A Novel C-Type Lysozyme from Mytilus galloprovincialis: Insight into Innate Immunity and Molecular Evolution of Invertebrate C-Type Lysozymes

A c-type lysozyme (named as MgCLYZ) gene was cloned from the mussel Mytilus galloprovincialis. Blast analysis indicated that MgCLYZ was a salivary c-type lysozyme which was mainly found in insects. The nucleotide sequence of MgCLYZ was predicted to encode a polypeptide of 154 amino acid residues with the signal peptide comprising the first 24 residues. The deduced mature peptide of MgCLYZ was of a calculated molecular weight of 14.4 kD and a theoretical isoelectric point (pI) of 8.08. Evolution analysis suggested that bivalve branch of the invertebrate c-type lysozymes phylogeny tree underwent positive selection during evolution. By quantitative real-time RT-PCR (qRT-PCR) analysis, MgCLYZ transcript was widely detected in all examined tissues and responded sensitively to bacterial challenge in hemocytes and hepatopancreas. The optimal temperature and pH of recombinant MgCLYZ (rMgCLYZ) were 20 degrees C and 4, respectively. The rMgCLYZ displayed lytic activities against Gram-positive bacteria including Micrococcus luteus and Staphyloccocus aureus, and Gram-negative bacteria including Vibrio anguillarum, Enterobacter cloacae, Pseudomonas putida, Proteus mirabilis and Bacillus aquimaris. These results suggest that MgCLYZ perhaps play an important role in innate immunity of M. galloprovincialis, and invertebrate c-type lysozymes might be under positive selection in a species-specific manner during evolution for undergoing adaptation to different environment and diverse pathogens.A c-type lysozyme (named as MgCLYZ) gene was cloned from the mussel Mytilus galloprovincialis. Blast analysis indicated that MgCLYZ was a salivary c-type lysozyme which was mainly found in insects. The nucleotide sequence of MgCLYZ was predicted to encode a polypeptide of 154 amino acid residues with the signal peptide comprising the first 24 residues. The deduced mature peptide of MgCLYZ was of a calculated molecular weight of 14.4 kD and a theoretical isoelectric point (pI) of 8.08. Evolution analysis suggested that bivalve branch of the invertebrate c-type lysozymes phylogeny tree underwent positive selection during evolution. By quantitative real-time RT-PCR (qRT-PCR) analysis, MgCLYZ transcript was widely detected in all examined tissues and responded sensitively to bacterial challenge in hemocytes and hepatopancreas. The optimal temperature and pH of recombinant MgCLYZ (rMgCLYZ) were 20 degrees C and 4, respectively. The rMgCLYZ displayed lytic activities against Gram-positive bacteria including Micrococcus luteus and Staphyloccocus aureus, and Gram-negative bacteria including Vibrio anguillarum, Enterobacter cloacae, Pseudomonas putida, Proteus mirabilis and Bacillus aquimaris. These results suggest that MgCLYZ perhaps play an important role in innate immunity of M. galloprovincialis, and invertebrate c-type lysozymes might be under positive selection in a species-specific manner during evolution for undergoing adaptation to different environment and diverse pathogens

Proteomic and metabolomic responses of Pacific oyster Crassostrea gigas to elevated pCO(2) exposure

The gradually increased atmospheric CO2 partial pressure (pCO(2)) has thrown the carbonate chemistry off balance and resulted in decreased seawater pH in marine ecosystem, termed ocean acidification (OA). Anthropogenic OA is postulated to affect the physiology of many marine calcifying organisms. However, the susceptibility and metabolic pathways of change in most calcifying animals are still far from being well understood. In this work, the effects of exposure to elevated pCO(2) were characterized in gills and hepatopancreas of Crassostrea gigas using integrated proteomic and metabolomic approaches. Metabolic responses indicated that high CO2 exposure mainly caused disturbances in energy metabolism and osmotic regulation marked by differentially altered ATP, glucose, glycogen, amino acids and organic osmolytes in oysters, and the depletions of ATP in gills and the accumulations of ATP, glucose and glycogen in hepatopancreas accounted for the difference in energy distribution between these two tissues. Proteomic responses suggested that OA could not only affect energy and primary metabolisms, stress responses and calcium homeostasis in both tissues, but also influence the nucleotide metabolism in gills and cytoskeleton structure in hepatopancreas. This study demonstrated that the combination of proteomics and metabolomics could provide an insightful view into the effects of OA on oyster C. gigas. Biological significance The gradually increased atmospheric CO2 partial pressure (pCO(2)) has thrown the carbonate chemistry off balance and resulted in decreased seawater pH in marine ecosystem, termed ocean acidification (OA). Anthropogenic OA is postulated to affect the physiology of many marine calcifying organisms. However, the susceptibility and metabolic pathways of change in most calcifying animals are still far from being understood. To our knowledge, few studies have focused on the responses induced by pCO(2) at both protein and metabolite levels. The pacific oyster C. gigas, widely distributed throughout most of the world's oceans, is a model organism for marine environmental science. In the present study, an integrated metabolomic and proteomic approach was used to elucidate the effects of ocean acidification on Pacific oyster C. gigas, hopefully shedding light on the physiological responses of marine mollusk to the OA stress. (C) 2014 Elsevier B.V. All rights reserved

