Ruiwen Cao

Combined metabolome and proteome analysis of the mantle tissue from Pacific oyster Crassostrea gigas exposed to elevated pCO2

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 pCO2 condition was investigated in mantle tissue of Crassostrea gigas by an integrated metabolomic and proteomic approach. Analysis of metabolome and proteome revealed that elevated pCO2 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 pCO2 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.

Molecular characterization, expression and antimicrobial activities of two c-type lysozymes from manila clam Venerupis philippinarum

&NA; Lysozymes play an important role in the innate immune responses with which mollusks respond to bacterial invasion through its lytic activity. In the present study, two c‐type lysozymes (designed as VpCLYZ‐1 and VpCLYZ‐2, respectively) were identified and characterized from the manila clam Venerupis philippinarum. The full‐length cDNA of VpCLYZ‐1 and VpCLYZ‐2 was of 629 and 736 bp, encoding a polypeptide of 156 and 153 amino acid residues, respectively. The deduced amino acid sequences of VpCLYZs showed high similarity to other known invertebrate c‐type lysozymes. Multiple alignments and phylogenetic relationship strongly suggested that VpCLYZ‐1 and VpCLYZ‐2 belonged to the c‐type lysozyme family. Both VpCLYZ‐1 and VpCLYZ‐2 transcripts were constitutively expressed in a wide range of tissues with different levels. The VpCLYZ‐1 transcript was dominantly expressed in hepatopancreas and hemocytes, while VpCLYZ‐2 transcript was mainly expressed in the tissues of hepatopancreas and gills. Both the mRNA expression of VpCLYZ‐1 and VpCLYZ‐2 was significantly up‐regulated at 12 h post Vibrio anguillarum challenge. The recombinant VpCLYZ‐1 and VpCLYZ‐2 (designed as rVpCLYZ‐1 and rVpCLYZ‐2) exhibited lytic activity against all tested bacteria, and rVpCLYZ‐1 showed higher activities than rVpCLYZ‐2 in killing Micrococcus luteus and V. anguillarum. Overall, our results suggested that VpCLYZ‐1 and VpCLYZ‐2 belonged to the c‐type lysozyme family, and played important roles in the immune responses of manila clam, especially in the elimination of pathogens. HighlightsTwo c‐type lysozymes were identified from the manila clam Venerupis philippinarum.The transcripts of VpCLYZs were constitutively expressed in all examined tissues.The mRNA expression levels of VpCLYZs were up‐regulated post bacterial challenge.rVpCLYZ‐1 showed higher activity than rVpCLYZ‐2 in killing Micrococcus luteus and Vibrio anguillarum.The muramidase and chitinase activities may contribute to the elimination of bacteria and fungi.

Molecular characterization and antibacterial activity of a phage-type lysozyme from the Manila clam, Ruditapes philippinarum

&NA; A phage‐type lysozyme, designed as RpPLYZ, was cloned and characterized from the clam Ruditapes philippinarum. The full‐length cDNA of RpPLYZ was of 699 bp with an open reading frame (ORF) of 534 bp, encoding a polypeptide of 177‐amino acid with a calculated molecular mass of 19.6 kDa and an isoelectric point of 9.05. Multiple alignments and phylogenetic analysis strongly suggested that RpPLYZ was a new member of the phage‐type lysozyme family. The mRNA transcript of RpPLYZ was found to be constitutively expressed in a wide range of tissues and mainly in hemocytes and mantle. The relative expression of RpPLYZ mRNA in hemocytes was significantly up‐regulated at 6, 24, 48 and 72 h after Vibrio anguillarum challenge. The recombinant RpPLYZ (rRpPLYZ) showed high activity against Entherobacter cloacae and Staphyloccocus aureus, and less effective towards Entherobacter aerogenes and V. anguillarum. Moreover, the optimal pH, temperature and ionic strength for rRpPLYZ activity was determined to be 5.5, 50 °C and 5 mM, respectively. These results suggested that RpPLYZ was a member of the phage‐type lysozyme family and perhaps played an important role in the immune responses against bacterial invasion. HighlightsA phage‐type lysozyme was identified in the Manila clam, Ruditapes philippinarum.RpPLYZ showed antibacterial activities against both gram‐negative and gram‐positive bacteria.The optimal pH, temperature and ionic strength for rRpPLYZ was of 5.5, 50 °C and 5 mM, respectively.

