Ammonia nitrogen has been a potential menace to aquatic animals along the coastline of China. Presently, the toxicological effects of ammonia nitrogen were mainly concentrated on fishes, while little attention has been paid to molluscs. In this study, the clam Ruditapes philippinarum was used as the target animal to investigate the toxic effects of ammonia nitrogen. Our results showed that ammonia exposure could significantly reduce the integrity of lysosomes in a dose-dependent manner. Metabolite analysis revealed that exposure doses and duration time of ammonia nitrogen could affect the variation profiles of gill metabolites. In detail, branched chain amino acids, glutamate, choline and phosphocholine were significantly decreased after a one-day exposure. Inosine and phenylalanine were found significantly increased and ATP was decreased after a three-day exposure. The changes of metabolites implied that metabolisms of muscle element, neurotransmission and cell apoptosis of gill tissues would be affected by ammonia exposure. Such inferences were supported by the diminished muscle element, decreased concentrations of catecholamines and increased apoptosis rates, respectively. Therefore, we take advantage of metabolomics integrated with conventional biological assays to find out that ammonia exposure could cause lysosome instability, metabolic disturbance, aberrant gill structures and changes to neurotransmitters, and would result in mollusk gill dysfunction in feeding, respiration and immunity.

•TCPP induced hormesis effect in L02 cells.•TCPP caused ROS overproduction in L02 cells.•The expressions of Bax, Hrk and Bax/Bcl-2 increased significantly in 10−4 M group.•Energy metabolism, signal transduction and cytoskeleton were determined by omics.Organophosphate flame retardants (OPFRs) are widely used as flame retardants which are ubiquitous in various environment media. As many of OPFRs are toxic and persistent, concerns have been raised in regards to their environmental impact. In this study, the toxicological effects of tris(2-chloropropyl) phosphate (TCPP) in human L02 cells was investigated by cell proliferation and apoptosis, oxidative stress, metabolomic and proteomic responses as well as gene expressions related to apoptosis. Results showed that TCPP did not significantly affect the L02 cell apoptosis, however, a significant increase of ROS production was observed in L02 cells with TCPP treatment compared with that in control group (p < 0.05). The expression levels of Bcl-2 family-encoding genes (Bax, Hrk and Bax/Bcl-2) were up-regulated significantly in 10−4 M group (p < 0.05). Metabolomic and proteomic responses indicated that TCPP mainly caused disturbance in cell growth/division and gene expression, energy and material metabolism, signal transduction, defense and cytoskeleton, which was further confirmed by the western blot analysis.

•High levels of OPs in the Bohai and Yellow Seas were detected.•TCPP, TCEP and TPPO were the most dominant OPs.•Levels of OPs were negatively correlated with salinities.•High levels of OPs were found in the surface water and nearshore sites.•Land sources and hydrodynamic condition influence the spatial distributions of OPs.Seawater samples from 50 sites in the BS and YS were collected to investigate the spatial distribution of 7 OPs. Concentrations of the total OPs (ƩOPs) in the BS and YS ranged from 8.12 ng L− 1 to 98.04 ng L− 1 with a geometric mean (GM) of 23.70 ng L− 1. Tris(1-chloro-2-propyl) phosphate (TCPP) was the dominant compound, followed by tris(2-chloroethyl) phosphate (TCEP) and triphenylphosphine oxide (TPPO). The ƩOPs together with the most commonly detected individual OPs (TCPP, TCEP, tris(1,3-dichloro-2-propyl) phosphate (TDCPP), tri-iso-butyl phosphate (TiBP), triphenyl phosphate (TPhP), and TPPO) tended to decrease from nearshore to offshore and from the surface to the bottom seawaters, indicating major land-based sources. Furthermore, the Yellow Sea Cold Water Mass (YSCWM), Changjiang Diluted Water (CDW), Taiwan Warm Current (TaWC), and Subei Coastal Water (SCW) influenced the horizontal and vertical distributions of the OPs in the study area.

