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Immunolocalization of yessotoxins in the mussel Mytilus galloprovincialis
Toxicon 41 (2003) 967–970 www.elsevier.com/locate/toxicon
Immunolocalization of yessotoxins in the mussel Mytilus galloprovincialis A. Franchinia, A. Milandrib, R. Polettib, E. Ottaviania,* a
Department of Animal Biology, University of Modena and Reggio Emilia, Via Campi 213/D, Modena 41100, Italy b Centro Ricerche Marine, Cesenatico, Italy Received 29 November 2002; accepted 12 February 2003
Abstract This study reports, for the first time, immunocytochemical evidence of the distribution of algal yessotoxins (YTXs) in the mussel Mytilus galloprovincialis. Immunopositivity to YTXs was found in immunocytes and in the digestive gland. With regards the gland, the positivity was mainly present in the lumen of both tubules and ducts. No YTXs were detected in the gonads, while the presence of toxins in the gills cannot be excluded. The data are supported by both HPLC analysis and functional assays. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Mussel; Mytilus galloprovincialis; Adriatic sea; Yessotoxins; Immunocytochemistry
1. Introduction The algal yessotoxin (YTX) and its analogues could be ingested by shellfish such as scallops and mussels. It has been demonstrated that YTX provokes cardiotoxic effects in mice (Terao et al., 1990; Ogino et al., 1997; Aune et al., 2002). Even if to date there have been no reports of the classical effects of intoxication in humans consuming contaminated shellfish, the substantial increase in the distribution of YTXs along the coasts of the world and their repeated appearance could be a major problem for the community. At present, the detection of YTXs is performed by a fluorescent HPLC method (Yasumoto and Takizawa, 1997) and, recently, by ELISA essay (Briggs et al., 2002). However, these quantitative approaches show a limitation, i.e. they do not reveal a precise distribution of the toxins in the molluscan tissue. In the present investigation we report, for the first time, immunocytochemical evidence of the distribution of YTXs in the mussel Mytilus galloprovincialis * Corresponding author. Tel.: þ39-59-205-5536; fax: þ 39-59205-5548. E-mail address: [email protected] (E. Ottaviani).
in order to gain further information about biological action of these toxins.
2. Materials and methods 2.1. Samples Contaminated specimens of the mussel M. galloprovincialis Lmk. were collected from shellfish aquaculture in the Adriatic sea two miles far from Cesenatico (FC, Italy), while non-contaminated animals were obtained from rocks in the Adriatic sea around Cattolica (RN, Italy). 2.2. HPLC method and mouse bioassay The presence of YTXs was detected on extracts from edible part of contaminated samples using the HPLC method with fluorescent detector following the procedure of Yasumoto and Takizawa (1997). The mouse bioassay was performed by i.p. administration of extracts in Swiss mice according to the method proposed by Yasumoto (2002). Both the tests were also performed on control mussels.
0041-0101/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0041-0101(03)00039-4
968
A. Franchini et al. / Toxicon 41 (2003) 967–970
2.3. Immunocytochemical procedure Specimens of digestive gland, gonad and gill from contaminated and non-contaminated animals fixed in Bouin’s mixture or in neutralized 10% formalin and embedded in paraffin were used. Both unfixed and fixed (10% formalin vapours) frozen tissue sections, and unfixed immunocytes were used. Immunocytes were obtained by cytocentrifugation of the hemolymph on a slide at 800 rpm for 5 min followed by air-drying. The hemolymph was collected from the posterior adductor muscle using a 2 ml syringe. The immunocytochemical assay was performed using a polyclonal antibody recognizing antigenic determinants of the YTX and its analogues (Briggs et al., 2002). Before incubation with the primary antibody (1:1000; 1:2000) overnight at 4 8C, the sections were treated as follows: inhibition of endogenous peroxidase with 0.3% H2O2 in methanol for 15 min at room temperature (RT); rinsing in running tap water for 10 min and in phosphate-buffered saline (PBS) (3 £ 5 min); incubation in normal serum (Vector Lab., USA) (1:5) for 30 min at RT. After incubation with the primary antibody and washing in PBS (3 £ 5 min), the sections were incubated in biotinylated, anti-sheep IgG immunoglobulins (Vector Lab.,) (1:200) for 30 min, washed in PBS (3 £ 5 min), incubated in avidin –biotin – peroxidase complex (ABC) (Vector Lab.,) (1:100) for 30 min at RT and washed in PBS (3 £ 10 min). Peroxidase activity was demonstrated by dissolving a tablet of 3,30 -diaminobenzidine tetrahydrochloride (Sigma, USA) (10 mg) in 40 ml of McIlvane buffer at pH 5.5 and adding 5 ml of H2O2. Nuclei were counterstained with Mayer’s hematoxylin. The antibody was also tested on sections from non-contaminated samples. Controls of immunocytochemical reactions were performed by substituting the primary antibody with non-immune sera.
