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Seasonal Variations of Metallothionein Concentrations in the Asiatic Clam (Corbicula fluminea)
Comp. Biochem. Physiol. Vol. 118C, No. 3, pp. 361–367, 1997 Copyright 1997 Elsevier Science Inc. All rights reserved.
ISSN 0742-8413/97/$17.00 PII S0742-8413(97)00157-6
Seasonal Variations of Metallothionein Concentrations in the Asiatic Clam (Corbicula fluminea) M. Baudrimont, S. Lemaire-Gony, F. Ribeyre, J. Me´tivaud and A. Boudou Laboratoire d’Ecotoxicologie, Universite´ Bordeaux I, Avenue des Faculte´s, 33405 Talence cedex, France ABSTRACT. Natural variations of metallothionein (MT) concentrations in the Asiatic clam Corbicula fluminea were analyzed over a 1-year period in specimens collected from an unpolluted site (Cazaux-Sanguinet lake, southwest France). Sampling was carried out from November 1994 to December 1995, one to three times per month, according to the season. At each sampling time, lake temperature was measured and concentrations of MTs, Cd, Hg, Zn and Cu were determined in the whole soft body and in four organs or tissue samples. A histological study was conducted simultaneously to follow the development of the gonads in relation to the reproductive cycle of this bivalve. Results showed very high fluctuations in MT concentrations over the whole year, with a maximum value measured in the middle of May and ratios of around 4 at the whole organism level between extreme MT values. The tissue compartment presenting the most important variations was the visceral mass, which contains the gonads. Metal accumulation in the organisms did not seem to be involved among the factors likely to account for these variations in MT concentrations; metal concentrations remained at low and relatively constant levels throughout the whole year. The histological study revealed one spawning period from late May to the middle of June, appearing just after the MT ‘‘peak,’’ with maximum incubation of the embryos in the gills in late June. Because MT biosynthesis can be induced by hormonal secretions implicated during reproductive phenomena, the variations in MT concentrations appeared to be directly related to the biological cycle of this freshwater mollusc rather than to the direct or indirect effects of metal bioaccumulation. comp biochem physiol 118C;3:361–367, 1997. 1997 Elsevier Science Inc. KEY WORDS. Asiatic clam, field study, fresh water, heavy metals, metallothionein, reproductive cycle, temperature
INTRODUCTION Metallothioneins (MTs) are low-molecular-weight proteins (6–7 kD), first discovered by Margoshes and Vallee in 1957 from horse kidney cortex (17,20). They were then described in a large number of animal species (mammals, reptiles, amphibians, invertebrates) and in plants (5,15,20,22). They present a high proportion of cysteine residues (30% of total amino acids), placed in specific sequences that allows their classification in three classes according to their homology with mammalian MTs (12,18). The primary role of MTs in cellular metabolism is believed to be their participation in the homeostasis of essential metals like Cu and Zn (23). They are also responsible for protective action against toxic metals like Cd, Ag and Hg because of their capacity to sequester these metals and decrease their bioavailability toward other cellular ligands. The metals present different afAddress reprint requests to: A. Boudou, Laboratoire d’Ecotoxicologie, Universite´ Bordeaux I, Avenue des Faculte´s, 33405 Talence cedex, France. Tel. 05-56-84-88-08; Fax 05-56-84-84-05; E-mail: [email protected]. Received 5 May 1997; revised 7 July 1997; accepted 23 July 1997.
finity constants for the thiol sites located in the two clusters of these globular proteins, following the sequence Hg(II) .. Cu(I), Ag(I), Bi(III) .. Cd(II) . Pb(II) . Zn(II) . Co(II) . Fe(II) (25). Many of these metals are able to induce the synthesis of MTs by a transcriptional activation system, using regulatory elements and factors, activated by Zn (23,24,26). These properties are at the origin of numerous research studies into the use of MTs as biomarkers in relation to the contamination of aquatic species by heavy metals. However, the use of these proteins comes up against the problem of the complexity of mechanisms regulating their biosynthesis. Indeed, MTs are inducible by a wide range of other factors, such as hormones, second messengers, cytotoxic agents and physical stress (17). In the present study, we investigated the fluctuations of MT concentrations in the Asiatic clam Corbicula fluminea during a 1-year field study at an unpolluted site. This bivalve species, originating from China, was first reported in the late 1930s in North America, where it developed invasive dynamics (7). It was then introduced into Europe around 1980 and is now present in very high densities in most freshwater systems in southwest France (10,21). This filter feeder
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has an average lifespan of 3 years, with shell sizes at the adult stage measuring from 1 to 5 cm. C. fluminea is a very interesting model from an ecotoxicological point of view because it lives buried in the upper layers of sediments, filtering large quantities of water, about 10 l/clam/day on average, for respiratory and nutritional purposes (28). This bivalve is thus able to accumulate very high quantities of metals. C. fluminea has been used for numerous studies both at the field level, on indigenous or transplanted populations and in the laboratory within indoor microcosms (2,14,16). Only a limited number of studies have been devoted to the analysis of MTs in this species and their roles in metal sequestration. Most research was conducted in the United States from samples exposed to Cd and Zn in artificial streams; results show the presence of MT-like heavy metal binding proteins, with an apparent molecular weight similar to that of rat liver and accounting for no more than 5% of the total amount of Cd in the clams (8,9). We recently made a comparative study of MT induction after experimental exposure to Cd and Hg(II) from the water column source. The results show a significant increase of MT concentrations in the gills and the visceral mass after Cd exposure; despite very high bioaccumulation of inorganic mercury in the organs, no significant MTs induction was revealed after the 45-day experiment (1). The objective of this study was to quantify variations in MT concentrations at the whole soft body level and in the principal organs or tissue groups of C. fluminea over a 1year cycle. The bivalves were collected on the banks of an unpolluted lake, selected as a reference site. Several abiotic and biotic factors were taken into account: water temperature, metal concentrations in the organisms (Cd, Hg, Zn, Cu), maturation levels of the gonads, presence of larvae in the ctenidia (gills), to investigate relationships between variations in these factors over the 1-year cycle and fluctuations in MT concentrations in the soft body tissues of the bivalves. MATERIALS AND METHODS Sampling Procedure Adult C. fluminea were collected from the banks of the Cazaux-Sanguinet freshwater lake (Aquitaine, France), at the Sanguinet site, located on the middle of the eastern shore of the lake. This is an oligotrophic lake, with very little agricultural and industrial activity in its catchment; the lake water provides drinking water to the southern part of the Arcachon Basin and to the Communes surrounding the lake (population of around 150,000); hence its designation as a reference site. The sampling area measured about 50 m 2; it was 20 m from the beach and at a depth of around 1 m. Average clam density was close to 500 individuals/m 2. Sampling was done in the mornings from November 1994 to December 1995, once a month during winter and two to three times per month from April–May to September–
