PMC:3820681 / 24108-30459 JSONTXT

{"project":"2_test","denotations":[{"id":"24244519-18550164-86861358","span":{"begin":1422,"end":1424},"obj":"18550164"},{"id":"24244519-18550164-86861359","span":{"begin":1868,"end":1870},"obj":"18550164"},{"id":"24244519-20549629-86861360","span":{"begin":1871,"end":1873},"obj":"20549629"},{"id":"24244519-19926125-86861361","span":{"begin":2249,"end":2251},"obj":"19926125"},{"id":"24244519-23277104-86861362","span":{"begin":2252,"end":2254},"obj":"23277104"},{"id":"24244519-1379544-86861363","span":{"begin":3281,"end":3283},"obj":"1379544"},{"id":"24244519-22771962-86861364","span":{"begin":3284,"end":3286},"obj":"22771962"},{"id":"24244519-22771962-86861365","span":{"begin":3357,"end":3359},"obj":"22771962"},{"id":"24244519-10099689-86861366","span":{"begin":3721,"end":3723},"obj":"10099689"},{"id":"24244519-23357950-86861367","span":{"begin":4127,"end":4129},"obj":"23357950"},{"id":"24244519-16257553-86861368","span":{"begin":4871,"end":4873},"obj":"16257553"},{"id":"24244519-21915694-86861368","span":{"begin":4871,"end":4873},"obj":"21915694"},{"id":"T71187","span":{"begin":1422,"end":1424},"obj":"18550164"},{"id":"T80645","span":{"begin":1868,"end":1870},"obj":"18550164"},{"id":"T33580","span":{"begin":1871,"end":1873},"obj":"20549629"},{"id":"T19267","span":{"begin":2249,"end":2251},"obj":"19926125"},{"id":"T21933","span":{"begin":2252,"end":2254},"obj":"23277104"},{"id":"T67730","span":{"begin":3281,"end":3283},"obj":"1379544"},{"id":"T66906","span":{"begin":3284,"end":3286},"obj":"22771962"},{"id":"T6362","span":{"begin":3357,"end":3359},"obj":"22771962"},{"id":"T97722","span":{"begin":3721,"end":3723},"obj":"10099689"},{"id":"T71739","span":{"begin":4127,"end":4129},"obj":"23357950"},{"id":"T90049","span":{"begin":4871,"end":4873},"obj":"16257553"},{"id":"T29648","span":{"begin":4871,"end":4873},"obj":"21915694"}],"text":"Identification and validation of putative biomarker transcriptomic sequences\nTo verify the assembly and annotation results and to identify potential environmental pollution biomarkers, 15 related assembled unigenes, including five antioxidase genes (Cu/Zn superoxide dismutase, Cu/Zn SOD), glutathione peroxidase A (GPx-A) and mu glutathione S-transferase (GST-mu), thioredoxin peroxidase 1(TPX1) and thioredoxin peroxidase 2 (TPX2), two cytochrome P450 genes (CYP4 and CYP30), three GABA receptor-related genes (GABA neurotransmitter transporter 1, GABAT1; GABAA receptor-associated protein, GABARAP; and GABAA receptor-associated protein-like 2, GABARAPL2) and five HSP genes (Hsp22, Hsp40, Hsp60, Hsp70 and Hsp90), were selected and subjected to RT-PCR and real-time PCR analyses (Table S6).\nThe antioxidant defense system (including SOD, GPx, GST and TPX) is very effective at reducing damage when oxidative stress has occurred. SOD catalyzes the transformation of superoxide radicals to H2O2 and O2 and represents the initial response to oxyradicals. GPx then catalyzes the reduction of H2O2. GST belongs to a multifunctional family of cytosolic enzymes involved in phase II biotransformation, which plays an important role in protecting tissues from oxidative stress. Eight classes of GST isoenzymes can be distinguished based on their substrate specificity, immunological properties and protein sequences homology [55]. Thioredoxin peroxidase (TPX) is a member of proteins that are conserved from yeast to mammals and to which natural killer enhancing factor belongs. These proteins are antioxidants that function as peroxidases only when coupled to a sulfhydryl reducing system.  Previous studies have suggested that SOD, CAT, Se-GPx and GST-pi transcript levels in Corbicula fluminea could be used as biomarkers of Cu and Cd exposure in aquatic environments [55,56]. The identification of Cu/Zn SOD, GPx-A, GST-mu, TPX1 and TPX2 genes in the present study would allow for the use of C. fluminea antioxidant defense system assays in pollutant monitoring.