Molecular identification and expression study of differentially regulated genes in the Pacific oyster Crassostrea gigas in response to pesticide exposure Arnaud Tanguy1, Isabelle Boutet1,2, Jean Laroche1 and Dario Moraga1 ´ ´ Laboratoire des Sciences de l’Environnement Marin (LEMAR), UMR-CNRS 6539, Institut Universitaire Europeen de la Mer, Universite de ´ Bretagne Occidentale, Plouzane, France ´ ´ ´ ` UMR CNRS-IFREMER 5171 ‘Genome, Populations, Interactions, Adaptation’, Station Mediterraneenne de l’Environnement Littoral, Sete, France Keywords Crassostrea gigas; environment; gene expression; pesticides; subtractive libraries Correspondence D Moraga, UMR-CNRS 6539 Laboratoire ´ LEMAR, Institut Universitaire Europeen de ´ la Mer, Universite de Bretagne Occidentale, ´ Place Nicolas Copernic, F-29280 Plouzane, France Fax: +33 98 49 86 45 Tel: +33 98 49 86 42 E-mail : Dario.Moraga@univ-brest.fr (Received August 2004, revised November 2004, accepted 12 November 2004) The effects of pesticide contamination on the metabolism of marine molluscs are poorly documented We investigated the response of a marine bivalve, the Pacific oyster, Crassostrea gigas, using a suppression subtractive hybridization method to identify up- and down-regulated genes after a 30-day exposure period to herbicides (a cocktail of atrazine, diuron and isoproturon, and to the single herbicide glyphosate) A total of 137 unique differentially expressed gene sequences was identified, as well as their associated physiological process The expression of 18 of these genes was analyzed by RT-PCR under laboratory experimental conditions The metabolic functions they are associated with include xenobiotic detoxification, energy production, immune system response and transcription This study provides a preliminary basis for studying the response of marine bivalves to long-term herbicide exposure in terms of regulated gene expression and characterizes new potential genetic markers of herbicide contamination doi:10.1111/j.1742-4658.2004.04479.x For several decades, coastal ecosystems have been subjected to increased pesticide contamination, mainly from agricultural practices For example, herbicide compounds such as atrazine, diuron, isoproturon, simazine, alachlor, metolachlor, and, more recently, glyphosate, are widely applied to cereal crops and reach coastal waters by runoff Moreover, the exposure of animals in these ecosystems to pesticides is never limited to a single pollutant, but to an assortment of chemicals from a variety of sources whose interactions could result in additive, synergistic or antagonistic effects with regard to toxic outcome Pesticides, similar to other xenobiotics, are metabolized by many enzymes, including those of the cytochrome P450dependent monooxygenase system, flavin-containing monooxygenase, prostaglandin synthetase, alcohol dehydrogenase, esterases, and a variety of transferases [1,2] Pesticides are also known to alter the progression of some human cancers by reducing immune defenses against cancer [3] They have also been reported to affect reproductive and developmental processes in wildlife, possibly by disrupting endocrine pathways [4–6] Some particular herbicides have been well studied in terms of toxicology effects in various organisms Atrazine [2-chlor-4-ethylamino-6-isopropylamino-1,3,5triazin] has a known toxicity to blood-forming organs and the immune system, and can induce the production of cytokinines such as interferon c or tumor necrosis factor a [7,8] In fish, atrazine affects different tissues, particularly liver tissue which shows a substantial increase in the size of lipid inclusions followed by lipoid degeneration, enlargment of the secondary Abbreviations AChE, acetylcholinesterase; SSH, suppression subtractive hybridization 390 FEBS Journal 272 (2005) 390–403 ª 2005 FEBS A Tanguy et al lysosomes, mitochondrial malformation and vacuolization, and a reduction in glycogen content [9] Isoproturon[3-(4-ospropylphenyl)-1,1-dimethylurea] is a nonhalogenated, lipophilic-substituted phenylurea herbicide that is used to protect cereal crops It has a strong affinity for interaction with membrane phospholipids [10] It has been shown recently that high concentrations of isoproturon could affect metall-othioneins by reducing their metal content in the aquatic oligochaete Tubifex tubifex [11] and could affect enzyme activities in amphibians [12] Diuron [3-(3,4dichlorphenyl)-1,1-dimethyl-harnstoff] is a phenylurea herbicide [13] used for pre- and post-emergence of weeds in agriculture The toxicity of this herbicide has mainly been studied in water phytoplankton [14] In aquatic organisms, LC50 (48 h) values for diuron range from 4.3 to 42 mgỈL)1 in fish and range from to 2.5 mgỈL)1 in aquatic invertebrates It was also shown that its principal biodegradation product, 3,4-dichloroaniline, exhibits a higher toxicity and is also persistent in soil, water and groundwater [15] Significant inhibitions (9–12%) of brain acetylcholinesterase (AChE) activity were also observed in response to diuron in the juvenile goldfish (Carassius auratus) [16] Glyphosate [N-(phosphonomethyl)glycine], also known as ‘Round-Up’, is a herbicide used to control grasses, herbaceous plants, including deep-rooted perennial weeds, brush, some broadleaf trees and shrubs, and some conifers Glyphosate acts by preventing the plant from producing an essential amino acid and by inhibiting the enzyme enolpyruvylshikimate-phosphate synthase which reduces the production of protein in the plant, thereby inhibiting plant growth Studies showed that glyphosate caused the appearance of myelin-like structures in Cyprinus carpio hepatocytes, swelling of mitochondria and disappearance of the internal mitochondrial membrane in carp at both exposure concentrations [17] However, little information about the effect of glyphosate on marine invertebrate species is available Despite the vast commercial use of these herbicides, there is little published data about their effects on marine molluscs In invertebrates, resistance to pesticides by detoxification has previously been directly correlated with some enzyme biomarkers, such as mixed-function oxidase activity, glutathione S-transferase activity, AChE or other esterase activity [18,19] Aquatic macro-invertebrates such as molluscs are widely used in biological monitoring programs, but their use in biomarker studies remains limited To identify biomarkers of exposure, it is first necessary to identify and describe the mechanisms of ecotoxicological damage and those involved in the response of the FEBS Journal 272 (2005) 390–403 ª 2005 FEBS Oyster response to pesticide exposure organisms to the pollutant In previous studies, we demonstrated that atrazine and isoproturon could select particular alleles at some enzymatic loci, such as adenylate kinase, phosphoglucomutase and phosphoglucoisomerase [20] In this paper, we report regulated genes involved in the molecular response induced by herbicides in C gigas As a first step, we identified the down and up-regulated genes after one month of exposure to a cocktail of globally employed herbicides using a suppression subtractive hybridization (SSH) method We then analyzed the regulation of specific herbicideregulated gene expression Results Identification of herbicide-regulated genes Two forward and reverse SSH libraries were made from pooled digestive glands and gills of C gigas after 30 days of exposure to a herbicide cocktail of atrazine, diuron and isoproturon Two other forward and reverse SSH libraries were made from the same tissue types from oysters exposed to glyphosate for 30 days The search for homology using the blastx program revealed a total of 137 different sequences, including 56 sequences corresponding to known genes and 81 sequences corresponding to new expressed sequence tags The sequences obtained from the various SSH libraries are listed in four tables: 30-days, up- and down-regulated after exposure to an atrazine ⁄ diuron ⁄ isoproturon (ADI) cocktail (Tables and 2), and 30-days, up-and down-regulated after exposure to glyphosate (Tables and 4) These genes regulated by herbicide exposure can be assigned to six major cellular physiological functions (a) Xenobiotic detoxification; (b) nucleic acid and protein regulation (including transcription, cell cycle regulation and metabolism of nucleic acid components); (c) respiration; (d) cell communication (including immune system and membrane receptors); (e) cytoskeleton production and maintenance; and (f) energy metabolism Several ribosomal proteins were also found in both the forward and reverse libraries from the two experiments There were more regulated genes from the libraries associated with exposure to the ADI cocktail than in the libraries associated with exposure to glyphosate, particularly among the down-regulated genes Expression of herbicide-regulated genes The time-dependent expression of 13 genes that were up-regulated by exposure to herbicide and five that 391 Oyster response to pesticide exposure A Tanguy et al Table Up-regulated genes identified in the SSH libraries of the atrazine ⁄ diuron ⁄ isoproturon experiment (after 30 days of exposure) with significant database matches BLASTX Homolog (protein) value Xenobiotics detoxification Glutamine synthetase 3e-33 Lysosomal associated protein Unknown function Clone 44 unnamed human protein product 6e-15 C380A1.2.2 Homo sapiens novel protein 0.048 ENSANGP00000014914 1e-6 ENSANGP00000024944 3e-10 Cellular cycle, protein regulation and transcription RNA helicase 1e-108 Hypertension-related 1e-18 calcium-regulated gene (HSCR) Elongation factor 3e-61 Respiratory chain Cytochrome oxidase 9e-23 NADH dehydrogenase subunit 8e-94 Metabolism ATP syntase beta subunit 1e-36 ATP syntase alpha subunit 8e-68 Cellular communication, membrane receptors and immune system b-1,3-Glucan binding protein 3e-6 Putative senescence-associated protein 5e-46 Cavortin 1e-5 Cytoskeleton Tubulin alpha chain 4e-17 Tubulin alpha chain 5e-20 Tubulin beta 1e-50 Actin cytoplasmic A3 3e-84 Ribosomal proteins Ribosomal protein L18a 6e-31 Ribosomal protein S18 5e-34 Ribosomal protein S7 2e-38 Ribosomal protein S17 3e-45 Ribosomal protein S6 4e-37 Unknown genes (27 sequences)a GenBank accession no CB617403 CF369139 CF369128 CF369136 CF369137 CF369140 CF369125 