. leading to efficient bacterial clearance.

The role of type 1 fimbriae in the interactions between Escherichia coli and Mytilus galloprovincialis Lam. hemocytes was evaluated. The association of fimbriated strain MG155 with hemocyte monolayers at 18 degrees C was 1.5- and 3- to 4-fold greater than the association of unfimbriated mutant AAEC072 in artificial seawater and in hemolymph serum, respectively. Such differences were apparently due to different adhesive properties since MG155 adhered more efficiently than AAEC072 when hemocytes were incubated at 4 degrees C to inhibit the internalization process. Hemolymph serum increased both association and adherence of MG155 two- to threefold but did not affect association and adherence of AAEC072. MG155 was also 1.5- to 1.7-fold more sensitive to killing by hemocytes than AAEC072, as evaluated by the number of culturable bacteria after 60 and 120 min of incubation. The role of type 1 fimbriae in MG155 interactions with hemocytes was confirmed by the inhibitory effect of D-mannose. In in vivo experiments MG155 cells were cleared from circulating hemolymph more rapidly than AAEC072 cells were cleared. These results confirm that surface properties are crucial in influencing bacterial persistence and survival within mussel hemolymph.

Occurrence of compounds estrogenic to freshwater mussels in surface waters in an urban area.

Estrogens play a major role in the sexual differentiation, gonad development, and oocyte growth of most oviparous organisms. They also stimulate vitellogenesis, the formation of high-density glycolipophosphoprotein that serves as an energy source for the developing embryo. Surface waters from the St. Lawrence River, obtained in the vicinity of an urban area (Montreal, Quebec, Canada), were studied with respect to their estrogenic potential to the freshwater mussel Elliptio complanata. Estrogenicity was measured in water extracts by means of a competitive assay of estradiol binding to cytosolic proteins and by the vitellin-inducing ability of mussel hemolymph following direct extract injection. Surface-water samples drawn downstream of a municipal outfall plume and in a river draining a large farming and agricultural area had high levels of total and fecal coliform bacteria. High levels of estrogen competitors were also found and were able to induce vitellins in injected mussels. Moreover, the estrogen-competing potential of the extracts was found to be significantly correlated with total and fecal coliform bacteria (R = 0.9, p < 0.01) and with the levels of vitellins in the hemolymph (R = 0.62, p = 0.03). The results indicate that water samples drawn from within the municipal effluent plume and from a river draining an agricultural area are estrogenic to freshwater mussels. Thus, the environmental inputs of estrogens are likely to be associated with human sewage and pesticide products.

In vitrostudy of phagocytic ability ofMytilus galloprovincialisLmk. haemocytes

In vitroassays were performed to assess the phagocytic ability of mussel haemolymph cell types. Among the haemocyte types, the granulocytes showed an important phagocytic activity whereas the hyalinocytes did not, thus indicating that mussel haemocytes are functionally heterogeneous. Phagocytic activity of acidophilic granulocytes was higher than basophilic ones. Ultrastructural study showed thatVibrioP1 cells were internalised in phagosomes and then digested. As a result of the digestive process, concentric lamellae and other degraded materials were produced inside the phagosomes, and non degraded materials gave rise to residual bodies which could be discharged from the cells. Similar events were observed in the phagocytic process of zymosan particles. The study of influence of temperature on phagocytosis activity revealed that the percentage of phagocytic cells was lower at 10°C than at 20°C and 30°C. Phagocytosis activity was higher in the assays performed at 15ppt than those at 25ppt and 36ppt. No significant differences on percentage of phagocytic cells were found between young and adult mussels. Generation of superoxide anion by haemocytes using zymosan as a phagocytosis stimulant was demonstrated through the nitroblue tetrazolium reduction test.

Characterization and immunocytochemical localization of actin and fibronectin in haemocytes of the mussel Mytilus galloprovincialis.