\nThe cytochrome P450s (CYPs) comprise one of the largest and most versatile protein families in living organisms and are involved in a variety of detoxification and endogenous functions [57,58]. In bivalve mollusks, the CYPs are poorly studied, as there only a few CYP genes with known regulatory mechanisms that have been identified in mussel and oyster, including CYP1-like and CYP3-like genes. The study of CYP1A in C. fluminea showed that this gene was a useful biomarker for exposure to PCBs [59]. The CfCYP4 and CfCYP30 genes were identified based on transcript sequences obtained using specific primers in this present work. The identification of these genes will facilitate an improved understanding of CYPs in bivalves.\nGABARAP, an important protein in the autophagy process, is evolutionarily conserved and is involved in innate immunity in eukaryotic cells. In invertebrates, GABARAP is the major inhibitory neurotransmitter in synapses of both the central and peripheral nervous systems [60]. The GABARAP subfamily consists of GABARAP, GABARAPL1, GABARAPL2 and GABARAPL3. Recently, the GABARAP and related genes have been isolated from several mollusks, such as Lymnaea stagnalis and Haliotis diversicolor [61,62]. However, the actual biological functions of GABARAP remain elusive [62]. In this study, we identified three C. fluminea GABARAPs: GABAT1, GABARAP and GABARAPL2. These results will help us gain a better understanding of the molecular and physiological processes involving GABARAPs in mollusks.\nHSPs are commonly used as biomarkers of exposure to various stressors (such as temperature, metal toxicity, toxic exposure and infection) [63]. These proteins comprise a group of highly conserved, yet highly diversified, proteins that primarily function as molecular chaperones, stabilizing protein folding and preventing indiscriminate protein interactions by sequestering unfolded proteins, which can be found in diverse organisms from bacteria to mammals [64]. The major HSP families are Hsp60, Hsp70, Hsp90 and the small HSPs family (sHsp) [65]. The sHsp family is a heterogeneous group of proteins of intermediate molecular weight (12-43kDa), such as Hsp22 and Hsp40.The Hsp70 protein family is considered to be the major HSP family and has been the most extensively studied. Under adverse environmental conditions, Hsp70 can improve expression levels and takes part in the defense, repair or detoxification machinery of the cell by tightly binding denatured proteins. In bivalve species, many studies have shown that the transcription of Hsp70 is simultaneously and differentially modulated upon exposure to environmental stressors [66,67]. Recent studies reported that Hsp90 can be regulated by a range of stressors such as food deprivation, heavy metal exposure and thermal shock [68-70]. In this study, we sought to identify sequences in the C. fluminea transcriptome that encode Hsp22, Hsp40, Hsp60, Hsp70 and Hsp90. Based on the results of unigenes annotation (BLASTx with the E \u003c1e-5), we identified five putative HSP (22, 40, 60, 70 and 90) sequences corresponding to unigenes loci.\nPartial sequences for all 15 of the aforementioned potential biomarker genes were cloned from adult female C. fluminea digestive gland cDNAs and compared with the assembled sequences using RT-PCR. The products of RT-PCR were analyzed using agarose gel electrophoresis (Figure 5), RT-PCR primers and Sanger sequencing data are shown in Table S6. The consensus sequence data for the selected genes have been deposited with GenBank under the accession numbers shown in Table 1. The sequence variation was minimal (\u003e99% nucleotide identity) and was associated with either the heterogeneity of the C. fluminea colony, which was annually outbred with local conspecifics, or with sequencing errors introduced during Sanger sequencing of the RT-PCR products.