CF369127 CF369143 AF177226 AF177226 CF369132 CF369142 CF369126 CF369134 CF369147 CF369131 CF369135 CF369141 CF369133 CF369129 CF369145 CF369146 CF369144 CF369138 CF369148 to CF369174 a Sequences with nonsignificant e-value (< 0.01) or with unknown proteins were down-regulated was analyzed by RT-PCR using RNA from both the gills and digestive glands of oysters after 0, 7, 14, 21 and 30 days of pesticide exposure First, an RNA pool of the eight oysters collected at each exposure time was used to identify the tissue in which the differential expression when compared to the control was significant RT-PCR was then carried out on each oyster sample for the target tissues, to estimate the variation in gene expression between samples A summary of these results is presented in Table and Fig Among the 18 genes analyzed, only 392 Table Down-regulated genes identified in the SSH libraries of the atrazine ⁄ diuron ⁄ isoproturon experiment (after 30 days of exposure) with significant database matches value GenBank accession no 5e-70 8e-33 6e-18 3e-9 2e-9 3e-10 CF369178 CF369179 CF369181 CF369185 CF369187 CF369182 2e-4 3e-15 7e-10 4e-40 CF369175 CF369184 CF369183 CF369176 1e-4 9e-6 2e-52 2e-29 CF369177 CF369180 CF369186 CF369188 CF369189 to CF369220 BLASTX Homolog (protein) Unknown function EbiP2667 Anopheles gambiae CG1524-PC Drosophila melanogaster Unnamed protein Mus musculus ENSANGP00000010310 AgCP1592 Anopheles gambiae EbiP2667 Anopheles gambiae Cellular communication, membrane receptors and immune system Tripartite motif protein TRIM2 Apoliphorin precursor protein Transport protein Sec61 alpha subunit Guanyl cyclase receptor Ribosomal proteins Ribosomal protein L38 Ribosomal protein L13 Ribosomal protein L7 Ribosomal protein S8 Unknown genes (32 sequences)a a Sequences with nonsignificant e-value (< 0.01) or with unknown proteins two showed no clear regulation – fucolectin and ribosomal L18A All of the other genes showed some regulation with exposure to herbicide The regulation of expression was strongly tissue-dependent for 10 genes, and most of these are chiefly regulated in the digestive gland (Table 5) Student’s t-test showed that despite clear patterns of increased or decreased expression at the different times of exposure, the differences in gene expression are most significant when we compare samples exposed for and 14 days with those exposed for 21–30 days A summary of the significant value of statistical test is presented in Tables and Less significant differences can be explained by the variation seen in expression among the samples In the ADI (Fig 1C) and glyphosate (Fig 1B) experiments, identified genes from the SSH libraries were expressed differentially compared to the control In the control oysters, no significant variation in gene expression was observed between samples from the different sampling periods, though high inter-individual variations were detected (Fig 1A) Discussion Despite the intensity of pesticide use along the marine coast, few studies have investigated the response to FEBS Journal 272 (2005) 390–403 ª 2005 FEBS A Tanguy et al Oyster response to pesticide exposure Table Up-regulated genes identified in the SSH libraries of the glyphosate experiment (after 30 days of exposure) with significant database matches BLASTX Homolog (protein) value Xenobiotics detoxification Glutamine synthetase 3e-33 Respiratory chain NADH dehydrogenase subunit 8e-94 NADH dehydrogenase subunit 6e-16 Cytochrome oxidase 9e-23 Metabolism ATP syntase beta subunit 1e-36 Cellular communication,membrane receptors and immune system b-1,3-Glucan binding protein 3e-6 Procathepsin L 1e-12 Meningioma expressed antigen 7e-19 Lipoprotein receptor related protein 2e-24 Fucolectin-1 6e-4 Cytoskeleton Tubulin alpha chain 4e-40 Tubulin beta 2e-96 Actin cytoplasmic A3 3e-84 Ribosomal proteins Ribosomal protein L6 2e-33 Ribosomal protein S25 2e-14 Ribosomal protein S8 2e-29 Unknown genes (16 sequences)a GenBank accession no CB617403 AF177226 AF177226 AF177226 CF369132 CF369126 CF369221 CF369226 CF369227 CF369228 CF369225 CF369141 CF369133 CF369223 CF369224 CF369222 CF369229 to CF369244 a Sequences with nonsignificant e-value (< 0.01) or with unknown proteins Table Down-regulated genes identified in the SSH libraries of the glyphosate experiment (after 30 days of exposure) with significant database matches BLASTX Homolog (protein) value Cellular cycle, protein regulation and transcription ADP-ribosylation factor 1e-9 Ribosomal proteins Ribosomal protein S3A 3e-25 Ribosomal protein L27 1e-22 Ribosomal protein L13 2e-52 Ribosomal protein 60S P2 2e-52 Ribosomal protein S3B 4e-45 Ribosomal protein 40S S2 2e-52 Unknown genes (nine sequences)a GenBank accession no CF369249 CF369245 CF369246 CF369247 CF369252 CF369251 CF369248 CF369253 to CF369261 a Sequences with nonsignificant e-value (< 0.01) or with unknown proteins exposure in marine organisms at the level of gene transcription In this study, we characterized the response of the Pacific oyster, C gigas, to herbicide exposure FEBS Journal 272 (2005) 390–403 ª 2005 FEBS under experimental conditions Using a suppression subtractive hybridization method, we obtained 