Cadmium (Cd) is one of the most important metal contaminants in the Bohai Sea. In this work, NMR-based metabolomics was used to investigate the toxicological effects of Cd at an environmentally relevant concentration (50 µg L−1) in three different life stages (D-shape larval, juvenile and adult) of mussels Mytilus galloprovincialis. Results indicated that the D-shape larval mussel was the most sensitive life stage to Cd. The significantly different metabolic profiles meant that Cd induced differential toxicological effects in three life stages of mussels. Basically, Cd caused osmotic stress in all the three life stages via different metabolic pathways. Cd exposure reduced the anaerobiosis in D-shape larval mussels and disturbed lipid metabolism in juvenile mussels, respectively. Compared with the D-shape larval and juvenile mussels, the adult mussels reduced energy consumption to deal with Cd stress.

•OA affects energy metabolism, stress responses and calcium homeostasis in oysters.•Tissue-specific responses were founded in oyster exposed to elevated pCO2.•Proteomics and metabolomics provided an insightful view into the effects of OA.The gradually increased atmospheric CO2 partial pressure (pCO2) 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 pCO2 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 significanceThe gradually increased atmospheric CO2 partial pressure (pCO2) 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 pCO2 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.

Organophosphate flame retardants (OPFRs) have caused widespread concern because of the harm to the environment. In this study, to better explain the mechanism for the binding of OPFRs with the tumor suppressor gene p53, an integrated experimental and in silico approach was used. The binding constants of 10 OPFRs were measured by surface plasmon resonance technology (SPR). The effect of OPFRs on p53 gene and protein expression in ZF4 cells was determined by quantitative real-time PCR and Western blotting. Molecular docking and dynamics simulation were explored to find that the H-bonds and hydrophobic interactions were the dominant interaction between OPFRs and p53. On the basis of the observed interactions, proper molecular structural descriptors were used to build the quantitative structure–activity relationship (QSAR) model. The current QSAR model provided robustness, predictive ability, and mechanism interpretability. The applicability domain of the QSAR was discussed by the Williams plot. The results showed that H-bonds and electrostatic interaction governed the binding affinities between OPFRs and p53.

•BDE 47 induced dose-dependent effects in E. fetida.•Some protein biomarkers were validated by metabolite biomarkers in same pathways.•Proteomics and metabolomics provided a broader view into pollutant-induced effects.As a class of brominated flame-retardants (BFRs), polybrominated diphenyl ethers (PBDEs) are widely used in industrial products. PBDEs have been detected in local terrestrial biota from the Laizhou Bay in China. They can induce various toxicities, such as hepatotoxicity, neurotoxicity, cytotoxicity, genotoxicity and endocrine disrupting effects in animals. In this work, we characterized the dose-responsive effects of 2,2′,4,4′-tetrabromodiphenyl ether (BDE 47) in earthworm Eisenia fetida using an integrated proteomic and metabolomic approach. Metabolic responses indicated that BDE 47 mainly caused disturbance in osmotic regulation and energy metabolism marked by differentially altered betaine, amino acids, ATP, glucose, maltose and succinate in E. fetida. Proteomic responses revealed that BDE 47 induced cell apoptosis (or injury), oxidative stress, disturbance in protein biosynthesis and energy metabolism in E. fetida in terms of differential proteomic biomarkers. Especially, the increased ATP was confirmed by up-regulated nucleoside diphosphate kinase A and ATP synthase in 1 and 100 μg/L of BDE 47-treated groups, respectively. In addition, several metabolic biomarkers including betaine, glycine and 2-hexyl-5-ethyl-3-furansulfonate were relatively stable in all BDE 47-exposed groups. This work demonstrated that proteomics and metabolomics could partially validate one another and their combination could better understand toxicological effects of environmental pollutants.Biological significanceAs a class of brominated flame-retardants (BFRs), polybrominated diphenyl ethers (PBDEs) are widely used in industrial products and have been detected in local terrestrial biota from the Laizhou Bay in China. Therefore a study on PBDE-induced toxicological effects is necessary. The earthworm E. fetida, a terrestrial sentinel animal, is the most frequently used bioindicator for terrestrial environmental contaminants. To our knowledge, however, very few studies have focused on the dose-dependent responses induced by PBDEs, in terrestrial sentinel animal at protein and metabolite levels. In the present study, an integrated metabolomic and proteomic approach was used to elucidate the dose-dependent toxicological effects of BDE 47 in E. fetida.

