What does the freshwater clam, Corbicula largillierti, have to tell us about chlorothalonil effects?

Chlorothalonil (CLT) is a broad spectrum, and non-systemic fungicide applied in foliar structures to prevent and treat pathogens. This compound reaches to aquatic environments and affects the biota. In this context, the main goal of this study was to assess the effects of CLT at biochemical, tissular, and individual levels of biological organization using the invasive bivalve Corbicula largillierti as a bioindicator species. Clams were exposed to different sublethal concentrations (0, 10, 20 and 50 µg. L-1 CLT) for 96 h. At biochemical level, the enzymatic activity (Glutathione-s-Transferase, Catalase, Acetyl-, Butiryl- and Carboxyl-esterases) and lipid peroxidation were measured in gills and the visceral mass. Also, the digestive gland morphometry through quantitative histological indexes was registered at the tissular level. Finally, filtering activity and burial behavior at the individual level were measured. At the highest CLT concentration, the most significant changes were observed in enzymatic activity (except for butyrylcholinesterase), lipid peroxidation and in digestive gland morphometry. It was also registered increases of the filtering activity and the latency time to burial. Most of the biomarkers assessed showed significant responses under CLT exposure. Therefore, taking into account that C. largillierti was affected by CLT, it can be expected that other species could be in a potential risk if this fungicide is present in freshwater systems.

Sediment quality assessment in the Guadalquivir River (SW, Spain) using caged Asian clams: A biomarker field approach.

This study assesses the sediment quality of the Guadalquivir River watercourse between the Alcalá del Río dam and the city of Seville. The main objective of this work is to address sediment quality in the area using an integrative approach that links sediment contamination and toxicity using the Asiatic clam (Corbicula fluminea) under field conditions. This is the first study conducted in the area that use of a battery of biomarkers from exposure (GST, GPx) to adverse biological effect (DNA and histopathological damage) to identify the contamination adverse effects in a river area affected by a cocktail of different anthropogenic activities (urban, industrial, agricultural, etc.). The sediment quality characterized in the area shows a significant biological stress related to metal(loid)s at station located in Alcalá del Río in the river upper part of the studied area, being this stress toxic when approaching the city of Seville. The sediments located nearby this city showed toxicity by means of positive values in the biomarkers of effects measured in the caged clams and related to contaminants with an industrial and urban discharge origins. These results have shown the useful and strength of the biomarker approach used in this study that combines biomarker responses from exposure to effects and allows identifying the contamination adverse effects by means of using caging individuals of the Asian clam. It has been proved in the different experiments how once the exposure biomarkers reach a maximum value of their system the detoxification ability of the organisms is collapsed and then the biomarkers of effect are measured significantly in the different tissues. The use of field surveys using tolerant specie such as the Asian clam is recommendable to determine sediment quality under an integrative point of view as here reported.

Effects of zinc pyrithione on biochemical parameters of the freshwater Asian clam Corbicula fluminea.

Zinc pyrithione (ZnPT) is an organometallic biocide with bactericide, algaecide, and fungicide activity. Considering this biological activity, ZnPT has been used in anti-fouling paints, and also in human therapeutics and cosmetics, in shampoos to treat dandruff and seborrhoea. Despite its potential uses and consequent presence in the aquatic environment, the ecotoxicological effects of ZnPT are poorly understood. This work aims to characterise the effects of ZnPT in biochemical parameters of the Asian clam, one of the most invasive bivalves known for its biofouling action in hydro-dependent industries, using a classical (LC50 determination) and a biomarker-based approach (quantification of the activities of catalase, GSTs, and acetylcholinesterase, and also the muscle glycogen content). The here determined LC50-96 h for zinc pyrithione was 2.17 mg/L. ZnPT caused significant increases in the activity of catalase and of cholinesterases. These findings evidence the pro-oxidative effects caused by the metabolism of ZnPT. Despite the absence of clear effects, it is important to stress that the presence of ZnPT in the wild is usually accompanied by other pyrithiones, whose co-existence can contribute to the exertion of considerable toxic effects.