Table 1 Values obtained by HPLC in two extracts of mussel tissue Samples
45-OH-YTX (mg/g e.p.)
YTX (mg/g e.p.)
Total YTXs (mg/g e.p.)
1 2 Mean
1.490 1.234 1.362
1.720 1.557 1.639
3.210 2.791 3.001
45-Hydroxyyessotoxin ¼ 45-OH-YTX; yessotoxin ¼ YTX; e.p. ¼ edible part.
animals did not contain YTXs and the mouse tests performed with extracts from these mussels gave a negative response. The immunocytochemical investigation performed on contaminated and non-contaminated mussels showed immunoreactivity to the antibody tested in the cytoplasm of the immunocytes of the contaminated animals (Fig. 1(1)), in contrast with the negative response of the non-contaminated specimens (Fig. 1(2)). The immunoreactivity to YTXs was mainly localized in the digestive gland, and in particular in the lumen of tubules and ducts (Fig. 1(3– 5)). The cytoplasm of the two cell types in the epithelium of gland tubules, i.e. the digestive and basophilic cells, was negative. No YTXs were found in the gonads (Fig. 1(6)), while the presence of toxins in the gills could not be excluded. Indeed, the background of the immunocytochemical reaction made it impossible to reveal any small amounts of toxins that may have been present. No positivity to YTXs was observed in tissue from non-contaminated mussels (Fig. 1(7)), and the control reactions performed with non-immune sera were always negative (Fig. 1(8)). Furthermore, it should be underlined that immunoreactivity was not detected in paraffin fixed or in frozen formalin vapour fixed sections, but only in frozen unfixed tissue samples.
3. Results 4. Discussion HPLC detection revealed the presence of 45-hydroxyyessotoxin (45-OH-YTX) and YTX in the contaminated mussels, as reported in the Table 1. Furthermore, the functional assays demonstrated that the extracts from contaminated mussels were able to provoke neurotoxic effects and the mice died within 24 h. As expected, control
HPLC analysis reveals that YTXs are more accumulated than 45-OH-YTX in contaminated mussels, and their toxicity is evidenced by the mouse test. As far as the tissue distribution of YTXs is concerned, the immunocytochemical investigation performed on
Fig. 1. Immunoreactivity to YTXs is observed in the cytoplasm of immunocytes from contaminated mussels (1), in contrast to the negative response found in immunocytes from non-contaminated specimens (2). Bar ¼ 10 mm. Immunocytochemical reaction to YTXs on unfixed frozen sections of digestive gland from contaminated mussels shows the positivity in the gland tubule (t) and duct (d) lumens and no reaction in the cytoplasm of the gland epithelial cells. Residual bodies are present in the lumen of the tubule (arrows) (3, bar ¼ 50 mm; 4, 5, bar ¼ 10 mm). Immunocytochemical reaction to YTXs on sections of digestive gland from non-contaminated mussels shows an overall negative response (6). Sections of gonad from a contaminated male mussel (7) and the control of the reaction (8, see a section of digestive gland from contaminated mussels) are also negative. Counterstaining was performed with hematoxylin. (6, bar ¼ 50 mm; 7, 8, bar ¼ 10 mm).