\n10.1371/journal.pone.0079516.g005 Figure 5 RT-PCR analyses of the 15 potential C. fluminea genes.\nPCR was performed using the cDNA prepared from adult clams. 1: Cf(Cu/Zn)SOD,234bp;2: CfGPx-A,372bp;3:CfGST-mu,369bp;4:CfTPX1,393bp;5:CfTPX2,273bp;6:CfCYP4,459bp;7:CfCYP30,612bp;8: CfGABAT1,510bp;9:CfGABARAP,276bp;10:CfGABARAPL2,264bp;11:CfHsp22,282bp;12: CfHsp40,672bp;13:CfHsp60,477bp;14:CfHsp70,467bp;15:CfHsp90,570bp.\n\nR"}

{"project":"NEUROSES","denotations":[{"id":"T202","span":{"begin":42,"end":51},"obj":"CHEBI_59163"},{"id":"T203","span":{"begin":256,"end":266},"obj":"CHEBI_18421"},{"id":"T204","span":{"begin":969,"end":979},"obj":"CHEBI_18421"},{"id":"T205","span":{"begin":290,"end":301},"obj":"CHEBI_16856"},{"id":"T206","span":{"begin":330,"end":341},"obj":"CHEBI_16856"},{"id":"T207","span":{"begin":290,"end":301},"obj":"CHEBI_57925"},{"id":"T208","span":{"begin":330,"end":341},"obj":"CHEBI_57925"},{"id":"T209","span":{"begin":302,"end":312},"obj":"CHEBI_8027"},{"id":"T210","span":{"begin":378,"end":388},"obj":"CHEBI_8027"},{"id":"T211","span":{"begin":1439,"end":1449},"obj":"CHEBI_8027"},{"id":"T212","span":{"begin":366,"end":377},"obj":"CHEBI_15033"},{"id":"T213","span":{"begin":401,"end":412},"obj":"CHEBI_15033"},{"id":"T214","span":{"begin":1427,"end":1438},"obj":"CHEBI_15033"},{"id":"T215","span":{"begin":366,"end":377},"obj":"CHEBI_15967"},{"id":"T216","span":{"begin":401,"end":412},"obj":"CHEBI_15967"},{"id":"T217","span":{"begin":1427,"end":1438},"obj":"CHEBI_15967"},{"id":"T218","span":{"begin":484,"end":488},"obj":"CHEBI_16865"},{"id":"T219","span":{"begin":513,"end":517},"obj":"CHEBI_16865"},{"id":"T220","span":{"begin":518,"end":534},"obj":"CHEBI_25512"},{"id":"T221","span":{"begin":573,"end":583},"obj":"PATO_0001668"},{"id":"T222","span":{"begin":621,"end":631},"obj":"PATO_0001668"},{"id":"T223","span":{"begin":584,"end":591},"obj":"CHEBI_16541"},{"id":"T224","span":{"begin":632,"end":639},"obj":"CHEBI_16541"},{"id":"T225","span":{"begin":1394,"end":1401},"obj":"CHEBI_16541"},{"id":"T226","span":{"begin":584,"end":591},"obj":"CHEBI_36080"},{"id":"T227","span":{"begin":632,"end":639},"obj":"CHEBI_36080"},{"id":"T228","span":{"begin":1394,"end":1401},"obj":"CHEBI_36080"},{"id":"T229","span":{"begin":1471,"end":1479},"obj":"CHEBI_36080"},{"id":"T230","span":{"begin":668,"end":671},"obj":"CHEBI_50430"},{"id":"T231","span":{"begin":765,"end":769},"obj":"PATO_0001309"},{"id":"T232","span":{"begin":765,"end":769},"obj":"PATO_0000165"},{"id":"T233","span":{"begin":799,"end":810},"obj":"CHEBI_22586"},{"id":"T234","span":{"begin":890,"end":896},"obj":"PATO_0001020"},{"id":"T235","span":{"begin":980,"end":988},"obj":"CHEBI_26519"},{"id":"T236","span":{"begin":992,"end":996},"obj":"CHEBI_30492"},{"id":"T237","span":{"begin":1092,"end":1096},"obj":"CHEBI_30492"},{"id":"T238","span":{"begin":992,"end":996},"obj":"CHEBI_16240"},{"id":"T239","span":{"begin":1092,"end":1096},"obj":"CHEBI_16240"},{"id":"T240","span":{"begin":256,"end":266},"obj":"CHEBI_18421"},{"id":"T241","span":{"begin":969,"end":979},"obj":"CHEBI_18421"},{"id":"T242","span":{"begin":290,"end":301},"obj":"CHEBI_16856"},{"id":"T243","span":{"begin":330,"end":341},"obj":"CHEBI_16856"},{"id":"T244","span":{"begin":290,"end":301},"obj":"CHEBI_57925"},{"id":"T245","span":{"begin":330,"end":341},"obj":"CHEBI_57925"},{"id":"T246","span":{"begin":302,"end":312},"obj":"CHEBI_8027"},{"id":"T247","span":{"begin":378,"end":388},"obj":"CHEBI_8027"},{"id":"T248","span":{"begin":1439,"end":1449},"obj":"CHEBI_8027"},{"id":"T249","span":{"begin":366,"end":377},"obj":"CHEBI_15033"},{"id