137 unique partial sequences of cDNA (56 corresponding to known genes) encoding proteins being transcribed in oysters after 30 days exposure to herbicides Use of this method in conjunction with differential display PCR has previously identified 242 differentially expressed genes in the zebra mussel, Dreissena polymorpha, treated with various contaminants such as Aroclor 1254, 3-methylcholanthrene, chrysene and atrazine [21] Our results are difficult to compare with those obtained in D polymorpha because of the higher concentrations of pesticides (between gỈL)1 and mgỈL)1, according to the herbicide) and shorter time of exposure (between and 24 h) used by the authors In another study, 258 differentially expressed genes were identified in C gigas in response to exposure to hydrocarbons for and 21 days [22], and some of these genes were also present in the herbicide SSH libraries (including glutamine synthetase and cathepsin) In our experiments, we tested a cocktail of the three more common herbicides detected in the French Atlantic estuaries and used concentrations that can be found in seawater The experiment using only glyphosate was conducted to test the effect of this newly introduced herbicide in French ecosystems Glyphosate is known to be not bioaccumulated, biomagnified or persisting in a biologically available form in the environment Its mechanism of action is specific to plants and it is relatively nontoxic to animals [23] But several studies have demonstrated that glyphosate and more especially the surfactants used to increase its efficacy showed that glyphosate could be toxic for many organisms [24–26] Isoproturon appears not to bioaccumulate in molluscs [27], and the LC50 at days in C gigas larvae has been estimated at 0.37 mgỈL)1 In acute tests, diuron was had limited toxicity to fish and invertebrates [28] Few results are available from chronic tests, especially for aquatic invertebrates The LC50 (at 48 or 96 h) for diuron varied from to 30 mgỈL)1 in fish and invertebrate species [29] Fish species data are primarily from acute exposures, and the lethal effects ranged from 2.8 to 31 mgỈL)1 in 1–4 day exposures [28,30,31] Glyphosate is considered relatively effective with little to no hazard to animals [32] However, at sublethal concentrations, glyphosate affected the reproduction and development of Pseudosuccinea columella snails [32] Glyphosate was also shown to be relatively nontoxic in certain animal species and presents virtually no effects in some aquatic organisms (the 96 h LC50 in rainbow trout, Oncorhynchus mykiss, and other fish ranges from 86 to 168 mgỈL)1, and the 48 h LC50 was 780 mgỈL)1 in Daphnia) [33] The LC50 for oyster 393 Oyster response to pesticide exposure A Tanguy et al Table Summary of the results of expression studies in the two tissues used in SSH experiments Gill Digestive gland Gene name SSH library ADI Glyphosate ADI Glyphosate Glutamine synthetase ATP syntase beta Coelomic factor RNA helicase Procathepsin L Meningioma associated protein Lipoprotein receptor HSCR Senescente associated prot Lysosomal associated protein Elongation factor Apolipophorin Tripartite motif protein TRIM2 Guanyl cyclase ADP ribo RiboL13 ADI and G (up) ADI and G (up) ADI and G (up) ADI (up) G (up) G (up) G (up) ADI and G (up) ADI (up) ADI (up) ADI (up) ADI (down) ADI (down) ADI (down) G (down) ADI (down) + + – – – – – + – + – – + + – + + + – – + – – – – – – – – – – + + + + + – – – + + + + + + – – + + + + – + + + + + – – – – – + + larvae has been estimated at more than 10 mgỈL)1 [34] Glyphosate closely resembles naturally occurring substances and does not possess chemical groups that would confer great reactivity or biological persistence, and its chemical properties indicate that it is not bioaccumulate [35] Although primarily aimed at reversibly inhibiting photosynthesis in plants [36], atrazine has also been found to affect a variety of physiological processes in aquatic animals Atrazine accumulation has been seen in a number of tissues [37,38] In freshwater invertebrates, atrazine affected hydromineral balance or gill function in crabs [39,40], as well as hemocyanin function [41] In fish it affects hematology [42,43] and metabolism [41,44,45] More recently, low levels of atrazine have been shown to impair sexual development in male frogs [6] In the mollusc C virginica, the 96 h LC50 has been estimated at mgỈL)1 The supposed low level of toxicity of glyphosate to oysters, and the weak concentration used in our experiment could partly explain the difference observed in the number of genes identified from the libraries (98 in the ADI SSH libraries vs 48 in the glyphosate SSH libraries) In previous experiments studying the effect of atrazine on fish, the authors showed that the concentrations quantified in water were about 70% less than the concentrations introduced in the tanks, probably due to adsorption of atrazine to the surfaces of the tanks and possibly due to removal by the fish themselves [46] Moreover, the authors suggested that some of the atrazine added may be liable to biotic (e.g bacteria) and abiotic (e.g light) degradation and therefore that the physiological