Molecular characterization of cu/Zn SOD gene in Asian clam Corbicula fluminea and mRNA expression and enzymatic activity modulation induced by metals.

The copper/zinc superoxide dismutase (Cu/Zn SOD) could effectively eliminate reactive oxygen species (ROS). In this study, the cDNA sequence of Cu/Zn SOD from Corbicula fluminea (designated as CfCu/Zn SOD) was cloned by the rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfCu/Zn SOD was of 1288 bp, including a 465 bp ORF encoding a protein of 154 amino acids. Two SOD family signatures were identified in CfCu/Zn SOD amino acids sequence. Multiple sequence alignments indicated that CfCu/Zn SOD amino acid sequences exhibited high similarities to those of other species. The tissue distribution of the CfCu/Zn SOD of C. fluminea was detected by fluorescent real-time quantitative PCR. The mRNA expression levels in digestive gland and gill were significantly higher than those of other four tissues. In response to metal ions (Cd2+, Cu2+, and Pb2+) challenge, the expression and enzymatic activities of CfCu/Zn SOD in the gills were measured after exposure for 0, 6, 12, 24, 48, and 72 h. The results showed that the mRNA expression and enzymatic activities of CfCu/Zn SOD could be induced by three metal ions. After Cd2+ and Cu2+ exposure, the mRNA expression levels increased gradually and reach the peak at 24 h, afterwards it slowed down. The change trends of enzymatic activities were similar to the mRNA expression. After Pb2+ exposure, the expression and activities CfCu/Zn SOD were raised up gradually and reach the highest value at 72 h. All these results indicated that the mRNA expression and enzymatic activity of CfCu/Zn SOD are sensitive to Cd2+, Cu2+ and Pb2+ ions and can be used as molecular biomarkers of metal pollution in water.

In vitro test systems supporting the development of improved pest control methods: a case study with chemical mixtures and bivalve biofoulers.

Using the bivalve macrofouler Corbicula fluminea, the suitability of in vitro testing as a stepping stone towards the improvement of control methods based on chemical mixtures was addressed in this study. In vitro cholinesterase (ChE) activity inhibition following single exposure of C. fluminea tissue to four model chemicals (the organophosphates dimethoate and dichlorvos, copper and sodium dodecyl phosphate [SDS]) was first assessed. Consequently, mixtures of dimethoate with copper and dichlorvos with SDS were tested and modelled; mixtures with ChE revealed synergistic interactions for both chemical pairs. These synergic combinations were subsequently validated in vivo and the increased control potential of these selected combinations was verified, with gains of up to 50% in C. fluminea mortality relative to corresponding single chemical treatments. Such consistency supports the suitability of using time- and cost-effective surrogate testing platforms to assist the development of biofouling control strategies incorporating mixtures.

Responses of cytochrome P450, GST, and MXR in the mollusk Corbicula fluminea to the exposure to hospital wastewater effluents.

Pharmaceutical products are a major group of chemical compounds that are continuously released into the environment. The primary pathway of pharmaceuticals to the aquatic environment is the discharge of wastewater effluents. The Psychiatric hospital of Montpon (Dordogne, France) operates with its wastewater treatment plant. We first evaluated the presence and concentrations of 27 pharmaceuticals compounds in these effluents. All of the 27 compounds were detected in these wastewater effluents at concentrations ranging between 37,500 ng L(-1) (paracetamol) and 150 ng L(-1) (citalopram). The aim of the study was then to evaluate the exposure effects of the effluents on cytochrome P450, GST, and MXR responses in Corbicula fluminea gills and digestive glands. Experiments on clams exposed during 1, 3, 7 14, and 21 days revealed a strong and continuous overexpression of mdr1 (multidrug resistant 1) gene expression in gills and transitory variations in pi-gst expression and GST activity. EROD activity increased also transitory after 1 day in the digestive gland of exposed clams. These results indicated that in the effluent, some molecules have undergone metabolism of phase 1 and/or phase 2.

Bioavailability of cadmium and biochemical responses on the freshwater bivalve Corbicula fluminea--the role of TiO2 nanoparticles.