":"T250","span":{"begin":401,"end":412},"obj":"CHEBI_15033"},{"id":"T264","span":{"begin":632,"end":639},"obj":"CHEBI_36080"},{"id":"T265","span":{"begin":1394,"end":1401},"obj":"CHEBI_36080"},{"id":"T266","span":{"begin":1471,"end":1479},"obj":"CHEBI_36080"},{"id":"T267","span":{"begin":668,"end":671},"obj":"CHEBI_50430"},{"id":"T268","span":{"begin":765,"end":769},"obj":"PATO_0001309"},{"id":"T269","span":{"begin":765,"end":769},"obj":"PATO_0000165"},{"id":"T270","span":{"begin":799,"end":810},"obj":"CHEBI_22586"},{"id":"T271","span":{"begin":890,"end":896},"obj":"PATO_0001020"},{"id":"T272","span":{"begin":980,"end":988},"obj":"CHEBI_26519"},{"id":"T273","span":{"begin":992,"end":996},"obj":"CHEBI_30492"},{"id":"T274","span":{"begin":1092,"end":1096},"obj":"CHEBI_30492"},{"id":"T275","span":{"begin":992,"end":996},"obj":"CHEBI_16240"},{"id":"T276","span":{"begin":1092,"end":1096},"obj":"CHEBI_16240"},{"id":"T277","span":{"begin":1171,"end":1176},"obj":"PATO_0000083"},{"id":"T278","span":{"begin":1224,"end":1228},"obj":"CHEBI_50906"},{"id":"T279","span":{"begin":1581,"end":1589},"obj":"CHEBI_36080"},{"id":"T280","span":{"begin":2138,"end":2145},"obj":"CHEBI_36080"},{"id":"T281","span":{"begin":2813,"end":2820},"obj":"CHEBI_36080"},{"id":"T282","span":{"begin":1594,"end":1606},"obj":"CHEBI_22586"},{"id":"T283","span":{"begin":2004,"end":2015},"obj":"CHEBI_22586"},{"id":"T284","span":{"begin":1612,"end":1620},"obj":"PATO_0000173"},{"id":"T285","span":{"begin":1730,"end":1733},"obj":"CHEBI_32402"},{"id":"T286","span":{"begin":1951,"end":1958},"obj":"PATO_0000467"},{"id":"T287","span":{"begin":2680,"end":2687},"obj":"PATO_0000467"},{"id":"T288","span":{"begin":2067,"end":2077},"obj":"CHEBI_4056"},{"id":"T289","span":{"begin":2138,"end":2145},"obj":"CHEBI_16541"},{"id":"T290","span":{"begin":2813,"end":2820},"obj":"CHEBI_16541"},{"id":"T291","span":{"begin":2327,"end":2330},"obj":"CHEBI_38559"},{"id":"T292","span":{"begin":2528,"end":2537},"obj":"CHEBI_59163"},{"id":"T293","span":{"begin":2688,"end":2692},"obj":"PATO_0001026"},{"id":"T294","span":{"begin":2838,"end":2845},"obj":"PATO_0000006"},{"id":"T295","span":{"begin":2981,"end":2997},"obj":"CHEBI_25512"},{"id":"T296","span":{"begin":3034,"end":3044},"obj":"PATO_0002107"},{"id":"T297","span":{"begin":3661,"end":3672},"obj":"PATO_0000146"},{"id":"T298","span":{"begin":3674,"end":3679},"obj":"CHEBI_33521"},{"id":"T299","span":{"begin":3732,"end":3740},"obj":"CHEBI_36080"},{"id":"T300","span":{"begin":3803,"end":3811},"obj":"CHEBI_36080"},{"id":"T301","span":{"begin":3919,"end":3926},"obj":"CHEBI_36080"},{"id":"T302","span":{"begin":3965,"end":3973},"obj":"CHEBI_36080"},{"id":"T303","span":{"begin":4176,"end":4184},"obj":"CHEBI_36080"},{"id":"T304","span":{"begin":4264,"end":4271},"obj":"CHEBI_36080"},{"id":"T305","span":{"begin":3752,"end":3757},"obj":"CHEBI_24433"},{"id":"T306","span":{"begin":4167,"end":4172},"obj":"CHEBI_24433"},{"id":"T307","span":{"begin":3827,"end":3835},"obj":"PATO_0000173"},{"id":"T308","span":{"begin":3919,"end":3926},"obj":"CHEBI_16541"},{"id":"T309","span":{"begin":4264,"end":4271},"obj":"CHEBI_16541"},{"id":"T310","span":{"begin":3904,"end":3918},"obj":"PATO_0001318"},{"id":"T311","span":{"begin":4056,"end":4059},"obj":"CHEBI_50430"},{"id":"T312","span":{"begin":4309,"end":4312},"obj":"CHEBI_50430"},{"id":"T313","span":{"begin":4101,"end":4106},"obj":"PATO_0000587"},{"id":"T314","span":{"begin":4211,"end":4217},"obj":"PATO_0000128"},{"id":"T315","span":{"begin":4546,"end":4554},"obj":"CHEBI_36080"},{"id":"T316","span":{"begin":4729,"end":4735},"obj":"PATO_0001484"},{"id":"T317","span":{"begin":4813,"end":4817},"obj":"CHEBI_33290"},{"id":"T318","span":{"begin":4831,"end":4836},"obj":"PATO_0000582"},{"id":"T319","span":{"begin":5108,"end":5111},"obj":"CHEBI_50430"},{"id":