•Bacteria induced disruption in osmotic regulation and energy metabolism in mussel.•Both bacteria induced oxidative stress in mussel.•V. anguillarum could cause more severe stress than M. luteus did in mussel.•Proteomics coupled with metabolomics can better understand stressor-caused effects.The outbreak of pathogens can induce diseases and lead to massive mortalities of aquaculture animals including fish, mollusk and shrimp. In this work, the responses induced by Micrococcus luteus and Vibrio anguillarum were investigated in hepatopancreas of mussel Mytilus galloprovincialis using proteomics and metabolomics. Metabolic biomarkers demonstrated that M. luteus and V. anguillarum injections could induce osmotic stress and disturbance in energy metabolism. And the uniquely and more markedly altered metabolic biomarkers (glutamine, succinate, aspartate, glucose, ATP, homarine and tyrosine) indicated that V. anguillarum could cause more severe disturbances in osmotic regulation and energy metabolism. The differentially altered proteins meant that M. luteus and V. anguillarum induced different effects in mussels. However, the common proteomic biomarkers, arginine kinase and small heat shock protein, demonstrated that these two bacteria induced similar effects including oxidative stress and disturbance in energy metabolism in M. galloprovincialis. In addition, some metabolic biomarkers, ATP and glutamine, were confirmed by related proteins including arginine kinase, ATP synthase, nucleoside diphosphate kinase and glutamine synthetase in bacteria-challenged mussels. This study demonstrated that proteomics and metabolomics could provide an insightful view into the effects of environmental pathogens to the marine mussel M. galloprovincialis.Biological significanceThe outbreak of pathogens can lead to diseases and massive mortalities of aquaculture animals including fish, mollusk and shrimp. The mussel M. galloprovincialis distributes widely along the Bohai coast and is popularly consumed as delicious seafood by local residents. This bivalve has become one of the important species in marine aquaculture industry in China. Therefore a study on pathogen-induced effects is necessary. In the present study, an integrated metabolomic and proteomic approach was used to elucidate the differential effects induced by the representative Gram-positive (M. luteus) and Gram-negative (V. anguillarum) bacteria in M. galloprovincialis.

As a pioneer halophyte, Suaeda salsa can grow in the intertidal zones, which are often polluted by heavy metals from both terrigenous wastewater and tidewater containing high concentrations of heavy metals. Therefore S. salsa is potentially suitable as a biomonitor for heavy-metal pollution in the intertidal zones. In this study, regulation of metabolites, gene expression, and antioxidant status of environmentally relevant lead and zinc were characterized using NMR-based metabolomics, real-time quantitative reverse transcription polymerase chain reaction, and antioxidant enzyme activities. In Pb-exposed S. salsa samples, only decreased tyrosine was observed, with statistical significance approaching 0.05. Metabolic biomarkers in Zn-exposed S. salsa samples included increased amino acids (valine, isoleucine, leucine, threonine, asparagine, and phenylalanine), and decreased acetate, glucose, ferulate, and fumarate. Increased succinate, aspartate, and malonate and decreased fructose were uniquely found in mixed Pb- and Zn-exposed samples in addition to the similar metabolic changes such as alanine, glucose, fumarate, and ferulate in Zn-exposed samples. Based on the metabolic biomarkers, gene expressions, and antioxidant enzyme activities, both Zn and mixed Pb and Zn induced significant oxidative stress and disturbances in energy metabolism, photosynthesis/glucogenesis, and protein biodegradation in S. salsa. However, environmentally relevant Pb could induce slight oxidative stress in S. salsa as indicated by increased catalase gene expression levels and catalase activities.

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.