October. The organisms were selected according to the maximum anteroposterior length of their shell, between 1.5 and 2.0 cm. Each sampling was based on 23 molluscs, 10 for MT quantification, 10 for metal determinations and 3 for histological studies. At each sampling time, the temperature of the lake water was measured. After collection, the molluscs sampled for MT analysis were dried on absorbent paper sheets and immediately sealed in a polyethylene bag filled with nitrogen and stored at 220°C. This freezing under anoxic conditions is necessary to avoid MT oxidation, which could result in the polymerization of the molecules and interfere with isolation and quantification procedures (19). Organisms collected for metal determinations were dried on absorbent paper sheets and frozen at 220°C. The three molluscs sampled for the histological studies were immediately dissected to isolate the visceral mass and the gills from the soft body; these samples were directly immersed in toto in Bouin-Hollande’s fixative. Metallothionein Determination MT concentrations were measured at the whole organism and organ or tissue group levels. After thawing, the soft bodies were dissected into four tissue samples: gills, mantle, foot with adductor muscles and visceral mass (labial palps, digestive tract and annex gland, gonad, kidney, heart). They were dried on absorbent paper and weighed (fresh weight, fw). A pool of five organisms was used per replicate, with two replicates for each sampling time. Data at the whole soft body level were deduced from the organ and tissue analysis. MT concentrations were measured with the mercurysaturation assay adapted from Dutton et al. (11) and Couillard et al. (6). Several important changes in the procedure were made, notably the replacement of 203 Hg by cold inorganic Hg; this avoids problems relating to the use of this radioelement (γ emittor) and also enables us to analyze MTs after exposure of the organisms to mercury (1). The mollusc tissues were homogenized in 20-ml polypropylene tubes with a tissue grinder (Ultra-Turrax T-25) in an ice-cold Tris–HCl 25 mM buffer (Sigma, St. Louis, USA, pH 7.2 at 20°C), with two dilution conditions: 2.5 ml of buffer for the gills (dilution of about 20, w/w basis) and 3 ml of buffer for the other organs (dilution of between 4 and 10, for the visceral mass, mantle and foot). This step was performed in a glove bag filled with nitrogen (Atmosbag, Aldrich Chemical Co., St. Louis, USA). The homogenized samples were kept on ice to inhibit protease activity. Aliquots of 1.5 ml of the homogenates were placed in microtubes (Eppendorf) and centrifuged at 20,000 g for 60 min at 4°C (Sigma 3K12, rotor 12154). To 200 µl of supernatant was added a 200 µl HgCl2 (Merck, Darmstadt, Germany) solution at 50 mg Hg/l in trichloroacetic acid 10% (Sigma, St. Louis, USA). The high-molecularweight proteins were precipitated and the MTs saturated by
Metallothionein Concentrations in the Asiatic Clam
Hg(II). After incubating for 10 min, a 400-µl solution of pig blood hemolysate was added to scavenge excess mercury not bound to the MTs and rapidly centrifuged at 20,000 g for 20 min to avoid displacement of the Hg(II) from the MTs to the hemoglobin. The final supernatant was quantitatively recovered and digested in 3 ml of pure HNO3 for 15 min before Hg determination. At the same time, three reference samples or ‘‘blanks’’— 200 µl Tris–HCl buffer 1 200 µl HgCl2 solution 1 400 µl pig blood hemolysate—were prepared to monitor the Hg complexation efficiency of the hemoglobin. Under our experimental conditions, an average burden of 12.3 6 6.7 ng Hg (n 5 30) was measured in these reference samples, compared with the 10,000 ng initially added (0.12%). The mean of the three blank values measured at each analytical run was deducted from the Hg burdens measured in each sample. A recovery percentage from purified rabbit liver MT (Sigma M-7641) was systematically determined. The standard MT solution was prepared in the homogenization buffer at a concentration of 10 µg MT/ml and stored in 1.5-ml polypropylene tubes under nitrogen at 220°C. This ‘‘internal standard’’ enabled us to determine the ratio between the binding sites measured after Hg saturation and the potential binding sites indicated by the supplier and previously verified by Cd and Zn determinations on purified MT solution samples. Under our experimental conditions, the average value of the recovery percentage was 97.4 6 7.7% (n 5 30). MT concentrations in C. fluminea samples were expressed as nmol Hg-binding sites per g (fw): [(ng Hg in sample)/ (ml of supernatant)] 3 [(tissue dilution)/(Hg molar mass)]. Because the exact quantity of Hg binding sites per MT molecule is unknown for this species, MT concentrations cannot be expressed in mol/g (fw).
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Zn and Cu determinations were carried out by flame atomic absorption spectrophotometry (Varian AA 20) on the same samples. The detection limits were 10 µg Zn/l and 40 µg Cu/l. Total Hg determination was carried out by flameless atomic absorption spectrometry (Varian AA 475) after dilution of the digestates up to 50 ml with ultra-pure water. A bromine salt treatment was applied before the addition of stannous chloride. The detection limit was 0.1 µg Hg/l. The validity of the three analytical methods was checked periodically by means of standard biological reference materials from BCR (Brussels, Belgium), KFA (Ju¨lich, Germany) or IEAE (Monaco). Values for Cd, Zn, Cu and Hg were consistently within the certified ranges for each element (data not shown). Histological Study After 48 hr of immersion in the Bouin-Hollande fixative at room temperature, the visceral mass and gills samples were rinsed and embedded in paraffin (Histomed, 58°C). They were then cut into 5-µm sections and stained with Toluidine Blue. To evaluate the gonadal seasonal changes, two tissues were distinguished in the gonad: the reproductive tissue, represented by the ovarian and spermatic cysts, and the connective tissue filling the spaces between the cysts. Their respective areas were measured using an ocular 100 3 100 grid, and the section was observed with a 310 lens. Three sections were measured for each individual with three individuals for each sampling time. RESULTS AND DISCUSSION MT concentrations measured at the whole organism level of C. fluminea presented large variations during the 1-year field study (Fig. 1). After relatively constant values from
Metal Determination Cadmium (Cd), mercury (Hg), zinc (Zn) and copper (Cu) determinations were conducted directly at the whole organism level. After thawing, the soft tissues were recovered from the shell, dried on absorbent paper and weighed (fw). Two replicates were analyzed for each sampling time, based on two composite samples of three organisms. Biological samples were first digested by a nitric acid attack (3 ml of pure HNO3) in a pressurized medium (borosilicate glass tubes) at 95°C for 3 hr. After dilution of the digestates up to 20 ml with ultra-pure water (MilliQ plus), Cd concentrations were measured with an atomic absorption spectrophotometer (Varian AA 20) equipped with a model GTA 96 graphite tube atomizer and autosampler. Samples of 10 µl were taken for the determination and mixed before atomization with 4 µl of a mixture of 50% Pd 1 50% Mg(NO3) 2 to facilitate removal of the matrix. The detection limit was 0.1 µg/l.
FIG. 1. MT concentrations in Corbicula fluminea measured
at the whole soft body level, over a 1-year cycle at an unpolluted field station, from November 1994 to December 1995, and temperature of the lake water (– – –). ——, Mean MT values; s, d, two replicates (two composite samples of five organisms for each sampling date).
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FIG. 2. MT concentrations measured in the four tissue
groups of Corbicula fluminea, over a 1-year cycle at an unpolluted field station, from November 1994 to December 1995. (A) Gills, G; visceral mass, VM. (B) Mantle, M; foot, F. The lines represent the mean values and the symbols the two replicates (two composite samples of five organisms for each sampling date).