The increasing and widespread applications of TiO2 engineered nanoparticles (nTiO2) led to the release of these materials into aquatic environments and consequently a change on the assessment of the environmental risk of trace metals. In this work, the role of two commercial nTiO2 with distinct crystalline phases and sizes (nTiO2-P25: 80% anatase+20% rutile, d=20nm; nTiO2-NA: 100% anatase, d=5 nm; 0.1 and 1.0 mg L(-1)) on Cd (112 µg L(-1)) speciation, biouptake and toxicity for the freshwater bivalve Corbicula fluminea was evaluated. The electroanalytical technique 'absence of gradients and Nernstian equilibrium stripping (AGNES)' was used to quantify the free Cd concentrations in the exposure medium in presence of both particles. Despite ca. 30-40% decrease of free Cd in the medium in presence of nTiO2, Cd uptake by C. fluminea was similar in the absence and presence of either of the particles. Superoxide dismutase and glutathione-S-transferase activities remained unchanged for Cd in absence and presence of nTiO2, whereas a significant increase of the catalase activity was obtained at the third day for Cd in presence of both nTiO2. Despite lipid peroxidation data shows that the presence of both nTiO2 seems to exert cells damage, a more quantitative description is not possible with the obtained data. The lack of clear-cut responses by the studied biomarkers, even when only in presence of Cd, do not allow insights into the effect of the presence of nTiO2 on the Cd toxicity to the bivalves. Notwithstanding, morphological changes in the digestive gland were clearly obtained in the presence of Cd, nTiO2 and Cd+nTiO2 indicating an inflammatory response.

Genotoxic and biochemical effects of atrazine and Roundup(®), alone and in combination, on the Asian clam Corbicula fluminea.

The present study aimed to evaluate biochemical and genotoxic effects of the herbicides atrazine (ATZ) and Roundup(®) (RD) separately, as well as their mixture, on the freshwater clam Corbicula fluminea after 96 h exposure. Animals were exposed to 2 and 10 ppb of ATZ (ATZ2 and ATZ10), 2 and 10 ppm of RD (RD2 and RD10) and the following mixtures: 2 ppb ATZ+2ppm RD (AR2) and 10 ppb ATZ+10 ppm RD (AR10). Activities of ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the multixenobiotic resistance mechanism (MXR), reduced glutathione concentrations (GSH) and lipid peroxidation (LPO) were measured in gills and digestive gland. DNA damage was determined in clams hemocytes through the comet assay. The gills were more susceptible to the action of the herbicides and the results showed that ATZ2 and ATZ10 caused a significant reduction in EROD and the mixture leads to a significant decrease in EROD and MXR. No significant change in the biotransformation parameters was observed in the digestive gland. Regarding the primary antioxidant defenses, SOD activity increased in the gills of clams exposed to ATZ10 and RD10 and in the digestive gland of animals exposed to RD2 and RD10, CAT activity was significantly reduced only in digestive gland of clams exposed RD10 while GPX increased in the gills after exposure to ATZ2 and RD10. The exposure to RD10 caused a significant increase in LPO in both gills and digestive gland. While the exposure to ATZ and RD separately did not increase DNA damage, the exposure to AR2 and AR10 caused a significant increase in the occurrence of DNA damage. In conclusion, this study showed that both herbicides applied alone caused effects on C. fluminea; ATZ interfered mostly in biotransformation while RD interfered mainly in antioxidant defenses leading to lipid peroxidation. The herbicides mixture showed antagonistic effects on the gills EROD and on lipid peroxidation in gills and digestive gland and synergistic effects on the gills MXR and on DNA damage in the hemocytes.

Proteomic profiling of cytosolic glutathione transferases from three bivalve species: Corbicula fluminea, Mytilus galloprovincialis and Anodonta cygnea.