Polychaetes have often been utilized as indicator species to investigate the impacts of pollutants, such as heavy metals. The uptake of Cd by the polychaete Nereis diversicolor was determined at varying Ca concentrations and with pre-exposure to Ca ion channel blockers and metabolic inhibitors in simulated sea water over 1 week period. The supply of Ca in simulated sea water inhibited Cd uptake and increased Ca concentration in N. diversicolor after 10 μM Cd exposure. Pre-exposure to a Ca-channel blocker (Lanthanum) significantly inhibited Cd uptake, suggesting that the uptake of Cd was exerted at a Ca channel. N-ethylmaleimide, which specifically binds to sulfhydryl groups, inhibited Cd uptake at 10 μM, implying that the transport of Cd is carrier-mediated by proteins or other SH-containing compounds. Subcellular Cd distribution analysis showed that more than 60% of the total Cd associated with the cytosolic fraction. The presence of higher concentration of Ca in simulated sea water did not impact the proportional subcellular distribution of Cd in N. diversicolor. Nevertheless, the supply of Ca could significantly lower Cd concentration in cytosol and cellular debris. The present study provides evidence that Cd transport by N. diversicolor was mediated mainly through lanthanum- sensitive Ca ion channels and accumulated by SH-containing compounds. These results help to understand the uptake mechanism and subcellular distribution of Cd in polychaetes.

Sequestration by metallothioneins and antioxidant defense are two kinds of important defense mechanisms employed by mollusks to minimize adverse effects caused by heavy metal contaminants in marine environment. In the present study, a novel metallothionein gene, CgMT-III, was cloned from Crassostrea gigas, consisting of eighteen conserved cysteine residues and encoding a MT III-like protein with two tandem β domains. The expression level of CgMT-III transcript induced by zinc was much higher than that induced by cadmium exposure. It suggested that CgMT-III was perhaps mainly involved in homeostatic control of zinc metabolism, which was distinct from previously identified MTs in C. gigas. Among the tested antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), SOD and GPx showed varying up-regulations in a tissue-specific manner, while CAT activities were inhibited in both gill and hepatopancreas from C. gigas exposed to heavy metals. It can be inferred that CgMT-III was mainly involved in zinc homeostasis, and CgMT-III gene together with CAT enzyme could be potential biomarkers to indicate heavy metal, especially zinc pollution in marine organisms.

Suaeda salsa is a native halophyte in saline soils. Salinity is the most important environmental constraint for plant productivity in the Yellow River Delta. In this work, we investigated the salt-induced effects in root of S. salsa exposed to two environmentally relevant salinities for 1 week and 1 month using nuclear magnetic resonance-based metabolomics. Our results indicated that salt stress inhibited the growth of S. salsa and induced significant metabolic responses including decreased amino acids, lactate, 4-aminobutyrate, malate, choline, phosphocholine, and increased betaine, sucrose, and allantoin in root tissues of S. salsa. In addition, salinity exposures upregulated the activities of superoxide dismutase, glutathione S-transferases, peroxidase, catalase, and glutathione peroxidase in the aboveground part of seedlings of S. salsa after exposures. Overall, these results demonstrated the osmotic and oxidative stresses, disturbances in protein biosynthesis/degradation, and energy metabolism in S. salsa exposed to salinities.

Mercury is a hazardous pollutant in the Bohai marine environments due to its high toxicity to the marine organisms and subsequent ecological risk. Manila clam Ruditapes philippinarum is one of important sentinel organisms in ‘Mussel Watch Program’ launched in China and therefore used as a bioindicator in marine and coastal ecotoxicology. There are dominantly distributed three pedigrees of clam (White, Liangdao Red and Zebra) in Yantai population endowed with different tolerances to environmental stressors. In this study, gill tissues were collected from both untreated and mercury exposed White, Liangdao Red and Zebra clams, and the extracts were analyzed by NMR-based metabolomics to compare the original metabolomes and the toxicological effects induced by mercury exposure in three pedigrees. The major abundant metabolites in White clam sample were branched-chain amino acids, lactate, alanine, arginine, acetoacetate, glutamate, succinate, citrate, malonate and taurine, while the metabolite profile of Liangdao Red clam sample comprises relative high levels of alanine, arginine, glutamate, succinate and glycogen. For Zebra clam sample, the metabolite profile exhibited relatively high amount of aspartate, acetylcholine and homarine. After 48 h exposure of 20 μg l−1 Hg2+, the metabolic profiles from all the three pedigrees of clams commonly showed significant increases in alanine, arginine, glutamate, aspartate, α-ketoglutarate, glycine and ATP/ADP, and decreases in citrate, taurine and homarine. The unique metabolic differences between the metabolomes of gill tissues from Hg2+-exposed White, Liangdao Red and Zebra clams were found, including elevated acetylcholine and branched-chain amino acids in White clams, and the declined succinate in both White and Liangdao Red samples as well as the declined betaine in Zebra and White clams. Overall, our findings showed the differential toxicological responses to mercury exposure and that White clams could be a preferable bioindicator for the metal pollution monitoring based on the metabolic changes from gill compared with other two (Liangdao Red and Zebra) pedigrees of clams.