November to January, MT concentrations increased progressively, with a maximum value of 18 nmol sites/g (fw) in the middle of May (ratio of 2 compared with the initial values). From this date to early August, a strong decrease in MT concentrations emerged, reaching very low levels of around 4–5 nmol sites/g. At the end of August, MT concentrations increased progressively once again and in November–December 1995 reached the same average values as measured in November–December 1994, around 9 nmol sites/g. Thus, the difference between extreme MT concentrations reached a factor of close to 4. MT determinations in the organs or tissue groups showed that the visceral mass presented the highest MT concentrations, with variations very close to those of the whole soft body; the ratio between minimum and maximum MT concentrations during the whole year cycle was close to 5 (Fig. 2). This tissue compartment represents 46% of the total fw of the whole soft body and contains up to 65% of total Hg binding sites on MTs. The other tissue groups presented approximately the same temporal evolution of MT concentrations. A small increase appeared from November to early April, followed by a progressive decrease until December,
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with some fairly marked fluctuations during these two periods. Ratios of around 4 were reached in the gills between extreme values of MT concentrations; they were close to three for the mantle and the foot. However, no clear maximum were observed in these three tissular compartments corresponding to the MTs peak obtained in the middle of May in the visceral mass and in the whole organism. These results clearly reveal marked differences between MT concentrations in C. fluminea according to the sampling period over a 1-year cycle. Complementary data collected during this study enabled us to analyze relations between these variations and several factors suspected of affecting, directly or indirectly, the biosynthesis of MTs and their concentrations measured at the whole soft body and organ levels. First, it is important to stress that the selection of organisms as they were collected in the field, based on the size of shell, produced homogenous batches for all 21 sampling dates. Analysis of the fws of the soft bodies revealed only slight variations, with an average value of 0.377 6 0.056 g (n 5 42). Thus, the variable fw has no significant action on the variations of MT concentrations at the whole organism level, via growth dilution effects, for instance. A similar conclusion can be reached at the organ level (data not shown). Although the bivalves were collected from an unpolluted site, metal determinations were systematically carried out to measure Cd, Hg, Zn and Cu concentrations at the whole soft body level. Results showed only very weak variations between the different sampling periods. Average concentrations for the four metals were 198 6 33 ng Cd/g (fw), 110 6 30 ng Hg/g (fw), 26.0 6 2.5 µg Zn/g (fw) and 6.6 6 1.1 µg Cu/g (fw). Analysis of variations in MT concentrations in the bivalves and in the lake water temperature (Fig. 1) shows a relative concordance between the progressive increase in temperature from January and the MT concentrations. Nevertheless, beyond the maximum value of MT concentrations (mid-May), the temperature continued to increase in a quasi-linear mode until the end of August, whereas MT concentrations decreased and reached their minimal level just as the temperature was maximal (26°C). During the autumn period and until December, on the other hand, the temperature decreased progressively, whereas MT concentrations increased. Data available in the literature indicate that C. fluminea is able to tolerate a wide range of temperatures, between 2 and 34°C, the thermal preferendum being between 20 and 25°C (4,10). Thus, fluctuations in MT concentrations observed after mid-May do not seem to be directly related to the marked variations. Among those factors able to induce MTs biosynthesis, apart from metals, hormones are frequently cited, especially in relation to reproductive phenomena. The histological study of the gonad, carried out on all the sample batches collected, enabled us to investigate qualitatively the repro-
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FIG. 4. Measurements of the area occupied by the reproductive tissue of Corbicula fluminea (as % of the total gonadal area) from November 1994 to June 1995 (pre-spawning and spawning periods). Each point represents mean 6 SEM (n 5 3).
FIG. 3. Histological sections of Corbicula fluminea’s soft tis-
sues (5 mm, stained with Toluidine Blue). (A) November 12th, 1994. Pre-spawning gonad. The gonadal area (arrow) is reduced to a thin layer located between the digestive gland and the external epithelium (346). (B) May 13th, 1995. Late pre-spawning gonad. (B1) The gonad has reached its maximum development and shows numerous ovocytes (ov) and spermatic cysts (sp) (346). (B2) Detail of ovocytes (ov) and spermatogonia (sp) (3162). (C) May 28th, 1995. Postspawning gonad. The gonad shows no more reproductive cells and is represented only by connective tissue with a high melanine content (arrow) (332). (D) June 10th, 1995. Postspawning period. Gill section displaying incubating embryos (arrow) (332).
ductive cycle of this bivalve. From November (beginning of the observations) to early May, the gonad presents a growing number of ovocytes. During the autumn (Fig. 3, panel A) and early winter, it represents only a very thin layer between the digestive gland and the visceral mass epithelium. In winter and spring, the gonad occupies a greater and greater part of the visceral mass and some spermatic cysts can be seen to be developing (Fig. 3, panels B1 and B2). During this period, the maturation of the gonad is also revealed by the greater proportion of reproductive tissue vs connective tissue, from 40% to 80% on average (Fig. 4). From late May to early June, two stages are seen among the different samples: the gonad is identical to that observed in early May, filled with ovocytes and spermatogonia and/or spermatozoa (Fig. 3, panels B1 and B2), and the gonad is completely empty (no more ovocytes or spermatozoa) and only the connective tissue remains, including a high melanine content (Fig. 3, panel C). Moreover, embryos can be observed in the gills (Fig. 3, panel D). No important change
occurs up to early September, when ovocytes again appear in the gonadal tissue. Thus, this histological study leads us to define three different periods in C. fluminea’s biological cycle: a pre-spawning period, from September to early May, when the gonad grows from a thin external layer to a real mass; a spawning period, from late May to early June, when the germ cells are released from the gonad and embryos incubated in the gills, and a post-spawning period, from late June to late August, when the gonad appears empty of any reproductive cell (resting period). These observations reveal only one spawning period per year; in effect, embryos were seen in the gills only from late May to late July, with a maximum of organisms incubating in late June (60% of the organisms collected). According to several authors, this mollusc is described as a bivoltine species, with two spawning periods and two phases of liberation of the juveniles, in June–July and in August–September (4,10). It is important to note that these earlier data are from observations made on populations living in running water; the ecological conditions in stagnant systems may be the reason for these differences. If we observe the chronology of events during this 1-year field study, it appears that the different phases of the reproductive cycle of C. fluminea are correlated slightly with the variations in MT concentrations measured in the whole soft body of this bivalve. The increase in MT concentrations until the maximum reached in the middle of May coincides with the gonadal maturation period, with a strong similarity between the curves relating to the increase of MT concentrations and the importance of the reproductive tissue vs the gonadal area. Note also that the increase in the relative surface area occupied by the reproductive tissue is correlated with variations in temperature, which plays an important part in initiating the reproductive activity of the molluscs. The post-spawning period, with the maximum incubation of eggs, corresponds to the minimum value of MT concen-