Suspension-feeding bivalves are considered efficient toxin vectors with a relative insensitivity to toxicants compared to other aquatic organisms. This fact highlights the potential role of detoxification enzymes, such as glutathione transferases (GSTs), in this bivalve resistance. Nevertheless, the GST system has not been extensively described in these organisms. In the present study, cytosolic GSTs isoforms (cGST) were surveyed in three bivalves with different habitats and life strategies: Corbicula fluminea, Anodonta cygnea and Mytilus galloprovincialis. GSTs were purified by glutathione-agarose affinity chromatography, and the collection of expressed cGST classes of each bivalve were identified using a proteomic approach. All the purified extracts were also characterized kinetically. Results reveal variations in cGST subunits collection (diversity and properties) between the three tested bivalves. Using proteomics, four pi-class and two sigma-class GST subunits were identified in M. galloprovincialis. C. fluminea also yielded four pi-class and one sigma-class GST subunits. For A. cygnea, two mu-class and one pi-class GST subunits were identified, these being the first record of GSTs from these freshwater mussels. The affinity purified extracts also show differences regarding enzymatic behavior among species. The variations found in cGST collection and kinetics might justify diverse selective advantages for each bivalve organism.

The impact of paracetamol on selected biomarkers of the mollusc species Corbicula fluminea.

The Asian clam Corbicula fluminea is an invasive bivalve that has recently spread in Europe and currently represents a large portion of the aquatic biomass in specific areas. Because of the impacts that the species may have in invaded ecosystems, increased knowledge on the physiologic features of the species life-cycle under different environmental scenarios (e.g., contamination events) is critical to understand the dynamics of the invasion and resulting ecosystem imbalance. The presence of pharmaceutical residues in the aquatic environment has recently received great attention since high levels of contamination have been found, not only in sewage treatment plant effluents, but also in open waters. The present article reports toxicological biochemical effects of paracetamol to Corbicula fluminea following short- and long-term exposures. Oxidative stress parameters were specially focused namely catalase (CAT), glutathione S-transferases (GSTs), and glutathione reductase (GRed). The effect of tested substances on lipid peroxidation was also investigated. Paracetamol did not induce alterations on CAT activity, caused a significant decrease of GSTs activity following short- and long-term exposure (LOEC values of 532.78 mg L(-1) and 30.98 µg L(-1) , respectively), and was responsible for a significant and dose-dependent decrease of GRed activity in short- and long-term exposures. These results indicate that exposure to paracetamol can provoke significant alterations on the cellular redox status of C. fluminea.

Bioavailability and oxidative stress of cadmium to Corbicula fluminea.

This work set out to study the effects of cadmium (Cd) in sediments on the antioxidant enzyme activities in the digestive gland of Asian clam Corbicula fluminea and to explore the potential for applying these responses to evaluate the Cd-contaminated sediment. Additionally, diffusive gradients in thin-films (DGT) technique was used to predict the response of its antioxidant defense system. The sediments, collected from Taihu Lake, were spiked with Cd at different concentrations (0.72, 0.91, 1.62, 2.59, 11.2, 20.4 and 40.6 mg kg(-1), dry weight). Asian clam was cultivated for 28 days. Concentrations of Cd in the body of Asian clam had a good relationship with concentrations of Cd in overlying water and sediments, as measured by DGT. Cd affected these biochemical parameters significantly, especially for superoxide (SOD), peroxidase (POD) and glutathione (GSH), which began to show higher sensitivity and have a significant difference in low dosage (0.91 mg Cd kg(-1)) compared with blank. Contents of MDA and MT, which were induced by Cd, increased with the increasing Cd concentration in sediments and reached peak values at 11.2 and 20.4 mg kg(-1), respectively, after 28 days exposure. All of these results suggested that biochemical responses cooperated in detoxifying and maintaining cellular metabolic homeostasis. The R(2) of regression analysis between the contents of MT and the concentrations of Cd measured by DGT, in sediments and soft body were 0.71, 0.94 and 0.88 after 28 days exposure. This suggested that DGT measurement could predict the response of MT. Cd accumulation, GSH and MT were indispensable biomarkers and the MDA content and DGT appeared to be promising biomarkers. The results clearly indicated that Cd could induce oxidative stress in the digestive gland of Asian clam. The combination of biomonitors with DGT can obtain different information about Cd bioavailability and confirm the significance of applying a suite of biomarkers rather than a selective index to assess the sublethal effect. It also offered theoretical methods for the prediction of sediment Cd pollution.