•Cd is an immune toxicant to D-shape larval mussels.•Cd induced oxidative stress in D-shape larval mussels.•D-shape larval mussels were not sensitive to As (V) at protein level.•Cd and As (V) disturbed energy metabolism and osmotic regulation.Cadmium (Cd) and arsenic (As) are the main metal/metalloid contaminants in the coastal environments of the Bohai Sea, China. In this work, a combined proteomic and metabolomic approach was applied to investigate the biological effects of Cd and As (V) in the early life stage (D-shape larvae) of mussel Mytilus galloprovincialis. Results indicated that Cd was a potential immune toxicant to D-shape larval mussel because of the numerous proteomic responses related to immune system. Additionally, Cd induced oxidative stress, cellular injury and disturbance in nucleic acid metabolism in D-shape larval mussels. However, only two identified proteins were significantly altered in As (V)-treated group, suggesting that D-shape larval mussel was less sensitive to As (V) than to Cd at protein level. These two proteins in response to As (V) suggested that As (V) influenced anti-oxidative system and cell proliferation in D-shape larval mussels. Metabolic responses indicated that Cd and As (V) induced disturbances in osmotic regulation and energy metabolism in D-shape larval mussels via different metabolic pathways. In addition, Cd reduced lipid metabolism as well. This work demonstrated that a combination of proteomics and metabolomics could provide an insightful view in the biological effects of pollutants in mussel M. galloprovincialis at an early life stage.

•We cloned a gene of an invertebrate defensin from the clam V. philippinarum.•VpDef was constitutively expressed in hemocytes and mantles of the clam.•VpDef transcripts were significantly induced after Vibrio anguillarum infection.•rVpDef showed the highest activity against Micrococcus luteus.•VpDef appears to kill microbes by inducing membrane lesions.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.

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.The toxicological effects in clam R. philippinarum induced by environmentally relevant concentrations of copper were characterized using NMR-based metabolomics.Download full-size imageHighlights► The metabolic responses induced by Cu were characterized in Manila clam. ► Copper induced disturbances in osmotic regulation and energy metabolism in clams. ► Our results showed applicability of metabolomics for metabolic biomarker discovery.

•Reduced salinities enhanced proteolysis in juvenile flounder P. olivaceus.•Reduced salinities disturbed osmotic regulation and energy metabolism in flounder.•Reduced salinities induced immune stress and oxidative stress in juvenile flounder.•The low salinity (15.6‰) enhanced anaerobic metabolism in juvenile flounder.•Reduced salinities promoted growth and gonadal differentiation in juvenile flounder.Seawater salinity is one of the most important changeable environmental factors influencing the behavior, survival, growth and production of marine organisms. In this work, metabolite and gene expression profiles were used to elucidate the biological effects of reduced salinities in juvenile flounder Paralichthys olivaceus. Metabolic profiling indicated that both reduced salinities (23.3‰ and 15.6‰) enhanced proteolysis and disturbed osmotic regulation and energy metabolism in juvenile flounder P. olivaceus. Furthermore, the low salinity (15.6‰) enhanced anaerobic metabolism indicated by the elevated lactate in flounder tissue extracts. Gene expression profiles exhibited that reduced salinities could induce immune stress and oxidative stress and disturb energy metabolism in juvenile flounder P. olivaceus. In addition, reduced salinities might promote the growth and gonadal differentiation in juvenile flounder P. olivaceus.

