Jie Meng

Genome and Transcriptome Analyses Provide Insight into the Euryhaline Adaptation Mechanism of Crassostrea gigas

Background The Pacific oyster, Crassostrea gigas, has developed special mechanisms to regulate its osmotic balance to adapt to fluctuations of salinities in coastal zones. To understand the oyster’s euryhaline adaptation, we analyzed salt stress effectors metabolism pathways under different salinities (salt 5, 10, 15, 20, 25, 30 and 40 for 7 days) using transcriptome data, physiology experiment and quantitative real-time PCR. Results Transcriptome data uncovered 189, 480, 207 and 80 marker genes for monitoring physiology status of oysters and the environment conditions. Three known salt stress effectors (involving ion channels, aquaporins and free amino acids) were examined. The analysis of ion channels and aquaporins indicated that 7 days long-term salt stress inhibited voltage-gated Na+/K+ channel and aquaporin but increased calcium-activated K+ channel and Ca2+ channel. As the most important category of osmotic stress effector, we analyzed the oyster FAAs metabolism pathways (including taurine, glycine, alanine, beta-alanine, proline and arginine) and explained FAAs functional mechanism for oyster low salinity adaptation. FAAs metabolism key enzyme genes displayed expression differentiation in low salinity adapted individuals comparing with control which further indicated that FAAs played important roles for oyster salinity adaptation. A global metabolic pathway analysis (iPath) of oyster expanded genes displayed a co-expansion of FAAs metabolism in C. gigas compared with seven other species, suggesting oyster’s powerful ability regarding FAAs metabolism, allowing it to adapt to fluctuating salinities, which may be one important mechanism underlying euryhaline adaption in oyster. Additionally, using transcriptome data analysis, we uncovered salt stress transduction networks in C. gigas. Conclusions Our results represented oyster salt stress effectors functional mechanisms under salt stress conditions and explained the expansion of FAAs metabolism pathways as the most important effectors for oyster euryhaline adaptation. This study was the first to explain oyster euryhaline adaptation at a genome-wide scale in C. gigas.

Molecular Basis for Adaptation of Oysters to Stressful Marine Intertidal Environments

Oysters that occupy estuarine and intertidal habitats have well-developed stress tolerance mechanisms to tolerate harsh and dynamically changing environments. In this review, we summarize common pathways and genomic features in oyster that are responsive to environmental stressors such as temperature, salinity, hypoxia, air exposure, pathogens, and anthropogenic pollutions. We first introduce the key genes involved in several pathways, which constitute the molecular basis for adaptation to stress. We use genome analysis to highlight the strong cellular homeostasis system, a unique adaptive characteristic of oysters. Next, we provide a global view of features of the oyster genome that contribute to stress adaptation, including oyster-specific gene expansion, highly inducible expression, and functional divergence. Finally, we review the consequences of interactions between oysters and the environment from ecological and evolutionary perspectives by discussing mass mortality and adaptive divergence among populations and related species of the genus Crassostrea. We conclude with prospects for future study.

Alternative splicing and immune response of Crassostrea gigas tumor necrosis factor receptor-associated factor 3

Diverse alternative splicing isoforms play an important role in immune diversity and specificity. Their role in molluscan host-defense is however poorly understood. We characterized two alternative isoforms of tumor necrosis factor receptor-associated factor 3 (TRAF3) in the Pacific oyster, Crassostrea gigas, which were named CgTRAF3-S and CgTRAF3-L. An intron was retained in CgTRAF3-L, introducing a premature termination codon. Comparison and phylogenetic analysis revealed that CgTRAF3 shared a higher identity with other species, suggesting the conservation of the two gene transcripts. Quantitative real-time PCR was performed and the expression levels of CgTRAF3 isoforms were found to be significantly changed after Vibrio anguillarum and ostreid herpesvirus 1 challenges. These two isoforms represented contrary trends, indicating that CgTRAF3-L might function as a negative regulator of CgTRAF3-S. We also investigated the expression level of the transcripts of the two CgTRAF3 isoforms, following the silence of C. gigas mitochondrial anti-viral signaling protein like gene (CgMAVS-like). We concluded that CgTRAF3 might be involved in a MAVS-mediated immune signaling pathway. This study suggests that CgTRAF3 may be a response to bacterial and viral stimulation and that the two isoforms may be involved in immune response pathways. It is also possible that the two alternative splicing isoforms could be inter-coordinated and may promote survival of these oysters under immune stress conditions.