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trations in August. Thus, the MTs peak, observed in the middle of May, could be related to the reproductive activity of C. fluminea, notably to the hormonal secretions coming into play during the gonadal maturation phase until the spawning period. This interpretative hypothesis is also in agreement with MT determinations at the organ level; indeed, the visceral mass, including the gonads, represents around 65% of total Hg binding sites of the MT molecules from the whole soft body. It is not possible, however, from these results to define the respective roles of the gonad and the other compartments of the visceral mass in MT production by C. fluminea. The gonad is probably implicated, notably via ovocyte maturation during the first part of the reproductive cycle. Nevertheless, the digestive gland, which is described as a major organ in the accumulation and detoxification of heavy metals in bivalves (27), could also represent a significant site for the biosynthesis of these proteins, according to its volume and its direct or indirect links with the gonad, via hormonal secretion, for example. CONCLUSION This study of the seasonal variations of MT concentrations in the soft body of C. fluminea, collected from an unpolluted site over a 1-year cycle, reveals very marked variations. The maximum MT concentrations were measured in mid-May, appearing just before the spawning period and corresponding to maximum gonadal development. After this date, MT concentrations decreased strongly, with ratios of between the minimal and maximal average values reaching a factor close to 4. Such differences are of great significance from an ecotoxicological point of view. On the one hand, these natural variations can lead to misreadings of data from field studies. Along a pollution gradient, for example, if the sampling stations do not include unpolluted reference sites under identical ecological conditions, differencies in MT concentrations may be due not only to the gradient in metal contamination but also to variations in the timing of the molluscs’ reproductive cycle at each sampling site (3,13). On the other hand, these field data show that the binding potential on MT molecules with regard to toxic metals bioaccumulated in the soft body of C. fluminea, especially in the visceral mass, varies greatly according to the time of year. If we consider, on a purely theoretical basis, that 50% of the MT binding sites are available for cadmium, which enters to the cytosol compartments (free sites, displacement of Zn atoms whose constant affinity for thiol groups is lower), about 2 nmol Cd/g (fw) or 200 ng Cd/g (fw) could be bound to MTs when the MT concentrations are at their lowest (Aug–Sept), whereas around 9 nmol Cd/g (fw) or 900 ng Cd/g (fw) could be sequestered when the maximal MT concentrations are reached (May). These findings could thus have practical consequences in a biomonitoring context; a significant increase in Cd bioaccumulation is not necessarily followed by an increase in MT concentrations
in the molluscs. Conversely, this can also lead to errors in interpretation with regard to the induction of MTs in response to experimental exposure of the clams to cadmium, depending on the investigation methods used. Cd determinations in the cytosolic protein fractions collected after gel filtration could reveal an important increase in metallic burdens corresponding to the MT fraction, interpreted as an MT induction, even though quantification of these proteins by metal-saturation assays or other methods (polarography, analysis of MT gene expression via the detection of MT mRNA) will not reveal a significant induction of MT biosynthesis. In fact, the period chosen to conduct the experimental study could correspond to the maximal MT synthesis phase (under hormonal control, according to the reproductive cycle). Under such conditions, an important sequestration of metals would occur, even though the metals themselves had played no part in triggering the increase in MT biosynthesis. References 1. Baudrimont, M.; Metivaud, J.; Maury-Brachet, R.; Ribeyre, F.; Boudou, A. Bioaccumulation and metallothionein response in the Asiatic clam Corbicula fluminea after experimental exposure to cadmium and inorganic mercury. Environ. Toxicol. Chem. (In press) 2. Belanger, S.E.; Farris, J.L.; Cherry, D.S.; Cairns, J., Jr. Growth of Asiatic clams (Corbicula sp.) during and after long-term zinc exposure in field-located and laboratory artificial streams. Arch. Environ. Contam. Toxicol. 15:427–434;1986. 3. Benson, W.H.; Baer, K.N.; Watson, C.F. Metallothionein as a biomarker of environmental metal contamination: Speciesdependent effects. In: McCarthy, J.F.; Shugart, L.R. (eds). Biomarkers of Environmental Contamination. Chelsea, MI: Lewis Publishers; 1990:255–265. 4. Britton, J.C.; Morton, B. A dissection guide, field and laboratory manual for the introducted bivalve Corbicula fluminea. Malacol. Rev. Suppl. 3:1–82;1982. 5. Carpene`, E. Metallothionein in marine molluscs. In: Dallinger, R. (ed). Ecotoxicology of Metals in Invertebrates. Boca Raton, FL: CRC Press; 1993:55–72. 6. Couillard, Y.; Campbell, P.G.C.; Tessier, A. Response of metallothionein concentrations in a freshwater bivalve (Anodonta grandis) along an environmental cadmium gradient. Limnol. Oceanogr. 38:299–313;1993. 7. Counts, C.L. The zoogeography and history of the invasion of the United States by Corbicula fluminea (Bivalvia: Corbiculidae). Am. Malacol. Bull. 2:7–39;1986. 8. Doherty, F.G.; Failla, M.L.; Cherry, D.S. Identification of a metallothionein-like, heavy metal binding protein in the freshwater bivalve, Corbicula fluminea. Comp. Biochem. Physiol. 87C:113–120;1987. 9. Doherty, F.G.; Failla, M.L.; Cherry, D.S. Metallothionein-like heavy metal binding protein levels in Asiatic clams are dependent on the duration and mode of exposure to cadmium. Water Res. 22:927–932;1988. 10. Dubois, C. Biologie et de´mo-e´cologie d’une espe`ce invasive, Corbicula fluminea (Mollusca: Bivalvia) originaire d’Asie: Etude in situ (canal late´ral a` la Garonne, France) et en canal expe´rimental. Ph.D. Thesis. Toulouse: University of Paul Sabatier; 1995. 11. Dutton, M.D.; Stephenson, M.; Klaverkamp, J.F. A mercury
Metallothionein Concentrations in the Asiatic Clam
12. 13.
14. 15. 16.
17. 18. 19.
saturation assay for measuring metallothioneins in fish. Environ. Toxicol. Chem. 12:1193–1202;1993. Fowler, B.A.; Hildebrand, C.E.; Kojima, Y.; Webb, M. Nomenclature of metallothionein. Experienta Suppl. 52:19–22; 1987. George, S.G.; Langston, W.J. Metallothionein as an indicator of water quality—assessment of the bioavailability of cadmium, copper, mercury and zinc in aquatic animals at the cellular level. In: Sutcliffe, D.W. (ed). Water Quality and Stress Indicators in Marine and Freshwater Ecosystems: Linking Levels of Organisation (Individuals, Populations, Communities). Cumbria: Freshwater Biological Association; 1994:138–153. Graney, R.L.; Cherry, D.S.; Cairns, J., Jr. Heavy metal indicator potential of the Asiatic clam (Corbicula fluminea) in artificial stream systems. Hydrobiologia 102:81–88;1983. Hamer, D.H. Metallothionein. Annu. Rev. Biochem. 55:913– 951;1986. Inza, B.; Ribeyre, F.; Maury-Brachet, R.; Boudou, A. Tissue distribution of inorganic mercury, methylmercury and cadmium in the Asiatic clam (Corbicula fluminea) in relation to the contamination levels of the water column and sediment. Chemosphere. (In press) Ka¨gi, J.H.R. Overview of metallothionein. Methods Enzymol. 205:613–626;1991. Kojima, Y. Definitions and nomenclature of metallothioneins. Methods Enzymol. 205:8–10;1991. Lobel, P.B.; Payne, J.F. The mercury-203 method for evaluating metallothioneins: Interference by copper, mercury, oxygen, silver and selenium. Comp. Biochem. Physiol. 86C:37– 39;1987.
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20. Maroni, G. Animal metallothioneins. In: Jonathan Shaw, A. (ed). Heavy Metal Tolerance in Plants: Evolutionary Aspects. Boca Raton, FL: CRC Press; 1990. 21. Mouthon, J. Sur la pre´sence en France et au Portugal de Corbicula (Bivalvia, Corbiculidae) originaire d’Asie. Basteria 45: 109–116;1981. 22. Riordan, J.F.; Vallee, B.L. Metallobiochemistry. Part B. Metallothionein and related molecules. Methods Enzymol. 205:1–681;1991. 23. Roesijadi, G. Metallothioneins in metal regulation and toxicity in aquatic animals. Aquat. Toxicol. 22:81–114;1992. 24. Roesijadi, G.; Robinson, W.E. Metal regulation in aquatic animals: Mechanisms of uptake, accumulation, and release. In: Malins, D.C.; Ostrander, G.K. (eds). Aquatic Toxicology: Molecular, Biochemical, and Cellular Perspectives. Boca Raton, FL: CRC Press; 1994:387–420. 25. Vasak, M. Metal removal and substitution in vertebrate and invertebrate metallothioneins. Methods Enzymol. 205:452– 458;1991. 26. Viarengo, A. Heavy metals in marine invertebrates: Mechanisms of regulation and toxicity at the cellular level. Aquat. Sci. 1:295–317;1989. 27. Viarengo, A.; Nott, J.A. Mechanisms of heavy metal cation homeostasis in marine invertebrates. Comp. Biochem. Physiol. 104C:355–372;1993. 28. Way, C.M.; Hornbach, D.J.; Miller-Way, C.A.; Payne, B.S.; Miller, A.C. Dynamics of filter feeding in Corbicula fluminea (Bivalvia: Corbiculidae). Can. J. Zool. 68:115–120;1990.
