Jiachao Xu

Soluble adenylyl cyclase mediates mitochondrial pathway of apoptosis and ATP metabolism in oyster Crassostrea gigas exposed to elevated CO2

Abstract Ocean acidification (OA) has deleterious impacts on immune response and energy homeostasis status of Mollusca. In the present study, the apoptosis ratio of hemocytes and the adenosine triphosphate (ATP) allocation in gill tissues were determined after Pacific oysters Crassostrea gigas were exposed to elevated CO2 environment (pH = 7.50) for 16 days.The apoptosis ratio in CO2 exposure group (35.2%) was significantly higher (p < 0.05) than that in the control group, and the increased apoptosis ratio induced by elevated CO2 could be significantly inhibited (p < 0.05) by KH7, a specific inhibitor of a bicarbonate sensor soluble adenylyl cyclase (sAC). After CO2 exposure, sAC in oyster (CgsAC) was found to be clustered with mitochondria in the cytoplasm, and the pro‐caspase‐3 was cleaved into two small fragments. Moreover, the activities of caspase‐3 and caspase‐9 also increased post CO2 exposure and these increases could be inhibited by KH7. However, the activities of caspase‐8 did not change significantly compared with that in the control group. After CO2 exposure, the ATP content in the gill increased significantly (p < 0.05) and such increase could also be inhibited by KH7. The ATP content in purified gill mitochondria decreased significantly (p < 0.05) after CO2 exposure, which was also inhibited by KH7. These results implied that the elevated CO2 could activate the mitochondria‐CgsAC pathway of apoptosis and ATP metabolism in oyster, and this pathway played essential roles in maintaining the homeostasis and the balance of energy metabolism in response to OA. HighlightsCgsAC mediates the increase of apoptosis rate caused by CO2 exposure.The signal of CgsAC was clustered with mitochondria after CO2 exposure.CgsAC mediates the increase of Caspase‐3 and caspase‐9 activity caused by CO2 exposure.CgsAC mediates the increase of ATP in gill tissue caused by CO2 exposure.CgsAC mediates the decrease of ATP in gill mitochondria caused by CO2 exposure.

The sequence variation and functional differentiation of CRDs in a scallop multiple CRDs containing lectin.

ABSTRACT A C‐type lectin of multiple CRDs (CfLec‐4) from Chlamys farreri was selected to investigate the sequence variation and functional differentiation of its CRDs. Its four CRDs with EPD/LSD, EPN/FAD, EPN/LND and EPN/YND key motifs were recombined separately. The recombinant proteins of CRD1 and CRD2 (designated as rCRD1 and rCRD2) could bind LPS and mannan, while the recombinant proteins of CRD3 and CRD4 (designated as rCRD3 and rCRD4) could bind LPS, PGN, mannan and glucan. Moreover, rCRD3 displayed broad microbe binding spectrum towards Gram‐positive bacteria Staphylococcus aureus and Micrococcus luteus, Gram‐negative bacteria Escherichia coli and Vibrio anguillarum, as well as fungi Pichia pastoris and Yarrowia lipolytica. These results indicated CRD3 contributed more to CfLec‐4s nonself‐recognition ability. Furthermore, CRD1, CRD3 and CRD4 functioned as opsonin participating in the clearance against invaders in scallops. The sequence variation in Ca2+ binding site 2 among CRDs was suspected to be associated with such functional differentiation. HighlightsThe sequences of Ca2+‐binding site 2 in CRDs of CfLec‐4 were diversified.Functional differentiation was observed among the CRDs of CfLec‐4.CRD3 and CRD4 were the dominant CRDs for CfLec‐4s PAMP recognition.CRD1, CRD3 and CRD4 contributed mainly for opsonization triggered by CfLec‐4.

Two novel LRR and Ig domain-containing proteins from oyster Crassostrea gigas function as pattern recognition receptors and induce expression of cytokines

ABSTRACT Leucine‐rich repeat (LRR) domain and immunoglobulin (Ig) domain are both competent immune recognition modules, and the immunological roles of LRR and Ig domain containing‐ proteins (LRRIGs) are speculated to be multifunctional and worth investigating. In the present study, two novel LRRIGs, CgLRRIG‐1 and CgLRRIG‐2, were identified and characterized from oyster Crassostrea gigas. Both of them contained an N‐terminal LRR region, an Ig domain, a transmembrane region, and a C‐terminal cytoplasmic tail. The mRNA transcripts of CgLRRIG‐1 and CgLRRIG‐2 were constitutively expressed in muscle, gill, hepatopancreas, mantle, gonad and hemocytes with the highest expression level in hepatopancreas. Their mRNA expression levels in hemocytes were significantly up‐regulated after the stimulations with four PAMPs including peptidoglycan (PGN), lipopolysaccharide (LPS), glucan (GLU) and polyinosinic‐polycytidylic acid (poly I:C) and one bacteria Vibrio anguillarum. The recombinant proteins, rCgLRRIG‐1 and rCgLRRIG‐2, could bind to PGN, LPS, GLU and poly I:C, and rCgLRRIG‐2 exhibited higher binding affinity. Additionally, rCgLRRIG‐1 and rCgLRRIG‐2 could significantly induce the expression of CgTNF‐1 and CgIL17‐5 in cultured oyster hemocytes, and the activity of rCgLRRIG‐2 was higher than that of rCgLRRIG‐1. All these results indicated that CgLRRIG‐1 and CgLRRIG‐2 could function as immune effectors or pro‐inflammatory factors as well as PRRs in oyster. HighlightsTwo LRR and Ig domain containing proteins with different structure and activity were identified from Crassostrea gigas.CgLRRIGs were expressed universally with the highest levels in hepatopancreas.CgLRRIGs could be induced by PAMPs and bacteria stimulations, and displayed bind high affinities towards PAMPs.CgLRRIGs could induce the expression of CgTNF‐1 and CgIL17‐5 in hemocytes.