Cholinesterase characterization in Corbicula fluminea and effects of relevant environmental contaminants: a pesticide (chlorfenvinphos) and a detergent (SDS).

This study characterizes the cholinesterasic forms present in the bivalve Corbicula fluminea, through the analysis of differential affinity towards various substrates and inhibitors. This approach also permitted the validation of cholinesterase inhibition in the mentioned species for monitoring purposes by evaluating the inhibitory activity of a detergent (SDS) and a pesticide (chlorfenvinphos), both in vitro and in vivo. The cholinesterasic form present in the tissues of C. fluminea showed intermediate properties in terms of its relation with substrates and inhibitors. Furthermore, it was not inhibited by the detergent SDS under any of the test conditions, but was clearly inhibited by the selected organophosphate. The data show clearly that the main cholinesterasic form in C. fluminea is responsive to the organophosphate chlorfenvinphos, thus supporting the use of this biomarker for the environmental monitoring of specific contaminants such as anticholinesterasic pesticides. Despite not being commonly attained in the wild, the levels of chlorfenvinphos required to elicit a significant ChE inhibition were only one order of magnitude above actual values of aquatic contamination by this pesticide.

Phylogeny and androgenesis in the invasive Corbicula clams (Bivalvia, Corbiculidae) in Western Europe.

BACKGROUND: The genus Corbicula is one of the most invasive groups of molluscs. It includes both sexual and androgenetic lineages. The present study re-assessed the different morphotypes and haplotypes of West European Corbicula in order to clarify their taxonomic identification and phylogenetic relationships with American and Asian Corbicula clams. We studied several populations from West European river basins (Meuse, Seine, Rhine and Rhône) through an "integrative taxonomy" approach. We combined morphology, partial mitochondrial COI and cyt b sequences and eleven microsatellite loci. Furthermore, we looked for discrepancies between mtDNA and nrDNA/morphology, indicative of androgenesis between lineages. RESULTS: There are three Corbicula morphotypes in Western Europe associated to three mitochondrial lineages and three genotypes. Form R shares the same COI haplotype as the American form A and the Japanese C. leana. Form S and the American form C have the same haplotype, although their morphologies seem divergent. The European form Rlc belongs to the same mitochondrial lineage as both the American form B and the Asian C. fluminea.Interestingly, within each haplotype/genotype or lineage, no genetic diversity was found although their invasive success is high. Moreover, we detected rare mismatches between mtDNA and nrDNA/morphology, indicative of androgenesis and mitochondrial capture between form R and form S and therefore challenging the phylogenetic relatedness and the species status within this genus. The global phylogenetic analysis revealed that the sexual Corbicula lineages seem restricted to the native areas while their androgenetic relatives are widespread and highly invasive. CONCLUSIONS: We clarified the discrepancies and incongruent results found in the literature about the European morphotypes of Corbicula and associated mitochondrial lineages. The three West European morphotypes belong to three distinct nuclear and mitochondrial lineages. However mitochondrial capture occurs in sympatric populations of forms R and S. The species status of the morphotypes therefore remains doubtful. Moreover the androgenetic lineages seem widely distributed compared to their sexual relatives, suggesting that androgenesis and invasive success may be linked in the genus Corbicula.

How to remain nonfolded and pliable: the linkers in modular α-amylases as a case study.

The primary structure of linkers in a new class of modular α-amylases constitutes a paradigm of the structural basis that allows a polypeptide to remain nonfolded, extended and pliable. Unfolding is mediated through a depletion of hydrophobic residues and an enrichment of hydrophilic residues, amongst which Ser and Thr are over-represented. An extended and flexible conformation is promoted by the sequential arrangement of Pro and Gly, which are the most abundant residues in these linkers. This is complemented by charge repulsion, charge clustering and disulfide-bridged loops. Molecular dynamics simulations suggest the existence of conformational transitions resulting from a transient and localized hydrophobic collapse, arising from the peculiar composition of the linkers. Accordingly, these linkers should not be regarded as fully disordered, but rather as possessing various discrete structural patterns allowing them to fulfill their biological function as a free energy reservoir for concerted motions between structured domains.