ISSN 0742-8413/97/$17.00 PII S0742-8413(97)00157-6
Seasonal Variations of Metallothionein Concentrations in the Asiatic Clam (Corbicula fluminea) M. Baudrimont, S. Lemaire-Gony, F. Ribeyre, J. Me´tivaud and A. Boudou Laboratoire d’Ecotoxicologie, Universite´ Bordeaux I, Avenue des Faculte´s, 33405 Talence cedex, France ABSTRACT. Natural variations of metallothionein (MT) concentrations in the Asiatic clam Corbicula fluminea were analyzed over a 1-year period in specimens collected from an unpolluted site (Cazaux-Sanguinet lake, southwest France). Sampling was carried out from November 1994 to December 1995, one to three times per month, according to the season. At each sampling time, lake temperature was measured and concentrations of MTs, Cd, Hg, Zn and Cu were determined in the whole soft body and in four organs or tissue samples. A histological study was conducted simultaneously to follow the development of the gonads in relation to the reproductive cycle of this bivalve. Results showed very high fluctuations in MT concentrations over the whole year, with a maximum value measured in the middle of May and ratios of around 4 at the whole organism level between extreme MT values. The tissue compartment presenting the most important variations was the visceral mass, which contains the gonads. Metal accumulation in the organisms did not seem to be involved among the factors likely to account for these variations in MT concentrations; metal concentrations remained at low and relatively constant levels throughout the whole year. The histological study revealed one spawning period from late May to the middle of June, appearing just after the MT ‘‘peak,’’ with maximum incubation of the embryos in the gills in late June. Because MT biosynthesis can be induced by hormonal secretions implicated during reproductive phenomena, the variations in MT concentrations appeared to be directly related to the biological cycle of this freshwater mollusc rather than to the direct or indirect effects of metal bioaccumulation. comp biochem physiol 118C;3:361–367, 1997. 1997 Elsevier Science Inc. KEY WORDS. Asiatic clam, field study, fresh water, heavy metals, metallothionein, reproductive cycle, temperature
INTRODUCTION Metallothioneins (MTs) are low-molecular-weight proteins (6–7 kD), first discovered by Margoshes and Vallee in 1957 from horse kidney cortex (17,20). They were then described in a large number of animal species (mammals, reptiles, amphibians, invertebrates) and in plants (5,15,20,22). They present a high proportion of cysteine residues (30% of total amino acids), placed in specific sequences that allows their classification in three classes according to their homology with mammalian MTs (12,18). The primary role of MTs in cellular metabolism is believed to be their participation in the homeostasis of essential metals like Cu and Zn (23). They are also responsible for protective action against toxic metals like Cd, Ag and Hg because of their capacity to sequester these metals and decrease their bioavailability toward other cellular ligands. The metals present different afAddress reprint requests to: A. Boudou, Laboratoire d’Ecotoxicologie, Universite´ Bordeaux I, Avenue des Faculte´s, 33405 Talence cedex, France. Tel. 05-56-84-88-08; Fax 05-56-84-84-05; E-mail: [email protected]. Received 5 May 1997; revised 7 July 1997; accepted 23 July 1997.
finity constants for the thiol sites located in the two clusters of these globular proteins, following the sequence Hg(II) .. Cu(I), Ag(I), Bi(III) .. Cd(II) . Pb(II) . Zn(II) . Co(II) . Fe(II) (25). Many of these metals are able to induce the synthesis of MTs by a transcriptional activation system, using regulatory elements and factors, activated by Zn (23,24,26). These properties are at the origin of numerous research studies into the use of MTs as biomarkers in relation to the contamination of aquatic species by heavy metals. However, the use of these proteins comes up against the problem of the complexity of mechanisms regulating their biosynthesis. Indeed, MTs are inducible by a wide range of other factors, such as hormones, second messengers, cytotoxic agents and physical stress (17). In the present study, we investigated the fluctuations of MT concentrations in the Asiatic clam Corbicula fluminea during a 1-year field study at an unpolluted site. This bivalve species, originating from China, was first reported in the late 1930s in North America, where it developed invasive dynamics (7). It was then introduced into Europe around 1980 and is now present in very high densities in most freshwater systems in southwest France (10,21). This filter feeder
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has an average lifespan of 3 years, with shell sizes at the adult stage measuring from 1 to 5 cm. C. fluminea is a very interesting model from an ecotoxicological point of view because it lives buried in the upper layers of sediments, filtering large quantities of water, about 10 l/clam/day on average, for respiratory and nutritional purposes (28). This bivalve is thus able to accumulate very high quantities of metals. C. fluminea has been used for numerous studies both at the field level, on indigenous or transplanted populations and in the laboratory within indoor microcosms (2,14,16). Only a limited number of studies have been devoted to the analysis of MTs in this species and their roles in metal sequestration. Most research was conducted in the United States from samples exposed to Cd and Zn in artificial streams; results show the presence of MT-like heavy metal binding proteins, with an apparent molecular weight similar to that of rat liver and accounting for no more than 5% of the total amount of Cd in the clams (8,9). We recently made a comparative study of MT induction after experimental exposure to Cd and Hg(II) from the water column source. The results show a significant increase of MT concentrations in the gills and the visceral mass after Cd exposure; despite very high bioaccumulation of inorganic mercury in the organs, no significant MTs induction was revealed after the 45-day experiment (1). The objective of this study was to quantify variations in MT concentrations at the whole soft body level and in the principal organs or tissue groups of C. fluminea over a 1year cycle. The bivalves were collected on the banks of an unpolluted lake, selected as a reference site. Several abiotic and biotic factors were taken into account: water temperature, metal concentrations in the organisms (Cd, Hg, Zn, Cu), maturation levels of the gonads, presence of larvae in the ctenidia (gills), to investigate relationships between variations in these factors over the 1-year cycle and fluctuations in MT concentrations in the soft body tissues of the bivalves. MATERIALS AND METHODS Sampling Procedure Adult C. fluminea were collected from the banks of the Cazaux-Sanguinet freshwater lake (Aquitaine, France), at the Sanguinet site, located on the middle of the eastern shore of the lake. This is an oligotrophic lake, with very little agricultural and industrial activity in its catchment; the lake water provides drinking water to the southern part of the Arcachon Basin and to the Communes surrounding the lake (population of around 150,000); hence its designation as a reference site. The sampling area measured about 50 m 2; it was 20 m from the beach and at a depth of around 1 m. Average clam density was close to 500 individuals/m 2. Sampling was done in the mornings from November 1994 to December 1995, once a month during winter and two to three times per month from April–May to September–