CO2-induced ocean acidification impairs the immune function of the Pacific oyster against Vibrio splendidus challenge: An integrated study from a cellular and proteomic perspective

Ocean acidification (OA) and pathogenic diseases pose a considerable threat to key species of marine ecosystem. However, few studies have investigated the combined impact of reduced seawater pH and pathogen challenge on the immune responses of marine invertebrates. In this study, Pacific oysters, Crassostrea gigas, were exposed to OA (~2000 ppm) for 28 days and then challenged with Vibrio splendidus for another 72 h. Hemocyte parameters showed that V. splendidus infection exacerbated the impaired oyster immune responses under OA exposure. An iTRAQ-based quantitative proteomic analysis revealed that C. gigas responded differently to OA stress and V. splendidus challenge, alone or in combination. Generally, OA appears to act via a generalized stress response by causing oxidative stress, which could lead to cellular injury and cause disruption to the cytoskeleton, protein turnover, immune responses and energy metabolism. V. splendidus challenge in oysters could suppress the immune system directly and lead to a disturbed cytoskeleton structure, increased protein turnover and energy metabolism suppression, without causing oxidative stress. The combined OA- and V. splendidus-treated oysters ultimately presented a similar, but stronger proteomic response pattern compared with OA treatment alone. Overall, the impaired oyster immune functions caused by OA exposure may have increased the risk of V. splendidus infection. These results have important implications for the impact of OA on disease outbreaks in marine invertebrates, which would have significant economic and ecological repercussions.

Seawater acidification aggravated cadmium toxicity in the oyster Crassostrea gigas: Metal bioaccumulation, subcellular distribution and multiple physiological responses

Mounting evidence has demonstrated the combined effects of ocean acidification (OA) and other environmental stressors on marine organisms. Although metal pollution is widely distributed in coasts and estuaries, the combined effects of OA and metal pollution have received little attention until recent years. In this study, the accumulation and subcellular distribution of cadmium (Cd) and the physiological responses of the oyster Crassostrea gigas were investigated after 31 days of exposure to OA and Cd, either alone or in combination. Increased Cd accumulation was found both in gills (about 57% increase at pH 7.8, 22% increase at pH 7.6) and digestive glands (about 38% increase at pH 7.8, 22% increase at pH 7.6) of C. gigas under elevated pCO2 exposure. Although a similar total Cd accumulation pattern was seen in oyster gills and digestive glands, a higher partition of Cd in the BIM (biologically inactive metal) fractions of gills (about 60%) was found in Cd-exposed treatments compared to the digestive glands (about 45%), which might correspond to the generally lower toxicity in gills. Moreover, synergetic effects of Cd and OA on the oxidative stresses, histopathological damage, and apoptosis of exposed oysters were observed in this study, which might be explained by significant interactions of these two factors on increased generation of ROS. These findings demonstrated that OA could aggravate the toxicity of metals in marine organisms, with significant implications for coastal benthic ecosystems regarding the widespread metal contamination and the concurrent increase of acidified seawater.

Microplastic pollution in sediments from the Bohai Sea and the Yellow Sea, China.

Microplastics are one of the most significant pollutants in the marine environment and accumulate in sediments all over the world. To assess the pollution level in the marine environment in China, the distribution and abundance of microplastics in sediments from the Bohai Sea and the Yellow Sea were investigated in this study. The sediment samples were collected from 72 different sites in the Bohai Sea and the Yellow Sea. Microplastics were separated from sediment through density flotation and categorized according to shape and size under a microscope. Additionally, polymer types were identified using Fourier-Transform Infrared Micro-spectroscopy (μ-FT-IR). Our study demonstrated that microplastics were consistently found in all samples, which emphasized their extensive distribution throughout the Bohai Sea and the Yellow Sea. The average microplastic abundance was 171.8, 123.6 and 72.0 items per kg of dry weight sediment for the Bohai Sea, Northern Yellow Sea and Southern Yellow Sea, respectively. Among the sampled microplastics, fiber (93.88%) and small microplastics (<1000 μm) (71.06%) were the most frequent types. Fourier transform infrared microspectroscopy (μ-FT-IR) analysis determined that the main types of microplastics were rayon (RY), polyethylene (PE) and polyethylene terephthalate (PET). Our results highlighted the widespread distribution of microplastics in sediments from the Bohai Sea and the Yellow Sea and provided useful information for evaluating the environmental risks of microplastics in China.