Dynamics of protein phosphatase gene expression in Corbicula fluminea exposed to microcystin-LR and to toxic Microcystis aeruginosa cells.

This study investigated the in vivo effects of microcystins on gene expression of several phosphoprotein phosphatases (PPP) in the freshwater clam Corbicula fluminea with two different exposure scenarios. Clams were exposed for 96 h to 5 µg L(-1) of dissolved microcystin-LR and the relative changes of gene expression of three different types of PPP (PPP1, 2 and 4) were analyzed by quantitative real-time PCR. The results showed a significant induction of PPP2 gene expression in the visceral mass. In contrast, the cyanotoxin did not cause any significant changes on PPP1 and PPP4 gene expression. Based on these results, we studied alterations in transcriptional patterns in parallel with enzymatic activity of C. fluminea for PPP2, induced by a Microcystis aeruginosa toxic strain (1 × 10(5) cells cm(-3)) during 96 h. The relative changes of gene expression and enzyme activity in visceral mass were analyzed by quantitative real-time PCR and colorimetric assays respectively. The clams exhibited a significant reduction of PPP2 activity with a concomitant enhancement of gene expression. Considering all the results we can conclude that the exposure to an ecologically relevant concentration of pure or intracellular microcystins (-LR) promoted an in vivo effect on PPP2 gene expression in C. fluminea.

Acetylcholinesterase activity in Corbicula fluminea Mull., as a biomarker of organophosphate pesticide pollution in Pinacanauan River, Philippines.

Organophosphates are known to inhibit the enzyme acetylcholinesterase. In this study, the AChE activity from the total soft tissues of Corbicula fluminea Mull. was used as a biomarker of organophosphate pollution in Pinacanauan River. Clams were collected from two different sites and at different seasons of the year. A colorimetric assay on the total soft tissues of the clams showed a directly proportional relationship between enzyme activity and condition of the riverine system. In vitro experiments on the total soft tissue, adductor muscles, digestive glands, and gills were conducted to assess the degree of localization of AChE as well as the sensitivity and tolerance of the enzymes in these tissues to varying concentrations of malathion. The degree of enzyme localization from highest to lowest is as follows: adductor muscle > gills > digestive gland whereas sensitivity to OP from greatest to least is: gills > adductor muscles > digestive gland.

A comparative study of cellulase and hemicellulase activities of brackish water clam Corbicula japonica with those of other marine Veneroida bivalves.

Corbicula japonica is a typical brackish water bivalve species belonging to the order Veneroida, and it is the most important inland fishery resource in Japan. Corbicula japonica has been suggested to assimilate organic matter from terrestrial plants, unlike Ruditapes philippinarum and Mactra veneriformis, which selectively assimilate organic matter of marine origin. This led us to hypothesize that C. japonica, despite being a suspension feeder, could assimilate cellulosic materials derived from terrestrial plants. In the present study, we measured cellulase and hemicellulase activities in the crystalline styles of C. japonica and other commercially important Veneroida bivalve species in Japan: Ruditapes philippinarum, Meretrix lamarckii and Meretrix lusoria. Corbicula japonica demonstrated notably higher cellulase, xylanase and beta-mannanase activities than the other marine bivalves, suggesting that this species possesses a far greater biochemical capacity to break down the structural polysaccharides of plant cell walls than the other species. In contrast, the beta-1,3-glucanase and pectinase activities of C. japonica were similar to or even lower than those of the others. This is possibly due to the presence of these polysaccharides in the cell walls of diatoms, a principal food of most marine bivalves. Although direct evidence is lacking, the high cellulase, xylanase and beta-mannanase activities of C. japonica may result from adaptation to an upstream estuarine environment where phytoplankton and diatoms are scarce, but plant-derived substances are abundant.