•Metal pollutions caused differential responses in oysters from Baijiao and Fugong.•Metal pollutions induced oxidative and immune stresses in oysters.•Cathepsin L might be used as a biomarker of As in oysters.•Ferritin GF1 might be the biomarker of Fe in oysters.•Proteomics is useful to study the effects induced by metal pollutions in organisms.The metal pollution has posed great risk on the coastal organisms along the Jiulongjiang Estuary in South China. In this work, two-dimensional electrophoresis-based proteomics was applied to the oysters Crassostrea hongkongensis from metal pollution sites to characterize the proteomic responses to metal pollution. Metal accumulation and proteomic responses indicated that the oysters from BJ site were more severely contaminated than those from FG site. Compared with those oyster samples from the clean site (JZ), metal pollution induced cellular injuries, oxidative and immune stresses in oyster heapatopancreas from both BJ and FG sites via differential metabolic pathways. In addition, metal pollution in BJ site induced disturbance in energy and lipid metabolisms in oysters. Results indicated that cathepsin L and ferritin GF1 might be the biomarkers of As and Fe in oyster C. hongkongensis, respectively. This study demonstrates that proteomics is a useful tool for investigating biological effects induced by metal pollution.

•YRE and LZB are mainly polluted by Cd and As, respectively.•Metal pollutions caused differential effects in C. affinis from different sites.•Metabolomics is useful to elucidate metal pollution-induced biological effects.Both cadmium and arsenic are the important metal/metalloid pollutants in the Bohai Sea. In this work, we sampled the dominant species, shrimp Crangon affinis, from three sites, the Middle of the Bohai Sea (MBS), the Yellow River Estuary (YRE) and the Laizhou Bay (LZB) along the Bohai Sea. The concentrations of metals/metalloids in shrimps C. affinis indicated that the YRE site was polluted by Cd and Pb, while the LZB site was contaminated by As. The metabolic differences between shrimps C. affinis from the reference site (MBS) and metal-pollution sites (YRE and LZB) were characterized using NMR-based metabolomics. Results indicated that the metal pollutions in YRE and LZB induced disturbances in osmotic regulation and energy metabolism via different metabolic pathways. In addition, a combination of alanine and arginine might be the biomarker of Cd contamination, while BCAAs and tyrosine could be the biomarkers of arsenic contamination in C. affinis.

•Metal pollutions caused differential metabolic changes in oysters from different sites.•Metal pollutions disturbed osmotic regulation and energy metabolism in oysters.•Metabolomcis is useful to study metal pollution-induced effects in organisms.Metal pollution has become a great threat to organisms in the estuaries in South China. In the present study, the oysters Crassostrea sikamea were collected from one clean (Jiuzhen) and five metal polluted sites (Baijiao, Fugong, Gongqian, Jinshan and Songyu). The tissue metal concentrations in oysters indicated that the five metal sites were polluted by several metals, including Cr, Ni, Co, Cu, Zn, Ag, Cd and Pb with different patterns. Especially, Cu and Zn were the major contaminants in Baijiao, Fugong and Jinshan sites. The metabolic responses in oysters C. sikamea indicated that the metal pollutions in BJ, FG, JS and SY sites induced disturbances in osmotic regulation and energy metabolism via different metabolic pathways. However, the metal pollution in GQ site mainly influenced the osmotic regulation in the oysters C. sikamea. This study demonstrates that NMR-based metabolomics is useful to characterize metabolic responses induced by metal pollution.

•The S6283 and S5283 sites were polluted by metals, such as Cd, As and Cu.•Metal pollution caused multiple biological effects in shrimp C. affinis.•Metal pollution in two sites induced osmotic stress via different pathways.Marine environment in the Laizhou Bay is potentially contaminated by metals from industrial discharges. In this study, metal concentrations in shrimps Crangon affinis indicated that two typical sites (S6283 and S5283) close to Longkou and Zhaoyuan cities along the Laizhou Bay have been contaminated by metals, including Cd, As, Cu, Ni, Co, and Mn. In particular, Cd and As were the main metal contaminants in S6283. In S5283, however, Cu was the most important metal contaminant. The metabolic responses in the shrimps indicated that the metal pollution in S6283 and S5283 induced disturbances in osmotic regulation and energy metabolism and reduced anaerobiosis, lipid metabolism, and muscle movement. However, alteration in the levels of dimethylglycine, dimethylamine, arginine, betaine, and glutamine indicated that the metal pollution in S5283 induced osmotic stress through different pathways compared to that in S6283. In addition, dimethylamine might be the biomarker of Cu in shrimp C. affinis.