A. Franchini et al. / Toxicon 41 (2003) 967–970
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970
A. Franchini et al. / Toxicon 41 (2003) 967–970
contaminated and non-contaminated mussels shows that the main targets are the immunocytes and the digestive gland. In the circulating hemolymph of mussels only one cell type in two different stages, young or old, is present. In spite of differences in morphology and number, as a consequence of aging, they show similar function, i.e. cell motility, chemotaxis and phagocytosis, and express common signal molecules (Ottaviani et al., 1998). Regarding the digestive gland, two cell types are reported in tubule organ: the pyramidal basophilic secretory cell and the columnar acidophilic lysosome-rich digestive cell (Owen, 1972; Cajaraville et al., 1995). The latter cell type is involved in the uptake and intracellular digestion of food materials and undergoes a sequence of morphological changes during the digestive cycle terminating in the breakdown of the apical cell region that is pinched off to form fragmentation spherules with digestion waste products (Morton, 1983). Given its implication in detoxification processes and digestive functions, the digestive gland is considered a model for ecotoxicological studies. It has been demonstrated that metals and organic pollutants induce structural changes in the major compartments of digestive cells, i.e. the size and number of lysosomes (Moore, 1988; Marigo´mez et al., 1996). The concentration of immunoreactivity to YTXs mainly in gland tubule lumens suggests that algal toxins are accumulated together with aggregates of material digested by lysosomal action as residual bodies. From the location of immunoreactivity in the lumen of both tubules and ducts, we surmize that after the extracellular digestive phase of the ingested alga in the stomach, the toxins reach the digestive tubules and are then discharged back unaffected into the stomach. The lack of immunoreactivity in the cytoplasm of the digestive cells suggests that the toxins might be discharged, not bound for instance, to cytoplasmic proteins, as seen in metallic pollutants (Frankenne et al., 1980; Viarengo et al., 1981).
Acknowledgements The authors are very grateful to Dr N. Towers and Dr L. Briggs (AgResearch Ruakura, Hamilton, New Zealand) for providing the antibody against YTXs. This work was supported by MIUR (Italy) grant to E.O.
References Aune, T., Sørby, R., Yasumoto, T., Ramstad, H., Landsverk, T., 2002. Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice. Toxicon 40, 77–82. Briggs, L., Fitzgerald, J., Garthwaite, L., Miles, C., Garthwaite, I., Ross, K., Towers, N., Samdal, I., Aasen, J., Torgersen, T., Aune, T., Peterson, D., 2002. Immunoassays for the analysis of yessotoxins. Fourth International Conference on Molluscan Shellfish Safety, Santiago de Compostela, Spain Cajaraville, M.P., Pal, S.G., Robledo, Y., 1995. Light and electron microscopical localization of lysosomal acid hydrolases in bivalve haemocytes by enzyme cytochemistry. Acta Histochem. Cytochem. 28, 409 –416. Frankenne, F., Noe¨l-Lambot, F., Disteche, A., 1980. Isolation and characterization of metallothioneins from cadmium loaded mussel Mytilus edulis. Comp. Biochem. Physiol. 66C, 179–182. Marigo´mez, I., Orbea, A., Olabarrieta, I., Etxeberria, M., Cajaraville, M.P., 1996. Structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress in mussel-watch programmes. Comp. Biochem. Physiol. 113C, 291 –297. Moore, M.N., 1988. Cytochemical responses of the lysosomal system and NADPH–ferrihemoprotein reductase in molluscan digestive cells to environmental and experimental exposure to xenobiotics. Mar. Ecol. Prog. Ser. 46, 7–15. Morton, B., 1983. Feeding and digestion in Bivalvia. In: Saleuddin, A.S.M., Wilbur, K.M. (Eds.), The Mollusca, Physiology Part 2, vol. 5. Academic Press, New York, pp. 65–147. Ogino, H., Kumagi, M., Yasumoto, T., 1997. Toxicological evaluation of yessotoxin. Nat. Toxins 5, 255 –259. Ottaviani, E., Franchini, A., Barbieri, D., Kletsas, D., 1998. Comparative and morphofunctional studies on Mytilus galloprovincialis hemocytes: presence of two aging-related hemocyte stages. Ital. J. Zool. 65, 349 –354. Owen, G., 1972. Lysosomes, peroxisomes and bivalves. Sci. Prog. 60, 299–318. Terao, K., Ito, E., Oarada, M., Murata, M., Yasumoto, T., 1990. Histopathological studies on experimental marine toxin poisoning-5. The effects in mice of yessotoxin isolated from Patinopecten yessoensis and of a desulfated derivative. Toxicon 9, 1095–1104. Viarengo, A., Pertica, M., Mancinelli, G., Palmero, S., Zanicchi, G., Orunesu, M., 1981. Synthesis of Cu-binding proteins in different tissues of mussels exposed to the metal. Mar. Pollut. Bull. 12, 347 –350. Yasumoto, T., 2002. European commission protocol based on that of Yasumoto T., Murata, M., Oshima, Y., Matsumoto, C.K., Clark, J., 1984. Diarrhetic shellfish poisoning. In: Ragelis, E.P., (Ed.), Seafood Toxins, ACS Symposium Series 262, Ameriacn Chemical Society, Washington, pp. 207–214. Yasumoto, T., Takizawa, A., 1997. Fluorometric measurement of yessotoxins in shellfish by high-pressure liquid chromatography. Biosci. Biotechnol. Biochem. 61, 1775–1777.