Comparative analysis of oyster (Crassostrea gigas) immune responses under challenge by different Vibrio strains and conditions

Screening a virulent Vibrio strain to study the interactions between bacterial infection and host immune defence is important to resolve large-scale summer oyster mortalities. Using adductor muscle injection we tested the oyster responses to phosphate-buffered saline (PBS), lipopolysaccharide (LPS), or Vibrio strains. Mortalities and stress gene expressions were used to characterize oyster immune responses. At 5 days post injection, the cumulative mortality rates in PBS, LPS, Vibrio tubiashii, Vibrio anguillarum, Vibrio alginolyticus and Vibrio aestuarianus groups were 8%, 16%, 36%, 40%, 40% and 76%, respectively, indicating that V. aestuarianus induced the highest death rate. Two-factor analyses of variance revealed that expression of SOD, CAT, GPX and HSP70 was influenced by bacterial injections in a time-dependent manner. The expression of all genes increased and reached their peak 3 or 12 h after bacterial injection and then decreased. These genes could be applied as immune responsive biomarkers to monitor early changes in oysters in response to bacterial infection. The greatest changes were observed in the V. aestuarianus-injected group, which may indicate that V. aestuarianus could be used as one of the more virulent strains for experimental infections.

Transcription factor CgMTF-1 regulates CgZnT1 and CgMT expression in Pacific oyster (Crassostrea gigas) under zinc stress

Oysters accumulate zinc at high tissue concentrations, and the metal response element (MRE)-binding transcription factor (MTF) functions as the cellular zinc sensor that coordinates the expression of genes involved in zinc efflux and storage, as well as those that protect against metal toxicity. In this study, we cloned MTF-1 in oysters and examined its regulation mechanism for its classic target genes, including MTs and ZnT1 under zinc exposure conditions. We cloned CgMTF-1 and determined the subcellular locations of its protein product in HEK293 cells. CgMTF-1 has a 2826bp open reading frame that encodes a predicted polypeptide with 707 amino acid residues, showing six well-conserved zinc finger domains that are required for metal binding. In HEK293 cell lines, CgMTF-1 primarily localizes in the cell nucleus under unstressed conditions and nuclear translocation was not critical for the activation of this gene. We searched for CgMTF-1-regulated genes in oysters using RNA interference. Decreased expression levels of CgMT1, CgMT4, and CgZnT1 were observed after CgMTF-1 interference (>70% inhibition) under zinc exposure, indicating the critical role of CgMTF-1 in the regulation of these genes. We searched for a direct regulation mechanism involving CgMTF-1 for CgMT1, CgMT4, and CgZnT1 in vitro. EMSA experiments indicated that CgMTF-1 can bind with the MREs found in the CgZnT1, CgMT1 and CgMT4 promoter regions. Additionally, luciferase reporter gene experiments indicated that CgMTF-1 could activate the CgMT1, CgMT4, and CgZnT1 promoters. Overall, our results suggest that CgMTF-1 directly coordinates the regulation of CgMTs and CgZnT1 expression and plays important roles in protecting oysters under zinc exposure conditions. To our knowledge, this is the first study to elucidate the function of MTF-1 in marine bivalves and provides new insights into the mechanisms of zinc accumulation and tolerance in mollusks.

Cadmium effects on DNA and protein metabolism in oyster ( Crassostrea gigas ) revealed by proteomic analyses

Marine molluscs, including oysters, can concentrate high levels of cadmium (Cd) in their soft tissues, but the molecular mechanisms of Cd toxicity remain speculative. In this study, Pacific oysters (Crassostrea gigas) were exposed to Cd for 9 days and their gills were subjected to proteomic analysis, which were further confirmed with transcriptomic analysis. A total of 4,964 proteins was quantified and 515 differentially expressed proteins were identified in response to Cd exposure. Gene Ontology enrichment analysis revealed that excess Cd affected the DNA and protein metabolism. Specifically, Cd toxicity resulted in the inhibition of DNA glycosylase and gap-filling and ligation enzymes expressions in base excision repair pathway, which may have decreased DNA repair capacity. At the protein level, Cd induced the heat shock protein response, initiation of protein refolding as well as degradation by ubiquitin proteasome pathway, among other effects. Excess Cd also induced antioxidant responses, particularly glutathione metabolism, which play important roles in Cd chelation and anti-oxidation. This study provided the first molecular mechanisms of Cd toxicity on DNA and protein metabolism at protein levels, and identified molecular biomarkers for Cd toxicity in oysters.

Characterization of the IRF2 proteins isolated from the deep-sea mussel Bathymodiolus platifrons and the shallow-water mussel Modiolus modiolus

ABSTRACT Interferon regulatory factors (IRFs) are transcription factors that play important roles in immune defense, stress response, hematopoietic differentiation, and cell apoptosis. IRFs of invertebrate organisms and their functions remain largely unexplored. In the present study, for the first time new IRFs (BpIRF2 and MmIRF2) were identified in the deep‐sea mussel Bathymodiolus platifrons and the shallow‐water mussel Modiolus modiolus. The open reading frame of BpIRF2 and MmIRF2 encoded putative proteins of 354 and 348 amino acids, respectively. Comparison and phylogenetic analysis revealed that both IRF2 proteins were new identified invertebrate IRF molecular. As transcriptional factors, both BpIRF2 and MmIRF2 could activate the interferon‐stimulated response element‐containing promoter and BpIRF2 could interact with itself. Moreover, both BpIRF2 and MmIRF2 were localized to the cytoplasm and nucleus. Collectively, these results demonstrated that IRF2 proteins might be crucial in the innate immunity of deep‐sea and shallow‐water mussels. HighlightsBpIRF2 and MmIRF2 were identified in the deep‐sea and the shallow‐water mussels.Both BpIRF2 and MmIRF2 could activate the ISRE‐containing promoter.BpIRF2 could form homodimers.Both BpIRF2 and MmIRF2 were localized to the cytoplasm and nucleus.