Molecular characterization of two glutathione peroxidase genes in Mytilus galloprovincialis and their transcriptional responses to sub-chronic arsenate and cadmium exposure

Glutathione peroxidases (GPxs) are key enzymes in the antioxidant defense system of living organisms, and protect organisms against oxidative stresses. In this study, the full-length cDNA sequences encoding cytosolic GPx (MgcGPx) and phospholipid-hydroperoxide GPx (MgGPx4) were identified from Mytilus galloprovincialis. The mussels were exposed to 0, 1, 10, and 100 mu g/L cadmium and arsenate for 30 days. The mRNA transcripts of these two genes and total GPx activity were examined in the gills and digestive gland after contaminants exposure. The mussels exposed to cadmium and arsenate responded mainly by down-regulating MgcGPx and MgGPx4 mRNA transcription in gills and up-regulating transcription in digestive gland. However, total GPx activities increased following cadmium exposure but decreased after arsenate stress in both tissues. These results suggest that MgcGPx and MgGPx4 perhaps play an important role in maintaining cellular redox homeostasis and protecting M. galloprovincialis against cadmium and arsenate toxicity. It can also be inferred that these genes have the potential to be used as molecular biomarkers for assessing cellular stress and toxicity of contaminants in this mussel.Glutathione peroxidases (GPxs) are key enzymes in the antioxidant defense system of living organisms, and protect organisms against oxidative stresses. In this study, the full-length cDNA sequences encoding cytosolic GPx (MgcGPx) and phospholipid-hydroperoxide GPx (MgGPx4) were identified from Mytilus galloprovincialis. The mussels were exposed to 0, 1, 10, and 100 mu g/L cadmium and arsenate for 30 days. The mRNA transcripts of these two genes and total GPx activity were examined in the gills and digestive gland after contaminants exposure. The mussels exposed to cadmium and arsenate responded mainly by down-regulating MgcGPx and MgGPx4 mRNA transcription in gills and up-regulating transcription in digestive gland. However, total GPx activities increased following cadmium exposure but decreased after arsenate stress in both tissues. These results suggest that MgcGPx and MgGPx4 perhaps play an important role in maintaining cellular redox homeostasis and protecting M. galloprovincialis against cadmium and arsenate toxicity. It can also be inferred that these genes have the potential to be used as molecular biomarkers for assessing cellular stress and toxicity of contaminants in this mussel

Identification and mRNA expression of two 17 beta-hydroxysteroid dehydrogenase genes in the marine mussel Mytilus galloprovincialis following exposure to endocrine disrupting chemicals

17 beta-Hydroxysteroid dehydrogenases (17 beta-HSDs) are multifunctional enzymes involved in the metabolism of steroids, fatty acids, retinoids and bile acid. In this study, two novel types of 17 beta-HSDs (named as MgHsd17b10 and MgHsd17b12) were cloned from Mytilus galloprovincialis by using rapid amplification of cDNA ends (RACE) approaches. Sequence analysis showed that MgHsd17b10 and MgHsd17b12 encoded a polypeptide of 259 and 325 amino acids, respectively. Phylogenetic analysis revealed that MgHsd17b10 and MgHsd17b12 were evolutionarily clustered with other invertebrate 17 beta-HSD type 10 and 17 beta-HSD type 12 homologues. The MgHsd17b10 and MgHsd17b12 transcripts could be detected in all examined tissues with higher expression levels in digestive glands and gonad. After exposed to endocrine disrupting chemicals (Bisphenol A or 2,2',4,4'-tetrabromodiphenyl ether), the expression of MgHsd17b10 and MgHsd17b12 transcripts was both down-regulated in digestive glands. These findings suggest that MgHsd17b10 and MgHsd17b12 perhaps play an important role in the endocrine regulation of M. galloprovincialis. (C) 2014 Elsevier B.V. All rights reserved.17 beta-Hydroxysteroid dehydrogenases (17 beta-HSDs) are multifunctional enzymes involved in the metabolism of steroids, fatty acids, retinoids and bile acid. In this study, two novel types of 17 beta-HSDs (named as MgHsd17b10 and MgHsd17b12) were cloned from Mytilus galloprovincialis by using rapid amplification of cDNA ends (RACE) approaches. Sequence analysis showed that MgHsd17b10 and MgHsd17b12 encoded a polypeptide of 259 and 325 amino acids, respectively. Phylogenetic analysis revealed that MgHsd17b10 and MgHsd17b12 were evolutionarily clustered with other invertebrate 17 beta-HSD type 10 and 17 beta-HSD type 12 homologues. The MgHsd17b10 and MgHsd17b12 transcripts could be detected in all examined tissues with higher expression levels in digestive glands and gonad. After exposed to endocrine disrupting chemicals (Bisphenol A or 2,2',4,4'-tetrabromodiphenyl ether), the expression of MgHsd17b10 and MgHsd17b12 transcripts was both down-regulated in digestive glands. These findings suggest that MgHsd17b10 and MgHsd17b12 perhaps play an important role in the endocrine regulation of M. galloprovincialis. (C) 2014 Elsevier B.V. All rights reserved