October. The organisms were selected according to the maximum anteroposterior length of their shell, between 1.5 and 2.0 cm. Each sampling was based on 23 molluscs, 10 for MT quantification, 10 for metal determinations and 3 for histological studies. At each sampling time, the temperature of the lake water was measured. After collection, the molluscs sampled for MT analysis were dried on absorbent paper sheets and immediately sealed in a polyethylene bag filled with nitrogen and stored at 220°C. This freezing under anoxic conditions is necessary to avoid MT oxidation, which could result in the polymerization of the molecules and interfere with isolation and quantification procedures (19). Organisms collected for metal determinations were dried on absorbent paper sheets and frozen at 220°C. The three molluscs sampled for the histological studies were immediately dissected to isolate the visceral mass and the gills from the soft body; these samples were directly immersed in toto in Bouin-Hollande’s fixative. Metallothionein Determination MT concentrations were measured at the whole organism and organ or tissue group levels. After thawing, the soft bodies were dissected into four tissue samples: gills, mantle, foot with adductor muscles and visceral mass (labial palps, digestive tract and annex gland, gonad, kidney, heart). They were dried on absorbent paper and weighed (fresh weight, fw). A pool of five organisms was used per replicate, with two replicates for each sampling time. Data at the whole soft body level were deduced from the organ and tissue analysis. MT concentrations were measured with the mercurysaturation assay adapted from Dutton et al. (11) and Couillard et al. (6). Several important changes in the procedure were made, notably the replacement of 203 Hg by cold inorganic Hg; this avoids problems relating to the use of this radioelement (γ emittor) and also enables us to analyze MTs after exposure of the organisms to mercury (1). The mollusc tissues were homogenized in 20-ml polypropylene tubes with a tissue grinder (Ultra-Turrax T-25) in an ice-cold Tris–HCl 25 mM buffer (Sigma, St. Louis, USA, pH 7.2 at 20°C), with two dilution conditions: 2.5 ml of buffer for the gills (dilution of about 20, w/w basis) and 3 ml of buffer for the other organs (dilution of between 4 and 10, for the visceral mass, mantle and foot). This step was performed in a glove bag filled with nitrogen (Atmosbag, Aldrich Chemical Co., St. Louis, USA). The homogenized samples were kept on ice to inhibit protease activity. Aliquots of 1.5 ml of the homogenates were placed in microtubes (Eppendorf) and centrifuged at 20,000 g for 60 min at 4°C (Sigma 3K12, rotor 12154). To 200 µl of supernatant was added a 200 µl HgCl2 (Merck, Darmstadt, Germany) solution at 50 mg Hg/l in trichloroacetic acid 10% (Sigma, St. Louis, USA). The high-molecularweight proteins were precipitated and the MTs saturated by
Metallothionein Concentrations in the Asiatic Clam
Hg(II). After incubating for 10 min, a 400-µl solution of pig blood hemolysate was added to scavenge excess mercury not bound to the MTs and rapidly centrifuged at 20,000 g for 20 min to avoid displacement of the Hg(II) from the MTs to the hemoglobin. The final supernatant was quantitatively recovered and digested in 3 ml of pure HNO3 for 15 min before Hg determination. At the same time, three reference samples or ‘‘blanks’’— 200 µl Tris–HCl buffer 1 200 µl HgCl2 solution 1 400 µl pig blood hemolysate—were prepared to monitor the Hg complexation efficiency of the hemoglobin. Under our experimental conditions, an average burden of 12.3 6 6.7 ng Hg (n 5 30) was measured in these reference samples, compared with the 10,000 ng initially added (0.12%). The mean of the three blank values measured at each analytical run was deducted from the Hg burdens measured in each sample. A recovery percentage from purified rabbit liver MT (Sigma M-7641) was systematically determined. The standard MT solution was prepared in the homogenization buffer at a concentration of 10 µg MT/ml and stored in 1.5-ml polypropylene tubes under nitrogen at 220°C. This ‘‘internal standard’’ enabled us to determine the ratio between the binding sites measured after Hg saturation and the potential binding sites indicated by the supplier and previously verified by Cd and Zn determinations on purified MT solution samples. Under our experimental conditions, the average value of the recovery percentage was 97.4 6 7.7% (n 5 30). MT concentrations in C. fluminea samples were expressed as nmol Hg-binding sites per g (fw): [(ng Hg in sample)/ (ml of supernatant)] 3 [(tissue dilution)/(Hg molar mass)]. Because the exact quantity of Hg binding sites per MT molecule is unknown for this species, MT concentrations cannot be expressed in mol/g (fw).
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Zn and Cu determinations were carried out by flame atomic absorption spectrophotometry (Varian AA 20) on the same samples. The detection limits were 10 µg Zn/l and 40 µg Cu/l. Total Hg determination was carried out by flameless atomic absorption spectrometry (Varian AA 475) after dilution of the digestates up to 50 ml with ultra-pure water. A bromine salt treatment was applied before the addition of stannous chloride. The detection limit was 0.1 µg Hg/l. The validity of the three analytical methods was checked periodically by means of standard biological reference materials from BCR (Brussels, Belgium), KFA (Ju¨lich, Germany) or IEAE (Monaco). Values for Cd, Zn, Cu and Hg were consistently within the certified ranges for each element (data not shown). Histological Study After 48 hr of immersion in the Bouin-Hollande fixative at room temperature, the visceral mass and gills samples were rinsed and embedded in paraffin (Histomed, 58°C). They were then cut into 5-µm sections and stained with Toluidine Blue. To evaluate the gonadal seasonal changes, two tissues were distinguished in the gonad: the reproductive tissue, represented by the ovarian and spermatic cysts, and the connective tissue filling the spaces between the cysts. Their respective areas were measured using an ocular 100 3 100 grid, and the section was observed with a 310 lens. Three sections were measured for each individual with three individuals for each sampling time. RESULTS AND DISCUSSION MT concentrations measured at the whole organism level of C. fluminea presented large variations during the 1-year field study (Fig. 1). After relatively constant values from
Metal Determination Cadmium (Cd), mercury (Hg), zinc (Zn) and copper (Cu) determinations were conducted directly at the whole organism level. After thawing, the soft tissues were recovered from the shell, dried on absorbent paper and weighed (fw). Two replicates were analyzed for each sampling time, based on two composite samples of three organisms. Biological samples were first digested by a nitric acid attack (3 ml of pure HNO3) in a pressurized medium (borosilicate glass tubes) at 95°C for 3 hr. After dilution of the digestates up to 20 ml with ultra-pure water (MilliQ plus), Cd concentrations were measured with an atomic absorption spectrophotometer (Varian AA 20) equipped with a model GTA 96 graphite tube atomizer and autosampler. Samples of 10 µl were taken for the determination and mixed before atomization with 4 µl of a mixture of 50% Pd 1 50% Mg(NO3) 2 to facilitate removal of the matrix. The detection limit was 0.1 µg/l.
FIG. 1. MT concentrations in Corbicula fluminea measured
at the whole soft body level, over a 1-year cycle at an unpolluted field station, from November 1994 to December 1995, and temperature of the lake water (– – –). ——, Mean MT values; s, d, two replicates (two composite samples of five organisms for each sampling date).
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FIG. 2. MT concentrations measured in the four tissue
groups of Corbicula fluminea, over a 1-year cycle at an unpolluted field station, from November 1994 to December 1995. (A) Gills, G; visceral mass, VM. (B) Mantle, M; foot, F. The lines represent the mean values and the symbols the two replicates (two composite samples of five organisms for each sampling date).