"T320","span":{"begin":5239,"end":5248},"obj":"CHEBI_59163"},{"id":"T321","span":{"begin":5278,"end":5284},"obj":"PATO_0000383"},{"id":"T322","span":{"begin":5418,"end":5425},"obj":"CHEBI_2511"},{"id":"T323","span":{"begin":5680,"end":5687},"obj":"PATO_0002123"},{"id":"T324","span":{"begin":5694,"end":5704},"obj":"CHEBI_36976"},{"id":"T325","span":{"begin":5750,"end":5763},"obj":"PATO_0002048"},{"id":"T251","span":{"begin":1427,"end":1438},"obj":"CHEBI_15033"},{"id":"T252","span":{"begin":366,"end":377},"obj":"CHEBI_15967"},{"id":"T253","span":{"begin":401,"end":412},"obj":"CHEBI_15967"},{"id":"T254","span":{"begin":1427,"end":1438},"obj":"CHEBI_15967"},{"id":"T255","span":{"begin":484,"end":488},"obj":"CHEBI_16865"},{"id":"T256","span":{"begin":513,"end":517},"obj":"CHEBI_16865"},{"id":"T257","span":{"begin":518,"end":534},"obj":"CHEBI_25512"},{"id":"T258","span":{"begin":573,"end":583},"obj":"PATO_0001668"},{"id":"T259","span":{"begin":621,"end":631},"obj":"PATO_0001668"},{"id":"T260","span":{"begin":584,"end":591},"obj":"CHEBI_16541"},{"id":"T261","span":{"begin":632,"end":639},"obj":"CHEBI_16541"},{"id":"T262","span":{"begin":1394,"end":1401},"obj":"CHEBI_16541"},{"id":"T263","span":{"begin":584,"end":591},"obj":"CHEBI_36080"}],"text":"Identification and validation of putative biomarker transcriptomic sequences\nTo verify the assembly and annotation results and to identify potential environmental pollution biomarkers, 15 related assembled unigenes, including five antioxidase genes (Cu/Zn superoxide dismutase, Cu/Zn SOD), glutathione peroxidase A (GPx-A) and mu glutathione S-transferase (GST-mu), thioredoxin peroxidase 1(TPX1) and thioredoxin peroxidase 2 (TPX2), two cytochrome P450 genes (CYP4 and CYP30), three GABA receptor-related genes (GABA neurotransmitter transporter 1, GABAT1; GABAA receptor-associated protein, GABARAP; and GABAA receptor-associated protein-like 2, GABARAPL2) and five HSP genes (Hsp22, Hsp40, Hsp60, Hsp70 and Hsp90), were selected and subjected to RT-PCR and real-time PCR analyses (Table S6).\nThe antioxidant defense system (including SOD, GPx, GST and TPX) is very effective at reducing damage when oxidative stress has occurred. SOD catalyzes the transformation of superoxide radicals to H2O2 and O2 and represents the initial response to oxyradicals. GPx then catalyzes the reduction of H2O2. GST belongs to a multifunctional family of cytosolic enzymes involved in phase II biotransformation, which plays an important role in protecting tissues from oxidative stress. Eight classes of GST isoenzymes can be distinguished based on their substrate specificity, immunological properties and protein sequences homology [55]. Thioredoxin peroxidase (TPX) is a member of proteins that are conserved from yeast to mammals and to which natural killer enhancing factor belongs. These proteins are antioxidants that function as peroxidases only when coupled to a sulfhydryl reducing system.  Previous studies have suggested that SOD, CAT, Se-GPx and GST-pi transcript levels in Corbicula fluminea could be used as biomarkers of Cu and Cd exposure in aquatic environments [55,56]. The identification of Cu/Zn SOD, GPx-A, GST-mu, TPX1 and TPX2 genes in the present study would allow for the use of C. fluminea antioxidant defense system assays in pollutant monitoring.\nThe cytochrome P450s (CYPs) comprise one of the largest and most versatile protein families in living organisms and are involved in a variety of detoxification and endogenous functions [57,58]. In bivalve mollusks, the CYPs are poorly studied, as there only a few CYP genes with known regulatory mechanisms that have been identified in mussel and oyster, including CYP1-like and CYP3-like genes. The study of CYP1A in C. fluminea showed that this gene was a useful biomarker for exposure to PCBs [59]. The CfCYP4 and CfCYP30 genes were identified based on transcript sequences obtained using specific primers in this present work. The identification of these genes will facilitate an improved understanding of CYPs in bivalves.