A defensin from clam Venerupis philippinarum: Molecular characterization, localization, antibacterial activity, and mechanism of action

Antimicrobial peptides (AMPs) are important mediators of the primary host defense system against microbial invasion. In the present study, we cloned and characterized a member of the invertebrate defensin from the clam Venerupis philippinarum, designated VpDef. Amino acid sequence analysis showed that VpDef was similar to defensins from marine mollusks and ticks. In non-stimulated clams, RT-PCR and immunohistochemical analysis revealed that both VpDef mRNA and the encoding peptide were constitutively expressed in hemocytes and mantles, as well as in other major tissues. VpDef transcripts were significantly induced in hemocytes at different time intervals post Vibrio anguillarum infection. The recombinant VpDef (rVpDef) showed the highest activity against Gram-positive bacteria Micrococcus luteus and less effective to Gram-negative bacteria. In addition, incubation of rVpDef with M. luteus at 1 x and 3 x MIC could induce an obvious decrease of the membrane potential and notable changes of membrane permeability in a dose-dependent manner. Membrane integrity and bacterial viability analysis also revealed that rVpDef increased the membrane permeability of M. luteus and then resulted in cell death at 2 x and 10 x MIC. Overall, these results suggest that VpDef has an important function in host defense against invasive pathogens, probably killing microbes by inducing membrane lesions. (C) 2015 Elsevier Ltd. All rights reserved

Combined metabolome and proteome analysis of the mantle tissue from Pacific oyster Crassostrea gigas exposed to elevated pCO(2)

Ocean acidification (OA) has been found to affect an array of normal physiological processes in mollusks, especially posing a significant threat to the fabrication process of mollusk shell. In the current study, the impact of exposure to elevated pCO(2) condition was investigated in mantle tissue of Crassostrea gigas by an integrated metabolomic and proteomic approach. Analysis of metabolome and proteome revealed that elevated pCO(2) could affect energy metabolism in oyster C. gigas, marked by differentially altered ATP, succinate, MDH, PEPCK and ALDH levels. Moreover, the up-regulated calponin-2, tropomyosins and myosin light chains indicated that elevated pCO(2) probably caused disturbances in cytoskeleton structure in mantle tissue of oyster C.gigas. This work demonstrated that a combination of proteomics and metabolomics could provide important insights into the effects of OA at molecular levels. (C) 2014 Elsevier Inc. All rights reserved.Ocean acidification (OA) has been found to affect an array of normal physiological processes in mollusks, especially posing a significant threat to the fabrication process of mollusk shell. In the current study, the impact of exposure to elevated pCO(2) condition was investigated in mantle tissue of Crassostrea gigas by an integrated metabolomic and proteomic approach. Analysis of metabolome and proteome revealed that elevated pCO(2) could affect energy metabolism in oyster C. gigas, marked by differentially altered ATP, succinate, MDH, PEPCK and ALDH levels. Moreover, the up-regulated calponin-2, tropomyosins and myosin light chains indicated that elevated pCO(2) probably caused disturbances in cytoskeleton structure in mantle tissue of oyster C.gigas. This work demonstrated that a combination of proteomics and metabolomics could provide important insights into the effects of OA at molecular levels. (C) 2014 Elsevier Inc. All rights reserved

Cloning and expression of a transcription factor activator protein-1 (AP-1) member identified from manila clam Venerupis philippinarum