November to January, MT concentrations increased progressively, with a maximum value of 18 nmol sites/g (fw) in the middle of May (ratio of 2 compared with the initial values). From this date to early August, a strong decrease in MT concentrations emerged, reaching very low levels of around 4–5 nmol sites/g. At the end of August, MT concentrations increased progressively once again and in November–December 1995 reached the same average values as measured in November–December 1994, around 9 nmol sites/g. Thus, the difference between extreme MT concentrations reached a factor of close to 4. MT determinations in the organs or tissue groups showed that the visceral mass presented the highest MT concentrations, with variations very close to those of the whole soft body; the ratio between minimum and maximum MT concentrations during the whole year cycle was close to 5 (Fig. 2). This tissue compartment represents 46% of the total fw of the whole soft body and contains up to 65% of total Hg binding sites on MTs. The other tissue groups presented approximately the same temporal evolution of MT concentrations. A small increase appeared from November to early April, followed by a progressive decrease until December,
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with some fairly marked fluctuations during these two periods. Ratios of around 4 were reached in the gills between extreme values of MT concentrations; they were close to three for the mantle and the foot. However, no clear maximum were observed in these three tissular compartments corresponding to the MTs peak obtained in the middle of May in the visceral mass and in the whole organism. These results clearly reveal marked differences between MT concentrations in C. fluminea according to the sampling period over a 1-year cycle. Complementary data collected during this study enabled us to analyze relations between these variations and several factors suspected of affecting, directly or indirectly, the biosynthesis of MTs and their concentrations measured at the whole soft body and organ levels. First, it is important to stress that the selection of organisms as they were collected in the field, based on the size of shell, produced homogenous batches for all 21 sampling dates. Analysis of the fws of the soft bodies revealed only slight variations, with an average value of 0.377 6 0.056 g (n 5 42). Thus, the variable fw has no significant action on the variations of MT concentrations at the whole organism level, via growth dilution effects, for instance. A similar conclusion can be reached at the organ level (data not shown). Although the bivalves were collected from an unpolluted site, metal determinations were systematically carried out to measure Cd, Hg, Zn and Cu concentrations at the whole soft body level. Results showed only very weak variations between the different sampling periods. Average concentrations for the four metals were 198 6 33 ng Cd/g (fw), 110 6 30 ng Hg/g (fw), 26.0 6 2.5 µg Zn/g (fw) and 6.6 6 1.1 µg Cu/g (fw). Analysis of variations in MT concentrations in the bivalves and in the lake water temperature (Fig. 1) shows a relative concordance between the progressive increase in temperature from January and the MT concentrations. Nevertheless, beyond the maximum value of MT concentrations (mid-May), the temperature continued to increase in a quasi-linear mode until the end of August, whereas MT concentrations decreased and reached their minimal level just as the temperature was maximal (26°C). During the autumn period and until December, on the other hand, the temperature decreased progressively, whereas MT concentrations increased. Data available in the literature indicate that C. fluminea is able to tolerate a wide range of temperatures, between 2 and 34°C, the thermal preferendum being between 20 and 25°C (4,10). Thus, fluctuations in MT concentrations observed after mid-May do not seem to be directly related to the marked variations. Among those factors able to induce MTs biosynthesis, apart from metals, hormones are frequently cited, especially in relation to reproductive phenomena. The histological study of the gonad, carried out on all the sample batches collected, enabled us to investigate qualitatively the repro-
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FIG. 4. Measurements of the area occupied by the reproductive tissue of Corbicula fluminea (as % of the total gonadal area) from November 1994 to June 1995 (pre-spawning and spawning periods). Each point represents mean 6 SEM (n 5 3).
FIG. 3. Histological sections of Corbicula fluminea’s soft tis-
sues (5 mm, stained with Toluidine Blue). (A) November 12th, 1994. Pre-spawning gonad. The gonadal area (arrow) is reduced to a thin layer located between the digestive gland and the external epithelium (346). (B) May 13th, 1995. Late pre-spawning gonad. (B1) The gonad has reached its maximum development and shows numerous ovocytes (ov) and spermatic cysts (sp) (346). (B2) Detail of ovocytes (ov) and spermatogonia (sp) (3162). (C) May 28th, 1995. Postspawning gonad. The gonad shows no more reproductive cells and is represented only by connective tissue with a high melanine content (arrow) (332). (D) June 10th, 1995. Postspawning period. Gill section displaying incubating embryos (arrow) (332).
ductive cycle of this bivalve. From November (beginning of the observations) to early May, the gonad presents a growing number of ovocytes. During the autumn (Fig. 3, panel A) and early winter, it represents only a very thin layer between the digestive gland and the visceral mass epithelium. In winter and spring, the gonad occupies a greater and greater part of the visceral mass and some spermatic cysts can be seen to be developing (Fig. 3, panels B1 and B2). During this period, the maturation of the gonad is also revealed by the greater proportion of reproductive tissue vs connective tissue, from 40% to 80% on average (Fig. 4). From late May to early June, two stages are seen among the different samples: the gonad is identical to that observed in early May, filled with ovocytes and spermatogonia and/or spermatozoa (Fig. 3, panels B1 and B2), and the gonad is completely empty (no more ovocytes or spermatozoa) and only the connective tissue remains, including a high melanine content (Fig. 3, panel C). Moreover, embryos can be observed in the gills (Fig. 3, panel D). No important change
occurs up to early September, when ovocytes again appear in the gonadal tissue. Thus, this histological study leads us to define three different periods in C. fluminea’s biological cycle: a pre-spawning period, from September to early May, when the gonad grows from a thin external layer to a real mass; a spawning period, from late May to early June, when the germ cells are released from the gonad and embryos incubated in the gills, and a post-spawning period, from late June to late August, when the gonad appears empty of any reproductive cell (resting period). These observations reveal only one spawning period per year; in effect, embryos were seen in the gills only from late May to late July, with a maximum of organisms incubating in late June (60% of the organisms collected). According to several authors, this mollusc is described as a bivoltine species, with two spawning periods and two phases of liberation of the juveniles, in June–July and in August–September (4,10). It is important to note that these earlier data are from observations made on populations living in running water; the ecological conditions in stagnant systems may be the reason for these differences. If we observe the chronology of events during this 1-year field study, it appears that the different phases of the reproductive cycle of C. fluminea are correlated slightly with the variations in MT concentrations measured in the whole soft body of this bivalve. The increase in MT concentrations until the maximum reached in the middle of May coincides with the gonadal maturation period, with a strong similarity between the curves relating to the increase of MT concentrations and the importance of the reproductive tissue vs the gonadal area. Note also that the increase in the relative surface area occupied by the reproductive tissue is correlated with variations in temperature, which plays an important part in initiating the reproductive activity of the molluscs. The post-spawning period, with the maximum incubation of eggs, corresponds to the minimum value of MT concen-