Transcriptomic and Quantitative Proteomic Analyses Provide Insights Into the Phagocytic Killing of Hemocytes in the Oyster Crassostrea gigas

As invertebrates lack an adaptive immune system, they depend to a large extent on their innate immune system to recognize and clear invading pathogens. Although phagocytes play pivotal roles in invertebrate innate immunity, the molecular mechanisms underlying this killing remain unclear. Cells of this type from the Pacific oyster Crassostrea gigas were classified efficiently in this study via fluorescence-activated cell sorting (FACS) based on their phagocytosis of FITC-labeled latex beads. Transcriptomic and quantitative proteomic analyses revealed a series of differentially expressed genes (DEGs) and proteins present in phagocytes; of the 352 significantly high expressed proteins identified here within the phagocyte proteome, 262 corresponding genes were similarly high expressed in the transcriptome, while 140 of 205 significantly low expressed proteins within the proteome were transcriptionally low expressed. A pathway crosstalk network analysis of these significantly high expressed proteins revealed that phagocytes were highly activated in a number of antimicrobial-related biological processes, including oxidation–reduction and lysosomal proteolysis processes. A number of DEGs, including oxidase, lysosomal protease, and immune receptors, were also validated in this study using quantitative PCR, while seven lysosomal cysteine proteases, referred to as cathepsin Ls, were significantly high expressed in phagocytes. Results show that the expression level of cathepsin L protein in phagocytes [mean fluorescence intensity (MFI): 327 ± 51] was significantly higher (p < 0.01) than that in non-phagocytic hemocytes (MFI: 83 ± 26), while the cathepsin L protein was colocalized with the phagocytosed Vibrio splendidus in oyster hemocytes during this process. The results of this study collectively suggest that oyster phagocytes possess both potent oxidative killing and microbial disintegration capacities; these findings provide important insights into hemocyte phagocytic killing as a component of C. gigas innate immunity.

A Prokineticin (PK)-like cytokine from Chinese mitten crab Eriocheir sinensis promotes the production of hemocytes via reactive oxygen species

ABSTRACT Astakine is a cytokine‐like factor containing a prokineticin domain, which directly participates in hematopoiesis and blood cell differentiation. In the present study, a novel Astakine gene was identified from Chinese mitten crab Eriocheir sinensis (designated as EsAst). The full‐length cDNA of EsAst was of 1163 bp, consisting of a 5′ untranslated region (UTR) of 120 bp, a 3′ UTR of 656 bp, and an open reading frame (ORF) of 387 bp encoding a polypeptide of 128 amino acids. There were a signal peptide and a prokineticin domain with nine conserved cysteine residues in the deduced amino acid sequence of EsAst. EsAst shared higher similarity with Astakines from Penaeus monodon and Pacifastacus leniusculus, and it was closely clustered with the Astakine from shrimp P. monodon in the phylogenetic tree. The EsAst mRNA transcript was higher expressed in hemocytes and hepatopancreas. The relative expression level of EsAst in hemocytes was continuously increased from 1.5 to 48 h after Vibro anguillarum challenge compared that in the untreated control group. After Pichia pastoris GS115 challenge, the relative expression level of EsAst in hemocytes was also up‐regulated. After rEsAst injection, ROS levels in HPT cells were also increased at 12 and 24 h, and the total hemocyte counts were also significantly increased at 6, 9, 12, and 24 h post rEsAst injection. The interference of EsAst expression with dsRNA injection could delay the recovery of hemocytes production post A. hydrophila stimulation. When mitochondrial complexes I was knock down by dsRNA, ROS levels were decreased and THCs were also decreased. Recovery of hemocyte production inducing by A. hydrophila stimulation and rEsAst injection were delayed with dsEsbc1 injection. When ROS levels were increased after RNAi of Lon protease, THCs were also increased. The expression levels of five genes (EsJNK, EsSTAT, EsPI3K, EsAKT1, EsP70S6K) involved in SAPK‐JNK and mTOR signaling pathways were up‐regulated at 12 and 24 h in rEsAst group and EsLon dsRNA group compared with that in EGFP dsRNA group, and were similar to the trend of ROS levels. These results collectively suggested that EsAst should be a novel Astakine to promote the production of hemocytes in a ROS‐dependent way in E. sinensis. HighlightsAn Astakine was identified in Chinese mitten crab Eriocheir sinensis.rEsAst treatment could induce production of hemocytes and increase ROS level in HPT.RNAi for ROS production genes could block the hemocyte production induced by EsAst.