Linbao Zhang

Metabolic responses in gills of Manila clam Ruditapes philippinarum exposed to copper using NMR-based metabolomics

Copper is an important heavy metal contaminant with high ecological risk in the Bohai Sea. In this study, the metabolic responses in the bioindicator, Manila clam (Ruditapes philippinarum), to the environmentally relevant copper exposures were characterized using NMR-based metabolomics. The significant metabolic changes corresponding to copper exposures were related to osmolytes, intermediates of the Krebs cycle and amino acids, such as the increase in homarine, branched chain amino acids and decrease in succinate, alanine and dimethylamine in the copper-exposed clam gills during 96 h exposure period. Overall, Cu may lead to the disturbances in osmotic regulation and energy metabolism in clams during 96 h experimental period. These results demonstrate that NMR-based metabolomics is applicable for the discovery of metabolic biomarkers which could be used to elucidate the toxicological mechanisms of marine heavy metal contaminants.

Benzo(a)pyrene-induced metabolic responses in Manila clam Ruditapes philippinarum by proton nuclear magnetic resonance ( 1 H NMR) based metabolomics

Benzo(a)pyrene is an important polycyclic aromatic hydrocarbon (PAH) which causes carcinogenic, teratogenic and mutagenic effects in various species and the level of contamination of this toxic agent in the marine environment is of great concern. In this study, metabolic responses induced by two doses (0.02 and 0.2μM) of BaP were characterized in the gill tissues of Manila clam Ruditapes philippinarum after exposure for 24, 48 and 96h. The high dose (0.2μM) of BaP induced the disturbances in energy metabolism and osmotic regulation based on the metabolic biomarkers such as succinate, alanine, glucose, glycogen, branched chain amino acids, betaine, taurine, homarine, and dimethylamine in clam gills after 24h of exposure. In addition, hormesis induced by BaP was found in clams exposed to both doses of BaP. Overall, our results demonstrated the applicability of metabolomics for the elucidation of toxicological effects of marine environmental contaminants in a selected bioindicator species such as the Manila clam.

Expression profiles of seven glutathione S-transferase (GST) genes from Venerupis philippinarum exposed to heavy metals and benzo(a)pyrene

Glutathione S-transferases (GSTs) are phase II enzymes that facilitate the detoxification of xenobiotics, and also play important roles in antioxidant defense. In this study, we reported the cloning and molecular characteristics of seven genes of the GST family (VpGSTS1, VpGSTS2, VpGSTS3, VpGSTO, VpGSTMi, VpGSTM and VpGSTR) from Venerupis philippinarum together with mRNA tissue distribution patterns and temporal expression profiles in response to cadmium, copper and benzo[a]pyrene (B[a]P) exposures. The deduced amino acid sequences of VpGSTs showed high similarities to counterparts of other species that clustered into the same clades in the phylogenetic analysis. At basal levels of tissue expression, most VpGSTs were highly expressed in hepatopancreas compared with other tissues. All VpGSTs showed differential response profiles depending on the concentrations of various toxicants and exposure times. More notably, the expressions of VpGSTS2 and VpGSTS3 transcripts were significantly up-regulated in hepatopancreas from Cu and B[a]P-exposed animals, indicating that these two sigma VpGSTs were highly sensitive to Cu and B[a]P exposure. However, the expressions of VpGSTM and VpGSTR were significantly induced by Cu or B[a]P exposure, respectively. These findings suggested the role of VpGSTS2, VpGSTS3, VpGSTM and VpGSTR in defense against oxidative stress and highlighted their potential as biomarkers to Cu or B[a]P exposure.