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trations in August. Thus, the MTs peak, observed in the middle of May, could be related to the reproductive activity of C. fluminea, notably to the hormonal secretions coming into play during the gonadal maturation phase until the spawning period. This interpretative hypothesis is also in agreement with MT determinations at the organ level; indeed, the visceral mass, including the gonads, represents around 65% of total Hg binding sites of the MT molecules from the whole soft body. It is not possible, however, from these results to define the respective roles of the gonad and the other compartments of the visceral mass in MT production by C. fluminea. The gonad is probably implicated, notably via ovocyte maturation during the first part of the reproductive cycle. Nevertheless, the digestive gland, which is described as a major organ in the accumulation and detoxification of heavy metals in bivalves (27), could also represent a significant site for the biosynthesis of these proteins, according to its volume and its direct or indirect links with the gonad, via hormonal secretion, for example. CONCLUSION This study of the seasonal variations of MT concentrations in the soft body of C. fluminea, collected from an unpolluted site over a 1-year cycle, reveals very marked variations. The maximum MT concentrations were measured in mid-May, appearing just before the spawning period and corresponding to maximum gonadal development. After this date, MT concentrations decreased strongly, with ratios of between the minimal and maximal average values reaching a factor close to 4. Such differences are of great significance from an ecotoxicological point of view. On the one hand, these natural variations can lead to misreadings of data from field studies. Along a pollution gradient, for example, if the sampling stations do not include unpolluted reference sites under identical ecological conditions, differencies in MT concentrations may be due not only to the gradient in metal contamination but also to variations in the timing of the molluscs’ reproductive cycle at each sampling site (3,13). On the other hand, these field data show that the binding potential on MT molecules with regard to toxic metals bioaccumulated in the soft body of C. fluminea, especially in the visceral mass, varies greatly according to the time of year. If we consider, on a purely theoretical basis, that 50% of the MT binding sites are available for cadmium, which enters to the cytosol compartments (free sites, displacement of Zn atoms whose constant affinity for thiol groups is lower), about 2 nmol Cd/g (fw) or 200 ng Cd/g (fw) could be bound to MTs when the MT concentrations are at their lowest (Aug–Sept), whereas around 9 nmol Cd/g (fw) or 900 ng Cd/g (fw) could be sequestered when the maximal MT concentrations are reached (May). These findings could thus have practical consequences in a biomonitoring context; a significant increase in Cd bioaccumulation is not necessarily followed by an increase in MT concentrations
in the molluscs. Conversely, this can also lead to errors in interpretation with regard to the induction of MTs in response to experimental exposure of the clams to cadmium, depending on the investigation methods used. Cd determinations in the cytosolic protein fractions collected after gel filtration could reveal an important increase in metallic burdens corresponding to the MT fraction, interpreted as an MT induction, even though quantification of these proteins by metal-saturation assays or other methods (polarography, analysis of MT gene expression via the detection of MT mRNA) will not reveal a significant induction of MT biosynthesis. In fact, the period chosen to conduct the experimental study could correspond to the maximal MT synthesis phase (under hormonal control, according to the reproductive cycle). Under such conditions, an important sequestration of metals would occur, even though the metals themselves had played no part in triggering the increase in MT biosynthesis. References 1. Baudrimont, M.; Metivaud, J.; Maury-Brachet, R.; Ribeyre, F.; Boudou, A. Bioaccumulation and metallothionein response in the Asiatic clam Corbicula fluminea after experimental exposure to cadmium and inorganic mercury. Environ. Toxicol. Chem. (In press) 2. Belanger, S.E.; Farris, J.L.; Cherry, D.S.; Cairns, J., Jr. Growth of Asiatic clams (Corbicula sp.) during and after long-term zinc exposure in field-located and laboratory artificial streams. Arch. Environ. Contam. Toxicol. 15:427–434;1986. 3. Benson, W.H.; Baer, K.N.; Watson, C.F. Metallothionein as a biomarker of environmental metal contamination: Speciesdependent effects. In: McCarthy, J.F.; Shugart, L.R. (eds). Biomarkers of Environmental Contamination. Chelsea, MI: Lewis Publishers; 1990:255–265. 4. Britton, J.C.; Morton, B. A dissection guide, field and laboratory manual for the introducted bivalve Corbicula fluminea. Malacol. Rev. Suppl. 3:1–82;1982. 5. Carpene`, E. Metallothionein in marine molluscs. In: Dallinger, R. (ed). Ecotoxicology of Metals in Invertebrates. Boca Raton, FL: CRC Press; 1993:55–72. 6. Couillard, Y.; Campbell, P.G.C.; Tessier, A. Response of metallothionein concentrations in a freshwater bivalve (Anodonta grandis) along an environmental cadmium gradient. Limnol. Oceanogr. 38:299–313;1993. 7. Counts, C.L. The zoogeography and history of the invasion of the United States by Corbicula fluminea (Bivalvia: Corbiculidae). Am. Malacol. Bull. 2:7–39;1986. 8. Doherty, F.G.; Failla, M.L.; Cherry, D.S. Identification of a metallothionein-like, heavy metal binding protein in the freshwater bivalve, Corbicula fluminea. Comp. Biochem. Physiol. 87C:113–120;1987. 9. Doherty, F.G.; Failla, M.L.; Cherry, D.S. Metallothionein-like heavy metal binding protein levels in Asiatic clams are dependent on the duration and mode of exposure to cadmium. Water Res. 22:927–932;1988. 10. Dubois, C. Biologie et de´mo-e´cologie d’une espe`ce invasive, Corbicula fluminea (Mollusca: Bivalvia) originaire d’Asie: Etude in situ (canal late´ral a` la Garonne, France) et en canal expe´rimental. Ph.D. Thesis. Toulouse: University of Paul Sabatier; 1995. 11. Dutton, M.D.; Stephenson, M.; Klaverkamp, J.F. A mercury
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12. 13.
14. 15. 16.
17. 18. 19.
saturation assay for measuring metallothioneins in fish. Environ. Toxicol. Chem. 12:1193–1202;1993. Fowler, B.A.; Hildebrand, C.E.; Kojima, Y.; Webb, M. Nomenclature of metallothionein. Experienta Suppl. 52:19–22; 1987. George, S.G.; Langston, W.J. Metallothionein as an indicator of water quality—assessment of the bioavailability of cadmium, copper, mercury and zinc in aquatic animals at the cellular level. In: Sutcliffe, D.W. (ed). Water Quality and Stress Indicators in Marine and Freshwater Ecosystems: Linking Levels of Organisation (Individuals, Populations, Communities). Cumbria: Freshwater Biological Association; 1994:138–153. Graney, R.L.; Cherry, D.S.; Cairns, J., Jr. Heavy metal indicator potential of the Asiatic clam (Corbicula fluminea) in artificial stream systems. Hydrobiologia 102:81–88;1983. Hamer, D.H. Metallothionein. Annu. Rev. Biochem. 55:913– 951;1986. Inza, B.; Ribeyre, F.; Maury-Brachet, R.; Boudou, A. Tissue distribution of inorganic mercury, methylmercury and cadmium in the Asiatic clam (Corbicula fluminea) in relation to the contamination levels of the water column and sediment. Chemosphere. (In press) Ka¨gi, J.H.R. Overview of metallothionein. Methods Enzymol. 205:613–626;1991. Kojima, Y. Definitions and nomenclature of metallothioneins. Methods Enzymol. 205:8–10;1991. Lobel, P.B.; Payne, J.F. The mercury-203 method for evaluating metallothioneins: Interference by copper, mercury, oxygen, silver and selenium. Comp. Biochem. Physiol. 86C:37– 39;1987.
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20. Maroni, G. Animal metallothioneins. In: Jonathan Shaw, A. (ed). Heavy Metal Tolerance in Plants: Evolutionary Aspects. Boca Raton, FL: CRC Press; 1990. 21. Mouthon, J. Sur la pre´sence en France et au Portugal de Corbicula (Bivalvia, Corbiculidae) originaire d’Asie. Basteria 45: 109–116;1981. 22. Riordan, J.F.; Vallee, B.L. Metallobiochemistry. Part B. Metallothionein and related molecules. Methods Enzymol. 205:1–681;1991. 23. Roesijadi, G. Metallothioneins in metal regulation and toxicity in aquatic animals. Aquat. Toxicol. 22:81–114;1992. 24. Roesijadi, G.; Robinson, W.E. Metal regulation in aquatic animals: Mechanisms of uptake, accumulation, and release. In: Malins, D.C.; Ostrander, G.K. (eds). Aquatic Toxicology: Molecular, Biochemical, and Cellular Perspectives. Boca Raton, FL: CRC Press; 1994:387–420. 25. Vasak, M. Metal removal and substitution in vertebrate and invertebrate metallothioneins. Methods Enzymol. 205:452– 458;1991. 26. Viarengo, A. Heavy metals in marine invertebrates: Mechanisms of regulation and toxicity at the cellular level. Aquat. Sci. 1:295–317;1989. 27. Viarengo, A.; Nott, J.A. Mechanisms of heavy metal cation homeostasis in marine invertebrates. Comp. Biochem. Physiol. 104C:355–372;1993. 28. Way, C.M.; Hornbach, D.J.; Miller-Way, C.A.; Payne, B.S.; Miller, A.C. Dynamics of filter feeding in Corbicula fluminea (Bivalvia: Corbiculidae). Can. J. Zool. 68:115–120;1990.