\nGABARAP, an important protein in the autophagy process, is evolutionarily conserved and is involved in innate immunity in eukaryotic cells. In invertebrates, GABARAP is the major inhibitory neurotransmitter in synapses of both the central and peripheral nervous systems [60]. The GABARAP subfamily consists of GABARAP, GABARAPL1, GABARAPL2 and GABARAPL3. Recently, the GABARAP and related genes have been isolated from several mollusks, such as Lymnaea stagnalis and Haliotis diversicolor [61,62]. However, the actual biological functions of GABARAP remain elusive [62]. In this study, we identified three C. fluminea GABARAPs: GABAT1, GABARAP and GABARAPL2. These results will help us gain a better understanding of the molecular and physiological processes involving GABARAPs in mollusks.\nHSPs are commonly used as biomarkers of exposure to various stressors (such as temperature, metal toxicity, toxic exposure and infection) [63]. These proteins comprise a group of highly conserved, yet highly diversified, proteins that primarily function as molecular chaperones, stabilizing protein folding and preventing indiscriminate protein interactions by sequestering unfolded proteins, which can be found in diverse organisms from bacteria to mammals [64]. The major HSP families are Hsp60, Hsp70, Hsp90 and the small HSPs family (sHsp) [65]. The sHsp family is a heterogeneous group of proteins of intermediate molecular weight (12-43kDa), such as Hsp22 and Hsp40.The Hsp70 protein family is considered to be the major HSP family and has been the most extensively studied. Under adverse environmental conditions, Hsp70 can improve expression levels and takes part in the defense, repair or detoxification machinery of the cell by tightly binding denatured proteins. In bivalve species, many studies have shown that the transcription of Hsp70 is simultaneously and differentially modulated upon exposure to environmental stressors [66,67]. Recent studies reported that Hsp90 can be regulated by a range of stressors such as food deprivation, heavy metal exposure and thermal shock [68-70]. In this study, we sought to identify sequences in the C. fluminea transcriptome that encode Hsp22, Hsp40, Hsp60, Hsp70 and Hsp90. Based on the results of unigenes annotation (BLASTx with the E \u003c1e-5), we identified five putative HSP (22, 40, 60, 70 and 90) sequences corresponding to unigenes loci.\nPartial sequences for all 15 of the aforementioned potential biomarker genes were cloned from adult female C. fluminea digestive gland cDNAs and compared with the assembled sequences using RT-PCR. The products of RT-PCR were analyzed using agarose gel electrophoresis (Figure 5), RT-PCR primers and Sanger sequencing data are shown in Table S6. The consensus sequence data for the selected genes have been deposited with GenBank under the accession numbers shown in Table 1. The sequence variation was minimal (\u003e99% nucleotide identity) and was associated with either the heterogeneity of the C. fluminea colony, which was annually outbred with local conspecifics, or with sequencing errors introduced during Sanger sequencing of the RT-PCR products.\n10.1371/journal.pone.0079516.g005 Figure 5 RT-PCR analyses of the 15 potential C. fluminea genes.\nPCR was performed using the cDNA prepared from adult clams. 1: Cf(Cu/Zn)SOD,234bp;2: CfGPx-A,372bp;3:CfGST-mu,369bp;4:CfTPX1,393bp;5:CfTPX2,273bp;6:CfCYP4,459bp;7:CfCYP30,612bp;8: CfGABAT1,510bp;9:CfGABARAP,276bp;10:CfGABARAPL2,264bp;11:CfHsp22,282bp;12: CfHsp40,672bp;13:CfHsp60,477bp;14:CfHsp70,467bp;15:CfHsp90,570bp.\n\nR"}