Immunolocalization of yessotoxins in the mussel Mytilus galloprovincialis A. Franchinia, A. Milandrib, R. Polettib, E. Ottaviania,* a
Department of Animal Biology, University of Modena and Reggio Emilia, Via Campi 213/D, Modena 41100, Italy b Centro Ricerche Marine, Cesenatico, Italy Received 29 November 2002; accepted 12 February 2003
Abstract This study reports, for the first time, immunocytochemical evidence of the distribution of algal yessotoxins (YTXs) in the mussel Mytilus galloprovincialis. Immunopositivity to YTXs was found in immunocytes and in the digestive gland. With regards the gland, the positivity was mainly present in the lumen of both tubules and ducts. No YTXs were detected in the gonads, while the presence of toxins in the gills cannot be excluded. The data are supported by both HPLC analysis and functional assays. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Mussel; Mytilus galloprovincialis; Adriatic sea; Yessotoxins; Immunocytochemistry
1. Introduction The algal yessotoxin (YTX) and its analogues could be ingested by shellfish such as scallops and mussels. It has been demonstrated that YTX provokes cardiotoxic effects in mice (Terao et al., 1990; Ogino et al., 1997; Aune et al., 2002). Even if to date there have been no reports of the classical effects of intoxication in humans consuming contaminated shellfish, the substantial increase in the distribution of YTXs along the coasts of the world and their repeated appearance could be a major problem for the community. At present, the detection of YTXs is performed by a fluorescent HPLC method (Yasumoto and Takizawa, 1997) and, recently, by ELISA essay (Briggs et al., 2002). However, these quantitative approaches show a limitation, i.e. they do not reveal a precise distribution of the toxins in the molluscan tissue. In the present investigation we report, for the first time, immunocytochemical evidence of the distribution of YTXs in the mussel Mytilus galloprovincialis * Corresponding author. Tel.: þ39-59-205-5536; fax: þ 39-59205-5548. E-mail address: [email protected] (E. Ottaviani).
in order to gain further information about biological action of these toxins.
2. Materials and methods 2.1. Samples Contaminated specimens of the mussel M. galloprovincialis Lmk. were collected from shellfish aquaculture in the Adriatic sea two miles far from Cesenatico (FC, Italy), while non-contaminated animals were obtained from rocks in the Adriatic sea around Cattolica (RN, Italy). 2.2. HPLC method and mouse bioassay The presence of YTXs was detected on extracts from edible part of contaminated samples using the HPLC method with fluorescent detector following the procedure of Yasumoto and Takizawa (1997). The mouse bioassay was performed by i.p. administration of extracts in Swiss mice according to the method proposed by Yasumoto (2002). Both the tests were also performed on control mussels.
0041-0101/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0041-0101(03)00039-4
968
A. Franchini et al. / Toxicon 41 (2003) 967–970
2.3. Immunocytochemical procedure Specimens of digestive gland, gonad and gill from contaminated and non-contaminated animals fixed in Bouin’s mixture or in neutralized 10% formalin and embedded in paraffin were used. Both unfixed and fixed (10% formalin vapours) frozen tissue sections, and unfixed immunocytes were used. Immunocytes were obtained by cytocentrifugation of the hemolymph on a slide at 800 rpm for 5 min followed by air-drying. The hemolymph was collected from the posterior adductor muscle using a 2 ml syringe. The immunocytochemical assay was performed using a polyclonal antibody recognizing antigenic determinants of the YTX and its analogues (Briggs et al., 2002). Before incubation with the primary antibody (1:1000; 1:2000) overnight at 4 8C, the sections were treated as follows: inhibition of endogenous peroxidase with 0.3% H2O2 in methanol for 15 min at room temperature (RT); rinsing in running tap water for 10 min and in phosphate-buffered saline (PBS) (3 £ 5 min); incubation in normal serum (Vector Lab., USA) (1:5) for 30 min at RT. After incubation with the primary antibody and washing in PBS (3 £ 5 min), the sections were incubated in biotinylated, anti-sheep IgG immunoglobulins (Vector Lab.,) (1:200) for 30 min, washed in PBS (3 £ 5 min), incubated in avidin –biotin – peroxidase complex (ABC) (Vector Lab.,) (1:100) for 30 min at RT and washed in PBS (3 £ 10 min). Peroxidase activity was demonstrated by dissolving a tablet of 3,30 -diaminobenzidine tetrahydrochloride (Sigma, USA) (10 mg) in 40 ml of McIlvane buffer at pH 5.5 and adding 5 ml of H2O2. Nuclei were counterstained with Mayer’s hematoxylin. The antibody was also tested on sections from non-contaminated samples. Controls of immunocytochemical reactions were performed by substituting the primary antibody with non-immune sera.