Toxicological responses to acute mercury exposure for three species of Manila clam Ruditapes philippinarum by NMR-based metabolomics

The Manila clam (Ruditapes philippinarum) has been considered a good sentinel species for metal pollution monitoring in estuarine tidal flats. Along the Bohai coast of China, there are dominantly distributed three species of clams (White, Liangdao Red and Zebra in Yantai population) endowed with distinct tolerances to environmental stressors. In this study, adductor muscle samples were collected from both control and acute mercury exposed White, Liangdao Red and Zebra clams, and the extracts were analyzed by NMR-based metabolomics to compare the metabolic profiles and responses to the acute mercury exposure to determine the most sensitive clam species capable of acting as abioindicator for heavy metal pollution monitoring. The major abundant metabolites in the White clam sample were branched-chain amino acids (leucine, isoleucine and valine), lactate, arginine, aspartate, acetylcholine, homarine and ATP/ADP, while the metabolite profile of Zebra clam sample comprised high levels of glutamine, acetoacetate, betaine, taurine and one unidentified metabolite. For the Liangdao Red clam sample, the metabolite profile relatively exhibited high amount of branched-chain amino acids, arginine, glutamate, succinate, acetylcholine, homarine and two unassigned metabolites. After 48h exposure of 20μgL(-1) Hg(2+), the metabolic profiles showed significant differences between three clam species, which included increased lactate, succinate, taurine, acetylcholine, betaine and homarine and decreased alanine, arginine, glutamine, glutamate, acetoacetate, glycine and ATP/ADP in White clam samples, and elevated succinate, taurine and acetylcholine, and declined glutamine, glycine, and aspartate in Liangdao Red clam samples, while the increased branched-chain amino acids, lactate, succinate, acetylcholine and homarine, and reduced alanine, acetoacetate, glycine and taurine were observed in the Zebra clam samples. Overall, our findings showed that White clams could be a preferable bioindicator for the metal pollution monitoring based on the more sensitive metabolic changes in the adductor muscle compared with other two (Liangdao Red and Zebra) clam species.

Transcriptional regulation of selenium-dependent glutathione peroxidase from Venerupis philippinarum in response to pathogen and contaminants challenge

Glutathione peroxidases (GPx) are key enzymes in the antioxidant systems of living organisms by catalyzing the reduction of peroxides to non-reactive products. In the present study, the full-length cDNA encoding a selenium-dependent GPx was identified from Venerupis philippinarum (designated as VpSe-GPx), and the spatial and temporal expression patterns post-Vibrio anguillarum, heavy metals and benzo[a]pyrene (B[a]P) challenge were also investigated. VpSe-GPx possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase. The VpSe-GPx mRNA was found to be most abundantly expressed in hepatopancreas. Vibrio challenge could significantly up-regulate the mRNA expression of VpSe-GPx, and the highest expression level was detected at 24 h post-infection with 6.5-fold increase compared with that in the control group. For heavy metals exposure, the expression of VpSe-GPx was significantly induced by 20, 40 μg L(-1) Cd and 10, 20 μg L(-1) Cu but depressed by 10 μg L(-1) Cd and 40 μg L(-1) Cu. With regards to B[a]P exposure, the expression of VpSe-GPx mRNA was significantly induced at 48 and 96 h post challenge. All these results suggested that VpSe-GPx was potentially involved in mediating the immune response and antioxidant defense in V. Philippinarum.

Two Goose-Type Lysozymes in Mytilus galloprovincialis: Possible Function Diversification and Adaptive Evolution