Characterization of CgHIFα-Like, a Novel bHLH-PAS Transcription Factor Family Member, and Its Role under Hypoxia Stress in the Pacific Oyster Crassostrea gigas

Hypoxia-inducible factor (HIF), a critical member of the basic-helix-loop-helix (bHLH)-containing Per-Arnt-Sim (PAS) protein family, is a master transcription factor involved in maintaining oxygen homeostasis. In the present study, we isolated and characterized a novel bHLH-PAS family member, CgHIFα-like gene, from the Pacific oyster Crassostrea gigas, and determined its importance during hypoxia stress. The 3020-bp CgHIFα-like cDNA encoded a protein of 888 amino acids. The predicted CgHIFα-like amino acid sequence was conserved in the N-terminal bHLH, PAS, and PAC domains (but not in the C-terminal domain) and was most closely related to the HIF family in the bHLH-PAS protein phylogenic tree. Similar to the mammalian HIF-1α, CgHIFα-like could be expressed as four mRNA isoforms containing alternative 5′-untranslated regions and different translation initiation codons. At the mRNA level, these isoforms were expressed in a tissue-specific manner and showed increased transcription to varying degrees under hypoxic conditions. Additionally, the western blot analysis demonstrated that CgHIFα-like was induced by hypoxia. Electrophoretic mobility shift assay indicated that CgHIFα-like could bind to the hypoxia responsive element (HRE), whereas dual-luciferase reporter analysis demonstrated that CgHIFα-like could transactivate the reporter gene containing the HREs. In addition to CgHIFα-like, we identified CgARNT from the C. gigas, analyzed its expression pattern, and confirmed its interaction with CgHIFα-like using a yeast two-hybrid assay. In conclusion, this is the first report on the cloning and characterization of a novel hypoxia transcription factor in mollusks, which could accumulate under hypoxia and regulate hypoxia related gene expression by binding to HRE and dimerizing with CgARNT. As only one member of HIF has been identified in invertebrates to date, our results provide new insights into the unique mechanisms of hypoxia tolerance in mollusks.

Population resequencing reveals candidate genes associated with salinity adaptation of the Pacific oyster Crassostrea gigas

The Pacific oyster Crassostrea gigas is an important cultivated shellfish. As a euryhaline species, it has evolved adaptive mechanisms responding to the complex and changeable intertidal environment that it inhabits. To investigate the genetic basis of this salinity adaptation mechanism, we conducted a genome-wide association study using phenotypically differentiated populations (hyposalinity and hypersalinity adaptation populations, and control population), and confirmed our results using an independent population, high-resolution melting, and mRNA expression analysis. For the hyposalinity adaptation, we determined 24 genes, including Cg_CLCN7 (chloride channel protein 7) and Cg_AP1 (apoptosis 1 inhibitor), involved in the ion/water channel and transporter mechanisms, free amino acid and reactive oxygen species metabolism, immune responses, and chemical defence. Three SNPs located on these two genes were significantly differentiated between groups, as was Cg_CLCN7. For the hypersalinity adaptation, the biological process for positive regulating the developmental process was enriched. Enriched gene functions were focused on transcriptional regulation, signal transduction, and cell growth and differentiation, including calmodulin (Cg_CaM) and ficolin-2 (Cg_FCN2). These genes and polymorphisms possibly play an important role in oyster hyposalinity and hypersalinity adaptation. They not only further our understanding of salinity adaptation mechanisms but also provide markers for highly adaptable oyster strains suitable for breeding.

Inducible variation in anaerobic energy metabolism reflects hypoxia tolerance across the intertidal and subtidal distribution of the Pacific oyster (Crassostrea gigas)

Pacific oyster (Crassostrea gigas) distribute a steep gradient of environmental stress between intertidal and subtidal habits and provide insight into population-scale patterns and underlying processes of variation in physiological tolerance. In this study, 1-year-old-F1 oysters, collected from subtidal and intertidal habitats, were obtained after common garden experiment. Genetic differentiation and physiological responses under air exposure were examined to determine whether they had evolved into local adapted subpopulations. Mortality rate, anaerobic glycolysis metabolism, and energy status indicated that oyster had initiated metabolism depression and anaerobic glycolysis metabolism in both intertidal and subtidal oysters under air exposure. However, the subtidal oysters displayed the larger energy metabolism depressions and the earlier anaerobic glycolysis responses. This may indicate that subtidal oysters were more sensitives to hypoxia stress, which may lead the higher mortality rate under long term of air exposure. Based on a common garden experimental design, we propose that this diversification may have a genetic background. Overall, the clear differences between intertidal and subtidal oysters under air exposure have provided an important reference for their aquaculture and transportation used in commercial production.