Seawater Acidification Reduced the Resistance of Crassostrea gigas to Vibrio splendidus Challenge: An Energy Metabolism Perspective

Negative physiological impacts induced by exposure to acidified seawater might sensitize marine organisms to future environmental stressors, such as disease outbreak. The goal of this study was to evaluate if ocean acidification (OA) could reduce the resistance capability of the Pacific oyster (Crassostrea gigas) to Vibrio splendidus challenge from an energy metabolism perspective. In this study, the Pacific oyster was exposed to OA (pH 7.6) for 28 days and then challenged by V. splendidus for another 72 h. Antioxidative responses, lipid peroxidation, metabolic (energy sensors, aerobic metabolism, and anaerobic metabolism) gene expression, glycolytic enzyme activity, and the content of energy reserves (glycogen and protein) were investigated to evaluate the environmental risk of pathogen infection under the condition of OA. Our results demonstrated that following the exposure to seawater acidification, oysters exhibited an energy modulation with slight inhibition of aerobic energy metabolism, stimulation of anaerobic metabolism, and increased glycolytic enzyme activity. However, the energy modulation ability and antioxidative regulation of oysters exposed to seawater acidification may be overwhelmed by a subsequent pathogen challenge, resulting in increased oxidative damage, decreased aerobic metabolism, stimulated anaerobic metabolism, and decreased energy reserves. Overall, although anaerobic metabolism was initiated to partially compensate for inhibited aerobic energy metabolism, increased oxidative damage combined with depleted energy reserves suggested that oysters were in an unsustainable bioenergetic state and were thereby incapable of supporting long-term population viability under conditions of seawater acidification and a pathogen challenge from V. splendidus.

Integrative Biomarker Assessment of the Influence of Saxitoxin on Marine Bivalves: A Comparative Study of the Two Bivalve Species Oysters, Crassostrea gigas, and Scallops, Chlamys farreri

Harmful algae blooms have expanded greatly in recent decades, and their secreted toxins pose a severe threat to human health and marine ecosystems. Saxitoxin (STX) is a main paralytic shellfish poison naturally produced by marine microalgae of the genus Alexandrium. Despite numerous studies have assessed the impacts of STX on marine bivalves, comparative in vivo study on the toxicity of STX on bivalves with distinct accumulation ability (such as oysters and scallops) has been seldom investigated. The aim of this study was to identify whether distinct sensitivity exists between oysters, Crassostrea gigas, and scallops, Chlamys farreri under the same amount of STX exposure using multiple biomarker responses. The responses of different biochemical markers including oxidative stress markers (catalase, superoxide dismutase, glutathione S-transferase, and lipid peroxidation) and immunotoxicity biomarkers (hemocyte phagocytosis rate, reactive oxidative species production, and DNA damages) were evaluated in bivalves after 12, 48, and 96 h of exposure to STX. The integrated biomarker responses value combined with two-way ANOVA analysis suggested that STX posed slightly severer stress on scallops than oysters for the extended period of time. This study provided preliminary results on the usefulness of a multi-biomarker approach to assess the toxicity associated with STX exposure in marine bivalves.

The impact of ocean acidification and cadmium on the immune responses of Pacific oyster, Crassostrea gigas

&NA; Seawater acidification (OA) and cadmium (Cd) has the potential to lead to immunosuppression effect on marine bivalves. However, the interaction between these two environmental stressors on immune system of marine bivalves has received limited attention. In order to evaluate the defense responses of oysters under the combined exposure to OA and cadmium, the oysters Crassostrea gigas were exposed to 10 &mgr;g/L Cd at three pH levels (8.1, 7.8 and 7.6) for 31 days. Results showed that OA exposure alone led to increased DNA damage, apoptosis rate and ROS production of hemocytes. However, inhibited phagocytosis rate, combined with increased DNA damage, apoptosis rate and ROS production of hemocytes were observed in oysters under exposure to Cd exposure alone or combined with OA. Significant interactive effects between OA and Cd were observed on ROS production and DNA damage of hemocytes. In addition, there is generally significant increase in the mRNA expression of genes related to immune‐related TLR pathway and two immune factors (TNF and integrin beta‐1B) in Cd‐exposed oysters at pH 7.6. The results revealed that even though the mRNA expression of genes related to immune responses (TLR pathway and immune factors) was stimulated to counteract the immunosuppression caused by acidified seawater and Cd, depressed hemocyte function perhaps sensitized oysters to potential pathogen infection. HighlightsInhibited phagocytosis, along with increased DNA damage, apoptosis rate and ROS production found in hemocytes of oysters exposed to Cd or Cd‐OA.Significant interactive effects between OA and Cd were observed on ROS production and DNA damage of hemocytes.mRNA expression of genes related to immune responses (TLR pathway and immune factors) was stimulated in oysters exposed to Cd and OA at pH 7.6.