responses of the fish to atrazine may be occurring at lower water concentrations than indicated Until a better understanding of atrazine dynamics can be applied to our experimental set-up, we prefer to discuss the physiological changes in C gigas in relation to the concentrations of pesticides introduced in our tanks More, the fact that sea-water was changed every day in our tanks and the corresponding herbicide concentration was added at each water Fig Analysis of differential expression of up- and down-regulated genes in C gigas exposed to pesticides Expression of the gene studied is presented as the calculated ratios ODCgGSII ⁄ OD28S after RT-PCR For each gene and each sampling time, the bar represents the average value of gene expression (ratios ODCgGSII ⁄ OD28S) for the eight samples and the error bars correspond to the standard deviation for the eight samples at the sampling time considered (A) Expression of the 17 studied genes in the control samples 1, senescence associated protein; 2, Trim2; 3, lysosomal associated protein; 4, elongation factor 2; 5, hypertension-related calcium-regulated gene (HSCR); 6, apoliphorin precursor protein; 7, glutamine synthetase; 8, ATP syntase; 9, coelomic factor; 10, RNA helicase; 11, guanyl cyclase receptor; 12, ribosomal protein L13; 13, lipoprotein receptor related protein; 14, meningioma expressed antigen 5; 15, ADP-ribosylation factor 2; 16, procathepsin L (B) Expression of the nine studied genes in the glyphosate experiment 1, lipoprotein receptor related protein; 2, HSCR; 3, meningioma expressed antigen 5; 4, glutamine synthetase; 5, ATP synthase; 6, coelomic factor; 7, ADP-ribosylation factor-2; 8, procathepsin L; 9, ribosomal protein L13 (C) Expression of the 12 studied genes in the ADI experiment 1, senescence associated protein; 2, Trim2; 3, lysosomal associated protein; 4, elongation factor-2; 5, HSCR; 6, apoliphorin precursor protein; 7, glutamine synthetase; 8, ATP syntase; 9, coelomic factor; 10, RNA helicase; 11, guanyl cyclase receptor; 12, ribosomal protein L13 394 FEBS Journal 272 (2005) 390–403 ª 2005 FEBS A Tanguy et al Oyster response to pesticide exposure A 14 12 10 12 10 11 12 13 14 15 16 B 10 T0 T7 T14 T21 T30 14 C 12 10 2 FEBS Journal 272 (2005) 390–403 ª 2005 FEBS 10 11 12 395 Oyster response to pesticide exposure change, allowed to maintain the oysters in a more or less homogenous concentration of contaminants Most of the genes we identified function in the respiratory chain, cell communication, the immune system, or the regulation of protein or the cytoskeleton Only a few are specific to xenobiotic detoxification One, the glutamine synthetase gene was previously shown to be associated with the response of various organisms to pesticide exposure The response seen in our libraries was that more genes are up-regulated by herbicides than are down-regulated This pattern was stronger in the ADI libraries compared to the glyphosate libraries Glyphosate seemed not to have a strong effect on C gigas metabolism However, some of the genes involved in respiration and energy production were highly expressed in response to herbicide exposure no matter which pesticide was used Similar results were observed in previous reports studying the effect of other stress such as hydrocarbons [22] or parasite infection [47], showing that any source of stress generates an increase in energy production A comparison of the two experiments shows that the same cellular functions were affected by herbicides with only a few genes in common to both the ADI and glyphosate response SSH libraries These were glutamine synthase, ATP synthetase, b-1,3-glucan binding protein and some housekeeping genes such as tubulin and actin Among the 18 genes studied, 16 produced patterns of differential time- or tissue-dependent expression Most of the significant differences observed in gene expression appeared after 21–30 days, suggesting that at the concentrations used herbicides only affect oyster metabolism after long periods of exposure Among all the genes regulated was b-1,3-glucan binding protein, also named coelomic factor, that has been widely studied in shrimp [48,49] and other crustaceans [50] Lipopolysaccharide and b-1,3-glucan binding protein combine to form LGBP, a pattern recognition protein, that plays an important role in the innate immune response of crustaceans and insects This gene was previously identified in up-regulated SSH libraries from C gigas exposed to hydrocarbons [22] and parasites [47] The binding of lipopolysaccharide and b-1,3-glucan binding protein to form LGBP has been shown to activate the prophenoloxidase cascade The apolipophorin precursor protein we identified is also involved in the activation of phenoloxidase in invertebrates [51] The activation of the phenoloxidase cascade appears to be a general response of C gigas to stress exposure whatever its nature, abiotic or biotic We also studied the expression of an RNA helicase that is known to be a multifunctional protein involved 396 A Tanguy et al in various nuclear processes such as transcription, ribosomal RNA biogenesis and RNA export Several tissue-specific RNA helicases have