Cell-extracellular matrix interactions are recognized to be important for human leucocyte functions, including chemotaxis and phagocytosis. These activities depend on a reorganization of the microfilament actin (F-actin) promoted by fibronectin, one of the major components of extracellular matrices. Although invertebrate haemocytes are, in many aspects, similar to the human granulocyte-monocyte-macrophage cell lineage, actin and fibronectin have not been well studied in these cells. Consequently, the characterization and structural organization of actin and fibronectin in mussel (Mytilus galloprovincialis) haemocytes was investigated using Western blotting analysis, indirect immunofluorescence and immunoelectron microscopy. Actin was immunocharacterized by an anti-total actin monoclonal antibody. Fibronectin was immunocharacterized by an autologous polyclonal antiserum directed against the protein of mussel haemolymph. Actin was mainly localized along the peripheral cytoplasm of the haemocyte. The distribution of the F-actin microfilaments was assayed with Rhodamine-labelled phalloidin. F-actin was associated mainly with stress-fibres of spreading haemocytes and with microspikes at the adhesion sites. The labelling by the anti-fibronectin antiserum of the haemocyte rough endoplasmic reticulum vesicles, revealed by immunoelectron microscopy, suggests that these cells are involved in fibronectin biosynthesis. Gold particles were also present along the outer surfaces of the cell plasma membrane and its protrusions. Mussel fibronectin was localized immunohistochemically at the adhesion sites and in the extracellular matrix fibrils. The relationships between fibronectin and the actin cytoskeleton in Mytilus galloprovincialis haemocytes are discussed.

Present knowledge on the molecular basis of cytotoxicity, antibacterial activity and stress response in marine bivalves

Abstract Mussel hemolymph possessed naturally occurring cytotoxic activity. Vertebrate erythrocytes, mouse tumor cell line and parasite Bonamia were killed, but not bacteria. Two step chromatography revealed a complex multimeric protein of 320 kDa, acting through a polymerization process on the target cell membrane. Of naive animals from the two oyster species Crassostrea gigas and Ostrea eclulis, a low proportion of plasmas and hemocyte lysates presented naturally occurring antibacterial activity. Induction assays by injection of saline solution or bacterial suspensions resulted in increased activity in virtually all oysters. Identification of active polypeptides included acidic solubility followed by reverse phase chroma‐tographies. Oyster hemocytes responded to heat‐shock by the synthesis of particular proteins. Treated in the same way, the protozoan parasite Perkinsus marinus also synthesised long‐lasting 70 to 30 kDa proteins which are different from the oyster ones. The question arises of the biolog...

Effects of Hg2+ and Cu2+ on the cytosolic Ca2+ level in molluscan blood cells evaluated by confocal microscopy and spectrofluorimetry

In the present work the effect of Hg2+ and Cu2+ on the level of cytosolic Ca2+ in mussel (Mytilus edulis L.) haemolymph cells were investigated by confocal microscopy and spectrofluorimetry utilizing the fluorescent dye Fluo3. In the blood cells of marine molluscs, exposure to Cu2+ and Hg2+ in the nanomolar and micromolar range causes a time-and concentration-dependent increase in the cytosolic Ca2+ level. Both the presence of a low-calcium containing medium and pretreatment of the cells with the channel blocker Verapamil greatly reduced the effects of higher (50 μM) Hg2+ concentrations, this indicating that Hg2+ enhances the influx of extracellular Ca2+ partly through activation of voltage-dependent Ca2+ channels. Low concentrations of Hg2+ (1 μM) and also of Cu2+ (0.5 μM), an “essential” element, were able to induce a sustained increase in cytosolic Ca2+, which was not affected either by Verapamil pretreatment or by lowering the extracellular calcium concentration. These data indicate that in mussel haemocytes heavy metal cations impair Ca2+ homeostasis not only by affecting Ca2+ channels, but also by interfering with other mechanisms of calcium transport across cellular membranes, such as the Ca2+-ATPases. The resulting increase in cytosolic Ca2+ could activate Ca-dependent processes which may be involved in many of the biochemical and physiological alterations observed in the cells of metal-exposed mussels. Specimens used in these experiments were collected from the river Linker near Plymouth, U.K. in June 1991.