Table 1 Values obtained by HPLC in two extracts of mussel tissue Samples
45-OH-YTX (mg/g e.p.)
YTX (mg/g e.p.)
Total YTXs (mg/g e.p.)
1 2 Mean
1.490 1.234 1.362
1.720 1.557 1.639
3.210 2.791 3.001
45-Hydroxyyessotoxin ¼ 45-OH-YTX; yessotoxin ¼ YTX; e.p. ¼ edible part.
animals did not contain YTXs and the mouse tests performed with extracts from these mussels gave a negative response. The immunocytochemical investigation performed on contaminated and non-contaminated mussels showed immunoreactivity to the antibody tested in the cytoplasm of the immunocytes of the contaminated animals (Fig. 1(1)), in contrast with the negative response of the non-contaminated specimens (Fig. 1(2)). The immunoreactivity to YTXs was mainly localized in the digestive gland, and in particular in the lumen of tubules and ducts (Fig. 1(3– 5)). The cytoplasm of the two cell types in the epithelium of gland tubules, i.e. the digestive and basophilic cells, was negative. No YTXs were found in the gonads (Fig. 1(6)), while the presence of toxins in the gills could not be excluded. Indeed, the background of the immunocytochemical reaction made it impossible to reveal any small amounts of toxins that may have been present. No positivity to YTXs was observed in tissue from non-contaminated mussels (Fig. 1(7)), and the control reactions performed with non-immune sera were always negative (Fig. 1(8)). Furthermore, it should be underlined that immunoreactivity was not detected in paraffin fixed or in frozen formalin vapour fixed sections, but only in frozen unfixed tissue samples.
3. Results 4. Discussion HPLC detection revealed the presence of 45-hydroxyyessotoxin (45-OH-YTX) and YTX in the contaminated mussels, as reported in the Table 1. Furthermore, the functional assays demonstrated that the extracts from contaminated mussels were able to provoke neurotoxic effects and the mice died within 24 h. As expected, control
HPLC analysis reveals that YTXs are more accumulated than 45-OH-YTX in contaminated mussels, and their toxicity is evidenced by the mouse test. As far as the tissue distribution of YTXs is concerned, the immunocytochemical investigation performed on
Fig. 1. Immunoreactivity to YTXs is observed in the cytoplasm of immunocytes from contaminated mussels (1), in contrast to the negative response found in immunocytes from non-contaminated specimens (2). Bar ¼ 10 mm. Immunocytochemical reaction to YTXs on unfixed frozen sections of digestive gland from contaminated mussels shows the positivity in the gland tubule (t) and duct (d) lumens and no reaction in the cytoplasm of the gland epithelial cells. Residual bodies are present in the lumen of the tubule (arrows) (3, bar ¼ 50 mm; 4, 5, bar ¼ 10 mm). Immunocytochemical reaction to YTXs on sections of digestive gland from non-contaminated mussels shows an overall negative response (6). Sections of gonad from a contaminated male mussel (7) and the control of the reaction (8, see a section of digestive gland from contaminated mussels) are also negative. Counterstaining was performed with hematoxylin. (6, bar ¼ 50 mm; 7, 8, bar ¼ 10 mm).
A. Franchini et al. / Toxicon 41 (2003) 967–970
969
970
A. Franchini et al. / Toxicon 41 (2003) 967–970
contaminated and non-contaminated mussels shows that the main targets are the immunocytes and the digestive gland. In the circulating hemolymph of mussels only one cell type in two different stages, young or old, is present. In spite of differences in morphology and number, as a consequence of aging, they show similar function, i.e. cell motility, chemotaxis and phagocytosis, and express common signal molecules (Ottaviani et al., 1998). Regarding the digestive gland, two cell types are reported in tubule organ: the pyramidal basophilic secretory cell and the columnar acidophilic lysosome-rich digestive cell (Owen, 1972; Cajaraville et al., 1995). The latter cell type is involved in the uptake and intracellular digestion of food materials and undergoes a sequence of morphological changes during the digestive cycle terminating in the breakdown of the apical cell region that is pinched off to form fragmentation spherules with digestion waste products (Morton, 1983). Given its implication in detoxification processes and digestive functions, the digestive gland is considered a model for ecotoxicological studies. It has been demonstrated that metals and organic pollutants induce structural changes in the major compartments of digestive cells, i.e. the size and number of lysosomes (Moore, 1988; Marigo´mez et al., 1996). The concentration of immunoreactivity to YTXs mainly in gland tubule lumens suggests that algal toxins are accumulated together with aggregates of material digested by lysosomal action as residual bodies. From the location of immunoreactivity in the lumen of both tubules and ducts, we surmize that after the extracellular digestive phase of the ingested alga in the stomach, the toxins reach the digestive tubules and are then discharged back unaffected into the stomach. The lack of immunoreactivity in the cytoplasm of the digestive cells suggests that the toxins might be discharged, not bound for instance, to cytoplasmic proteins, as seen in metallic pollutants (Frankenne et al., 1980; Viarengo et al., 1981).