been described in the literature, such as the myocyte enhancer factor-2 protein that acts as an inhibitor of cell proliferation and cardiomyocyte hypertrophy and may also be involved in cell cycle progression [52] In previous studies, we also showed that this gene was up-regulated by exposure to hydrocarbons, and that its over-expression was greatest in the first week after PAH exposure [22] We saw strong inhibition of guanyl cyclase receptor, particularly in the gills The inhibition of guanyl cyclase has been shown to prevent increased enzyme activity associated with the nitric oxide-mediated damage recovery process [53] Nitric oxide (NO) is a potent, bioactive molecule produced in the presence of NO synthase, which constitutively mediates numerous physiological functions Over-production of NO (and NO-reaction products) can be induced, and typically leads to cell cycle arrest and apoptosis In vertebrate blood cells, an increase of extracellular levels of NO was detected after exposure to the herbicide paraquat [54] Other studies have likewise shown that pesticides could generate increasing NO levels which led to unrepaired damage caused by free radicals [55] Our experiments were not designed to quantify the amount of NO reaction products; however, the strong down-regulation of guanyl cyclase receptor that was measured suggests an activation of physiological process involved in free radical scavenging, especially NO, that could have been generated during the herbicide exposure in oysters ATPase is a large family of genes coding for different enzymes that all participate in the formation of ATP The enzyme identified from our libraries could not be specifically identified from the partial sequence Nevertheless, over-expression of this gene was observed in both experiments and in both tissue types analysed after 15 days of pesticide exposure In another study, increased Mg2+-ATPase and Na+ ⁄ K+ATPase activity was detected in the liver and erythrocytes of rats exposed to the insecticides malathion and anilofos [56] Similar results were obtained in snails exposed to fungicide and herbicides [57] Because ATPase is constitutively expressed and probably regulated by multiple environmental and ⁄ or physiological factors, a more complete study of the regulation of its expression has to be done before its potential use as a biomarker for pesticide-exposure monitoring can be determined Other interesting genes that were identified from our libraries are the lysosomal associated protein and procathepsin L, a protein protease Both are involved in FEBS Journal 272 (2005) 390–403 ª 2005 FEBS A Tanguy et al Oyster response to pesticide exposure Table Summary of the statistical test (Student’s t-test) performed on gene expression in the glyphosate experiment The gene expressions are compared by pair of sampling time *, Significant value at 0.05% T0 Lipoprotein receptor T7 T14 * T21 * T30 * HSCR T7 T14 * T21 * T30 * Meningioma antigen T7 T14 * T21 * T30 * Glutamine synthetase T7 T14 T21 * T30 * ATP synthase T7 T14 * T21 * T30 * Coelomic factor T7 T14 T21 T30 * ADP-ribosylation factor-2 T7 T14 T21 T30 * Procathepsin L T7 T14 * T21 * T30 * Ribosomal protein L13 T7 T14 T21 * T30 * T7 T14 T21 * * * * * * * * * * * * * * * * * * * * * * * * * * lysosomal functions Earlier studies have shown that pesticides can affect lysosome membrane stability [58] In insecticide-resistant Musca domestica strains, the mechanism by which proteases may confer advantages to insecticide resistant insects could involve providing an increased supply of precursor amino acids from FEBS Journal 272 (2005) 390–403 ª 2005 FEBS proteolytic degradation products to the intracellular pool, prior to the de novo synthesis of detoxifying enzymes [59] We previously showed that the lysosomal associated protein was up-regulated in response to parasite exposure [47] suggesting that membrane stability seems to be a target for stress factors The lysosomal membrane destabilization is a common parameter used to to investigate the impact of environmental pollution in disturbed ecosystems and is considered as a general biomarker of stress [60,61] We also identified the glutamine synthetase (GS) gene as being up-regulated with pesticide exposure GS performs the fundamental functions of ammonia fixation and glutamine biosynthesis, but also plays a role in the central nervous system where it clears the excitatory neurotransmitters of glutamic acid [62] In Arabidopsis thaliana, GS was shown to be induced under amino acid starvation conditions as well as by exposure to the herbicide acifluorfen [63] In the fish, Cyprinus carpio, an increase in GS was observed after several days of exposure to cypermethrin At the same time, there was a decrease in the amount of free amino acids coincident with changes that occurred in the transamination process, again, related to the formation of nitrogenous end products [64] For the other differentially transcribed genes identified in our libraries, we found no information in the literature concerning their regulation by pesticides Some of these genes, such as meningioma expressed antigen, hypertension-related calcium-regulated