The transcription factor activator protein-1 (AP-1) proteins are implicated to play a major role in the regulation of numerous genes involved in the function and development of the immune system, cell differentiation, proliferation, apoptosis, etc. It can bind to promoter of its target genes in a sequence-specific manner to transactivate or repress them. In this study, the full-length cDNA of an AP-1 was identified from Venerupis philippinarum (denoted as VpAP-1) by EST analysis and RACE approaches. Phylogenetic analysis indicated that VpAP-1 had higher evolutional conservation to invertebrate than vertebrate counterparts and should be a new member of the AP-1 protein family. Spatial expression analysis found that VpAP-1 transcript was most abundantly expressed in the hemocytes and hepatopancreas, weakly expressed in the tissues of the gills, mantle and muscle. After Vibrio anguillarum challenge, the expression of VpAP-1 transcript in overall hemocyte population was up-regulated in the first 6 h, and then decreased to 1.5-fold of the control group at 12 h. As time progressed, a second peak of VpAP-1 expression was detected at 24 h post-infection, which was 5-fold compared with that of the control group (P < 0.01). After that, the expression level was sharply decreased and dropped to 0.5-fold of the control at 96 h. The above results indicated that VpAP-1 was perhaps involved in the immune responses against microbe infection and might be contributed to the clearance of bacterial pathogens. (C) 2014 Published by Elsevier B.V.The transcription factor activator protein-1 (AP-1) proteins are implicated to play a major role in the regulation of numerous genes involved in the function and development of the immune system, cell differentiation, proliferation, apoptosis, etc. It can bind to promoter of its target genes in a sequence-specific manner to transactivate or repress them. In this study, the full-length cDNA of an AP-1 was identified from Venerupis philippinarum (denoted as VpAP-1) by EST analysis and RACE approaches. Phylogenetic analysis indicated that VpAP-1 had higher evolutional conservation to invertebrate than vertebrate counterparts and should be a new member of the AP-1 protein family. Spatial expression analysis found that VpAP-1 transcript was most abundantly expressed in the hemocytes and hepatopancreas, weakly expressed in the tissues of the gills, mantle and muscle. After Vibrio anguillarum challenge, the expression of VpAP-1 transcript in overall hemocyte population was up-regulated in the first 6 h, and then decreased to 1.5-fold of the control group at 12 h. As time progressed, a second peak of VpAP-1 expression was detected at 24 h post-infection, which was 5-fold compared with that of the control group (P < 0.01). After that, the expression level was sharply decreased and dropped to 0.5-fold of the control at 96 h. The above results indicated that VpAP-1 was perhaps involved in the immune responses against microbe infection and might be contributed to the clearance of bacterial pathogens. (C) 2014 Published by Elsevier B.V

Effects of ocean acidification on immune responses of the Pacific oyster Crassostrea gigas

Ocean acidification (OA), caused by anthropogenic CO(2)emissions, has been proposed as one of the greatest threats in marine ecosystems. A growing body of evidence shows that ocean acidification can impact development, survival, growth and physiology of marine calcifiers. In this study, the immune responses of the Pacific oyster Crassostrea gigas were investigated after elevated pCO(2) exposure for 28 days. The results demonstrated that OA caused an increase of apoptosis and reactive oxygen species (ROS) production in hemocytes. Moreover, elevated pCO(2) had an inhibitory effect on some antioxidant enzyme activities and decreased the GSH level in digestive gland. However, the mRNA expression pattern of several immune related genes varied depending on the exposure time and tissues. After exposure to pCO(2) at 2000 ppm for 28 days, the mRNA expressions of almost all tested genes were significantly suppressed in gills and stimulated in hemocytes. Above all, our study demonstrated that elevated pCO(2) have a significant impact on the immune systems of the Pacific oyster, which may constitute as a potential threat to increased susceptibility of bivalves to diseases. (C) 2015 Elsevier Ltd. All rights reserved

Molecular diversity and evolution of defensins in the manila clam Ruditapes philippinarum

Four types of defensins were identified in Manila clam and designated as Rpdef1, Rpdef2, Rpdef3 and Rpdef4, which encoded a polypeptide of 49, 46, 45 and 42 amino acids, respectively. Sequence alignments indicated that Rpdef1 shared 46.9% identity with Rpdef2, 40.8% with Rpdef3, and 34.7% with Rpdef4. Analysis of transcript polymorphism showed that Rpdef3 accounted for about 60% frequency of Rpdefs occurrence in clams from three geographic origins (Dalian, Qingdao and Hangzhou). By quantitative real-time RT-PCR (qRT-PCR) analysis, the transcripts of Rpdefs were mainly detected in hemocytes and they responded sensitively to bacterial challenge in hemocytes. Evolutionary analysis indicated that all Rpdefs were under positive selection with positively selected basic amino acid residues detected in the C-terminal regions, which perhaps have a functional relevance by modifying the charge distribution of Rpdefs. The results also showed some lineages with dN/dS > 1, suggesting positive selection pressures existed in some lineages of phylogeny tree constructed by mollusk defensins. Overall, our results suggest that Rpdefs perhaps played important roles in host defense and positive selection is the major driving force in generating high diversity of defensins in the Manila clam. (C) 2015 Elsevier Ltd. All rights reserved