Acknowledgements The authors are very grateful to Dr N. Towers and Dr L. Briggs (AgResearch Ruakura, Hamilton, New Zealand) for providing the antibody against YTXs. This work was supported by MIUR (Italy) grant to E.O.
References Aune, T., Sørby, R., Yasumoto, T., Ramstad, H., Landsverk, T., 2002. Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice. Toxicon 40, 77–82. Briggs, L., Fitzgerald, J., Garthwaite, L., Miles, C., Garthwaite, I., Ross, K., Towers, N., Samdal, I., Aasen, J., Torgersen, T., Aune, T., Peterson, D., 2002. Immunoassays for the analysis of yessotoxins. Fourth International Conference on Molluscan Shellfish Safety, Santiago de Compostela, Spain Cajaraville, M.P., Pal, S.G., Robledo, Y., 1995. Light and electron microscopical localization of lysosomal acid hydrolases in bivalve haemocytes by enzyme cytochemistry. Acta Histochem. Cytochem. 28, 409 –416. Frankenne, F., Noe¨l-Lambot, F., Disteche, A., 1980. Isolation and characterization of metallothioneins from cadmium loaded mussel Mytilus edulis. Comp. Biochem. Physiol. 66C, 179–182. Marigo´mez, I., Orbea, A., Olabarrieta, I., Etxeberria, M., Cajaraville, M.P., 1996. Structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress in mussel-watch programmes. Comp. Biochem. Physiol. 113C, 291 –297. Moore, M.N., 1988. Cytochemical responses of the lysosomal system and NADPH–ferrihemoprotein reductase in molluscan digestive cells to environmental and experimental exposure to xenobiotics. Mar. Ecol. Prog. Ser. 46, 7–15. Morton, B., 1983. Feeding and digestion in Bivalvia. In: Saleuddin, A.S.M., Wilbur, K.M. (Eds.), The Mollusca, Physiology Part 2, vol. 5. Academic Press, New York, pp. 65–147. Ogino, H., Kumagi, M., Yasumoto, T., 1997. Toxicological evaluation of yessotoxin. Nat. Toxins 5, 255 –259. Ottaviani, E., Franchini, A., Barbieri, D., Kletsas, D., 1998. Comparative and morphofunctional studies on Mytilus galloprovincialis hemocytes: presence of two aging-related hemocyte stages. Ital. J. Zool. 65, 349 –354. Owen, G., 1972. Lysosomes, peroxisomes and bivalves. Sci. Prog. 60, 299–318. Terao, K., Ito, E., Oarada, M., Murata, M., Yasumoto, T., 1990. Histopathological studies on experimental marine toxin poisoning-5. The effects in mice of yessotoxin isolated from Patinopecten yessoensis and of a desulfated derivative. Toxicon 9, 1095–1104. Viarengo, A., Pertica, M., Mancinelli, G., Palmero, S., Zanicchi, G., Orunesu, M., 1981. Synthesis of Cu-binding proteins in different tissues of mussels exposed to the metal. Mar. Pollut. Bull. 12, 347 –350. Yasumoto, T., 2002. European commission protocol based on that of Yasumoto T., Murata, M., Oshima, Y., Matsumoto, C.K., Clark, J., 1984. Diarrhetic shellfish poisoning. In: Ragelis, E.P., (Ed.), Seafood Toxins, ACS Symposium Series 262, Ameriacn Chemical Society, Washington, pp. 207–214. Yasumoto, T., Takizawa, A., 1997. Fluorometric measurement of yessotoxins in shellfish by high-pressure liquid chromatography. Biosci. Biotechnol. Biochem. 61, 1775–1777.