gene, and tripartite motif protein TRIM2, have been described as being regulated in cell proliferation [65] Their presence in our SSH libraries could suggest an effect of pesticides on these physiological processes Interestingly, no sequence corresponding to AChE was identified in our libraries The monitoring of AChE activity, particularly its inhibition, is commonly used as a biomarker of pesticide exposure in experiments on marine species such as the mussel, Mytilus sp [66,67], the shore crab, Carcinus maenas [68] and other invertebrates [69] This result can be explained in several ways First, AChE has different distributions and physiological roles in different species [70,71], which results in a highly variable degree of inhibition associated with toxic effects [68] Also, AChE activity is modulated by seasonal and nutritional variables [66] Finally, in bivalves, only a few studies have been published on the use of AChE activity as a biomarker of pesticide exposure, reflecting both low endogenous activity and a relative insensitivity to inhibition by these pollutants compared with other species [72–75] The results given here provide a preliminary basis for further study of detoxification processes and other physiological responses to herbicides in the marine 397 Oyster response to pesticide exposure A Tanguy et al Table Summary of the statistical test (Student test) performed on gene expression in the atrazine ⁄ diuron ⁄ isoproturon experiment The gene expressions are compared by pair of sampling time *, Significant value at 0.05% T0 Senescence associated protein T7 * T14 * T21 * T30 * Trim2 T7 T14 T21 * T30 * Lysosomal associated protein T7 T14 * T21 * T30 * Elongation factor-2 T7 * T14 * T21 * T30 * HSCR T7 T14 * T21 * T30 * Apoliphorin precursor T7 T14 * T21 * T30 * Glutamine synthetase T7 * T14 * T21 * T30 * ATP syntase T7 T14 * T21 * T30 * Coelomic factor T7 T14 T21 * T30 * RNA helicase T7 T14 * T21 * T30 * Guanyl cyclase receptor T7 * T14 * 398 T7 T14 * T21 Table (Continued) T0 T21 * T30 * Ribosomal protein L13 T7 T14 T21 T30 * T7 T14 T21 * * * * * * * * bivalve, C gigas This is the first published investigation into the mechanisms of these responses at the molecular level in this species We will focus our next efforts on a more complete study of the regulation of these genes with respect to pollutant concentration, their use in surveys of wild populations of oysters, and also in the search for functional polymorphism Experimental procedures * * Oyster conditioning and treatment * * * * * * * * * * * * * * * * * * * Adult Crassostrea gigas were collected from La Pointe du Chateau (Brittany, France) After an acclimatization perˆ iod of days in aerated 0.22 lm filtered seawater at constant temperature and salinity (15 °C and 34&, respectively), oysters were challenged as follows Two groups of 40 oysters were exposed to two experimental conditions One group was exposed to a cocktail of herbicides (ADI) composed of atrazine (2 lgỈL)1), diuron (1 lgỈL)1), and isoproturon (0.5 lgỈL)1) that represent the three most used and toxic herbicides detected in all French Bay waters The other group is exposed to lgỈL)1 of glyphosate, a herbicide that has been recently introduced in marine ecosystems, and for which no information about its toxicity at low concentration is known in marine molluscs Another group of 40 oysters was maintained in aerated 0.22 lm filtered seawater without contaminant as a control The experiment lasted for weeks and no mortality was observed Herbicide concentrations were chosen based on data reported by the Bay of Brest monitoring program [76] and by the National Observation Network of IFREMER-France The concentrations used in our experiment correspond to the highest concentrations observed in the various French Bay waters and correspond to about ⁄ 100 of the LC50 values observed for the herbicides tested [77–79] * * * Suppression subtractive hybridization After weeks, total RNA was extracted from the digestive gland and gills of a pool of 10 control and 10 exposed FEBS Journal 272 (2005) 390–403 ª 2005 FEBS A Tanguy et al Oyster response to pesticide exposure oysters using RNAble (Eurobio, les Ulis, France) according to the manufacturer’s instructions Poly(A+) mRNA was isolated from total RNA using the PolyATtractÒ mRNA Isolation System (Promega, Madison, WI, USA) according to the manufacturer’s instructions Both forward and reverse subtracted libraries were made on lg of mRNA extracted from the 10 oysters collected after 30 days of exposure and pooled for RNA extraction mRNA from the gill and the digestive gland (1 lg from each) were used for the construction of SSH libraries First and second strand cDNA synthesis, RsaI endonuclease enzyme digestion, adapter ligation, hybridization and PCR amplification were performed as described in the PCR-select cDNA subtraction manual (Clontech, Palo Alto, CA, USA) The differentially expressed PCR products were cloned into pGEM-T vector (Promega, Madison, WI, USA) Two hundred white colonies per library were cultured in Luria–Bertani medium (with 100 mgỈL)1 ampicillin) from which the vector was extracted using an alkaline lysis