Putative endogenous xylanase from brackish-water clam Corbicula japonica.

Xylan digestion by Corbicula japonica was investigated according to the hypothesis that C. japonica can breakdown xylan as well as cellulose. C. japonica showed relatively high xylanase activity compared to other bivalve species. Molecular cloning of a xylanase gene was performed in order to determine whether C. japonica possesses an endogenous xylanase, and resulted in the isolation of cDNA with an ORF of 2523 bp corresponding to 840 amino acids (CjXyn10A). CjXyn10A has a Glycoside Hydrolase Family 10 (GHF10) catalytic domain, N-terminal family 4 carbohydrate binding domain and novel C-terminal cysteine-rich domain. Phylogenetic analysis implies that this gene has common evolutional origin with other GHF10 genes of animal origin. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjXyn10A is likely to be expressed in the secretory cells in the digestive gland, suggesting that this enzyme is produced in the same site as previously reported endogenous cellulases of C. japonica. These findings suggest that CjXyn10A is an endogenous xylanase gene of this species. The occurrence of an endogenous xylanase gene in addition to cellulase genes in C. japonica strongly supports our hypothesis that this species can decompose plant-derived structural polysaccharides.

Molecular cloning of endogenous beta-glucosidase from common Japanese brackish water clam Corbicula japonica.

Studies on the cellulose utilization by animals have been conducted in keeping with the recent developments in molecular biology. In mollusks, endogenous cellulases have been reported from blue mussel, abalone, and freshwater snail. We previously reported the possibility of cellulose assimilation by Corbicula japonica, a representative bivalve dominant in brackish water environments in Japan, and the cloning of its endogenous cellulase (beta-1,4-glucanase) gene (Sakamoto, K., Touhata, K., Yamashita, M., Kasai, A. and Toyohara, H., 2007. Cellulose digestion by common Japanese freshwater clam Corbicula japonica. Fish. Sci. 73, 675-683). However, the gene of beta-glucosidase, another enzyme essential for the complete cellulose decomposition to glucose units, has not yet been isolated from the mollusk. Therefore, we attempted the molecular cloning of endogenous beta-glucosidase from C. japonica and succeeded in the isolation of a cDNA with a 2832-bp open reading frame (ORF) encoding 943 amino acid residues (CjCel1A). CjCEL1A has 2 repeated GHF-1(Glycosyl Hydrolase family 1)-like domains and showed high similarity with known insect beta-glucosidases and mammalian lactase-phlorizin-hydrolases. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjCEL1A is likely to be produced in the secretory cells in the digestive gland, suggesting that CjCEL1A is a digestive beta-glucosidase of C. japonica and is not derived from symbionts.

Molecular cloning of glycoside hydrolase family 45 cellulase genes from brackish water clam Corbicula japonica.

We previously reported endogenous Glycoside Hydrolase Family (GHF) 9 beta-1,4-glucanase gene, CjCel9A, from common Japanese freshwater clam Corbicula japonica. Here we identified another endogenous beta-1,4-glucanase genes which belong to GHF45 (CjCel45A, CjCel45B). Both genes encode ORF of 627 bp corresponding to 208 amino acids. CjCel45A and CjCel45B are different in 5' and 3'-untranslated regions and six nucleotides in the ORF. CjCEL45 has only one GHF45 catalytic domain without any carbohydrate binding modules as is the case with other molluskan GHF45 enzymes. Phylogenetic analysis and genomic structure of CjCel45 gene implies that this gene is likely to be acquired from fungi by common ancestor of mollusks. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjCel45A is likely to be expressed in the secretory cells in the digestive gland, suggesting that this cellulase is produced in the same site as CjCEL9A. CjCEL45A was successfully expressed in E. coli cells and zymographic analysis of the recombinant CjCEL45A showed that CjCEL45A is a functional beta-1,4-glucanase. The finding of multiple cellulase genes in C. japonica strongly supports our hypothesis that this species function as a cellulose decomposer in estuarine environments.