Two goose-type lysozymes (designated as MGgLYZ1 and MGgLYZ2) were identified from the mussel Mytilus galloprovincialis. MGgLYZ1 mRNA was widely expressed in the examined tissues and responded sensitively to bacterial challenge in hemocytes, while MGgLYZ2 mRNA was predominately expressed and performed its functions in hepatopancreas. However, immunolocalization analysis showed that both these lysozymes were expressed in all examined tissues with the exception of adductor muscle. Recombinant MGgLYZ1 and MGgLYZ2 could inhibit the growth of several Gram-positive and Gram-negative bacteria, and they both showed the highest activity against Pseudomonas putida with the minimum inhibitory concentration (MIC) of 0.95–1.91 µM and 1.20–2.40 µM, respectively. Protein sequences analysis revealed that MGgLYZ2 had lower isoelectric point and less protease cutting sites than MGgLYZ1. Recombinant MGgLYZ2 exhibited relative high activity at acidic pH of 4–5, while MGgLYZ1 have an optimum pH of 6. These results indicated MGgLYZ2 adapted to acidic environment and perhaps play an important role in digestion. Genomic structure analysis suggested that both MGgLYZ1 and MGgLYZ2 genes are composed of six exons with same length and five introns, indicating these genes were conserved and might originate from gene duplication during the evolution. Selection pressure analysis showed that MGgLYZ1 was under nearly neutral selection while MGgLYZ2 evolved under positive selection pressure with three positively selected amino acid residues (Y102, L200 and S202) detected in the mature peptide. All these findings suggested MGgLYZ2 perhaps served as a digestive lysozyme under positive selection pressure during the evolution while MGgLYZ1 was mainly involved in innate immune responses.

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°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 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 × and 3 × 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 × and 10 × MIC. Overall, these results suggest that VpDef has an important function in host defense against invasive pathogens, probably killing microbes by inducing membrane lesions.

Effects of Salinity on Metabolic Profiles, Gene Expressions, and Antioxidant Enzymes in Halophyte Suaeda salsa

Halophyte Suaeda salsa is native to the saline soil in the Yellow River Delta. Soil salinity can reduce plant productivity and therefore is the most important factor for the degradation of wetlands in the Yellow River Delta. In this work we characterized the salinity-induced effects in S. salsa in terms of metabolic profiling, antioxidant enzyme activities, and gene expression quantification. Our results showed that salinity inhibited plant growth of S. salsa and upregulated gene expression levels of myo-inositol-1-phosphate synthase (INPS), choline monooxygenase (CMO), betaine aldehyde dehydrogenase (BADH), and catalase (CAT), and elevated the activities of superoxide dismutase (SOD), peroxidase (POD), CAT, and glutathione peroxidase (GPx). The significant metabolic responses included the depleted amino acids malate, fumarate, choline, phosphocholine, and elevated betaine and allantoin in the aboveground part of S. salsa seedlings as well as depleted glucose and fructose and elevated proline, citrate, and sucrose in root tissues. Based on these significant biological markers, salinity treatments induced clear osmotic stress (for example, INPS, CMO, BADH, betaine, proline) and oxidative stress (for example, SOD, POD, CAT, GPx activities), disturbed protein biosynthesis/degradation (amino acids and total protein) and energy metabolism (for example, glucose, sucrose, citrate) in S. salsa.

Identification of two small heat shock proteins with different response profile to cadmium and pathogen stresses in Venerupis philippinarum

Small heat shock proteins (sHSPs) encompass a widespread and diverse class of proteins with molecular chaperone activity. In the present study, two sHSP isoforms (VpsHSP-1 and VpsHSP-2) were cloned from Venerupis philippinarum haemocytes by Rapid Amplification of cDNA Ends (RACE) approaches. The expression profiles of these two genes under Vibrio anguillarum challenge and cadmium exposure were investigated by quantitative real-time reverse transcriptase polymerase chain reaction. The bacterial challenge could significantly up-regulate the mRNA expression of both VpsHSP-1 and VpsHSP-2, with the increase of VpsHSP-2 expression occurred earlier than that of VpsHSP-1. During the cadmium exposure experiment, the expression level of both VpsHSP-1 and VpsHSP-2 decreased significantly with larger amplitude in VpsHSP-2. As time progressed, the expression levels of both genes were up-regulated with more increment in the low-chemical exposure groups. The differences in the response to pathogen stimulation and cadmium exposure indicated that there were functional diversity between the two structurally different molecules, VpsHSP-1 and VpsHSP-2, and they probably played distinct roles in mediating the environmental stress and immune responses in calm.