plasmid minipreparation, and screened by size after digestion A total of 250 clones from forward and reverse libraries were sequenced using a Li-COR IR2 (Sciencetech, Lincoln, NE, USA) and Thermo Sequenase Primer Cycle Sequencing Kit (Amersham Bioscience, Uppsala, Sweden) and a AB3100 sequencer (PerkinElmer, Boston, MA, USA) and Big Dye Terminator V3.1 Kit (PerkinElmer) All sequences were subjected to a homology search through the blastx program (http://www.ncbi.nlm.nih.gov/ BLAST/) Pesticide detoxification gene expression analysis by semiquantitative RT-PCR Total RNA was extracted from the gill and digestive gland of eight control and eight exposed oysters at days 0, 7, 15, 21 and 30 days using a method based on extraction in guanidium isothiocyanate [80] For each oyster, 50 lg of total RNA were reverse transcribed using the oligo(dT) anchor primer 5¢-GACCACGCGTATCGATGTCG>ACT16V-3¢ and M-MLV reverse transcriptase (Promega, Madison, WI, USA) Amplification of 18 regulated genes was carried out using cDNA from both the control and exposed samples in mm MgCl2, with pmol of each primer The names and primer sequences for the genes studied are listed in Table For an internal PCR control, 28S ribosomal DNA was amplified under the same conditions with sense (5¢-AAGG GCAGGAAAAGAAACTAAC-3¢) and antisense (5¢-GT TTCCCTCTAAGTGGTTTCAC-3¢) primers The resulting PCR products were electrophoresed in a 0.5· TBE ⁄ 1.5% agarose gel, and visualized with UV light after staining with ethidium bromide To minimize differences in RT efficiency, template for all the PCRs were done on lg of the same reverse transcribed product The number of PCR cycles needed to show differential expression between the control and exposed samples was the same (40 cycles) for each gene except for the 28S, where 25 cycles were used to avoid band intensity saturation for optical determination Band intensities were quantified using the gene profiler software (version 4.03, Scanalytics, Inc., Lincoln, NE, USA) Table Sequences of the primers used in the expression study Gene name SSH library Sense primer Antisense primer Glutamine synthetase ATP syntase beta Coelomic factor RNA helicase Procathepsin L Meningioma associated protein Lipoprotein receptor Fucolectin RiboL18A HSCR Senescente associated prot Lysosomalassociated protein Elongation factor2 Apolipophorin Tripartite motif protein TRIM2 Guanyl cyclase ADP ribo RiboL13 ADI and G (up) ADI and G (up) ADI and G (up) ADI (up) G (up) G (up) 5¢-GTGCATCAAAGAATTTTGGATAC-3¢ 5¢-AGAGAAGTGGCAGCTTTCGCTCAGTTTGG-3¢ 5¢-CTCGGCAAAGAAACCGCTGGTTCCTCCCA-3¢ 5¢-GAGACGTCCAGGAAATCTTCCGCAACACC-3¢ 5¢-CAGAGTGTGCACTAGCATGCGGTCCCGT-3¢ 5¢-AGGTGTCCTAGATACCGGCCATGTACCA-3¢ 5¢-TGCAATAATTTTTGAAGCCCCGG-3¢ 5¢-TTAGCATCTGTGGCCTCTGTGATTTGTCC-3¢ 5¢-GCCCCTACCATAACATAGAGGACCCCTGG-3¢ 5¢-CAAATCTACCTGCACGAGCCACTCTGTGC-3¢ 5¢-CACAACCTGGTCGCCGACCGCGGGGACT-3¢ 5¢-TGGACACCTTAGAGACGGTGGCCAGAC-3¢ G (up) ADI and G (up) ADI and G (up) ADI and G (up) ADI (up) 5¢-AGCCTTGATGAGCCAAGGGCAGTGACCT-3¢ 5¢-CATGGCTTCGAATTGATCTTGGAGCTGT-3¢ 5¢-ATACCGTGACCTGACATCCGCTGGTGCT-3¢ 5¢-CCTTTTTAGCAGTGACTTTCCGTTGCAA-3¢ 5¢-TTGCAACGACTGCAGTCATCAGTAGGGT-3¢ 5¢-GGCCCGACGGGTGTCTCTCCAGACCCGT-3¢ 5¢-TAACCTCCAAAAACTTGCACGTCGGCAA-3¢ 5¢-GCACAGTTCCCCTACAGTCCCGCTTTAG-3¢ 5¢-CACGTCTCAGCAGGGAGAATAATCCCGA-3¢ 5¢-GAGCTCAGCGAGGACGGAAACCTCGCGT-3¢ ADI (up) 5¢-CCAATCAGGTAGGCCTTCATGGAGAGGA-3¢ 5¢-CCCAGAGATCCTCCAAGAGACAGCCAGT-3¢ ADI (up) ADI (down) ADI (down) 5¢-ATCTGGAGAGCACATCATTGCTGGTGCA-3¢ 5¢-ACATCGAGGAAGAGTTTTCTATCCTGGA-3¢ 5¢-ACATCGCTGAGAATGTCAACGGGGATAT-3¢ 5¢-CTTTCTGGCCTCTCCAACATCCATGCCA-3¢ 5¢-ATGCCAAGGTAGTTTATGATGATCGAGA-3¢ 5¢-TCTCCTACGATCATCTCACCGTCACCGA-3¢ ADI (down) G (down) ADI (down) 5¢-GGTTGTCAATTTATTGAATGATCTCTACA-3 5¢-CATTTTGACTCGTCCCGATAAACAGGCA-3¢ 5¢-AACTCAGTTGATTGGCCTAGTTATGCCA-3¢ 5¢-CCTCGGATCTTGAGACCCTCGACTGGA-3¢ 5¢-GTTTTACCGGCAGCATCCAACCCCACCA-3¢ 5¢-TTGGGCTTGTTGGCCTCCTCCTGTGCCT-3¢ FEBS Journal 272 (2005) 390–403 ª 2005 FEBS 399 Oyster response to pesticide exposure Statistical analysis To compare mRNA expression between treatment and time of exposure, a statistical analysis using Student’s t-test was performed on the expression data For each treatment and each gene, differences were tested by sampling date pairs (first date vs second date, third date vs second date, etc.) Acknowledgements This research program was financially supported by ´ the Region Bretagne, the interregional program MOREST (Summer Mortality of juvenile oyster Crassostrea gigas, Grant number 02-2-500022) and the ´ ´ Conseil General du Finistere The 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(between and 24 h) used by the authors In another study, 258 differentially expressed genes were identified in C gigas in response to exposure to hydrocarbons for and 21 days [22], and some of these genes. .. among the down -regulated genes Expression of herbicide -regulated genes The time-dependent expression of 13 genes that were up -regulated by exposure to herbicide and five that 391 Oyster response to. .. genes in common to both the ADI and glyphosate response SSH libraries These were glutamine synthase, ATP synthetase, b-1,3-glucan binding protein and some housekeeping genes such as tubulin and