Yishuai Du

Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR.

Hatchery-reared larvae of the Pacific oyster (Crassostrea gigas) often suffer from massive mortality induced by Ostreid herpesvirus 1 (OsHV-1) infection, indicating the importance of better understanding of oyster immune defense systems. The accuracy of measurements of gene expression levels based on quantitative real-time PCR assays relies on the use of housekeeping genes as internal controls; however, few studies have focused on the selection of such internal controls. In this study, we conducted a comprehensive investigation of internal control genes during oyster development in virus-infected and uninfected samples. Transcriptome data for 38 developmental stages were downloaded and the gene expression patterns were classified into 30 clusters. A total of 317 orthologs of classical housekeeping genes in the oyster genome were annotated. After combining the expression profiles and oyster housekeeping gene dataset, 14 candidate internal controls were selected for further investigation: Elongation factor-1α (EF-1α), 18S rRNA (18S), 28S rRNA (28S), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin (ACT), Ribosomal protein L7 (RL7), Ribosomal protein L27 (RL27), Ribosomal protein L36 (RL36), Ribosomal protein S18 (RS18), Heterogeneous nuclear ribonucleoprotein A2/B1 (RO21), Eukaryotic translation elongation factor 2 (EF2), Ubiquitin-conjugating enzyme E2D2 (UBCD1), S-phase kinase-associated protein 1 (SKP1) and Heterogeneous nuclear ribonucleoprotein Q (HNRPQ). RNA was extracted from oyster larvae infected with OsHV-1 (group A; GA), and OsHV-1 free larvae (group B; GB). The expression levels of the 14 candidate internal controls were studied in GA and GB larvae by real-time PCR. Their expression stabilities were further analyzed using the GeNorm program. RL7 and RS18 were the most stable genes in both OsHV-1 infected (GA) and uninfected (GB) larvae. These results suggest that RL7 and RS18 could be used as internal controls for studying gene expression in normal growing oyster larvae and in OsHV-1 infected larvae. These high quality internal controls will be a valuable resource in future studies of oyster larval mortality.

Identification and functional characterization of two executioner caspases in Crassostrea gigas.

Caspase-3 and caspase-7 are two key effector caspases that play important roles in apoptotic pathways that maintain normal tissue and organ development and homeostasis. However, little is known about the sequence, structure, activity, and function of effector caspases upon apoptosis in mollusks, especially marine bivalves. In this study, we investigated the possible roles of two executioner caspases in the regulation of apoptosis in the Pacific oyster Crassostrea gigas. A full-length capase-3–like gene named Cgcaspase-3 was cloned from C.gigas cDNA, encoding a predicted protein containing caspase family p20 and p10 domain profiles and a conserved caspase active site motif. Phylogenetic analysis demonstrated that both Cgcaspase-3 and Cgcaspase-1 may function as effector caspases clustered in the invertebrate branch. Although the sequence identities between the two caspases was low, both enzymes possessed executioner caspase activity and were capable of inducing cell death. These results suggested that Cgcaspase-3 and Cgcaspase-1 were two effector caspases in C. gigas. We also observed that nucleus-localized Cgcaspase-3, may function as a caspase-3–like protein and cytoplasm-localized Cgcaspase-1 may function as a caspase-7–like protein. Both Cgcaspase-3 and Cgcaspase-1 mRNA expression increased after larvae settled on the substratum, suggesting that both caspases acted in several tissues or organs that degenerated after oyster larvae settlement. The highest caspase expression levels were observed in the gills indicating that both effector caspases were likely involved in immune or metabolic processes in C. gigas.

Oyster Shell Proteins Originate from Multiple Organs and Their Probable Transport Pathway to the Shell Formation Front

Mollusk shell is one kind of potential biomaterial, but its vague mineralization mechanism hinders its further application. Mollusk shell matrix proteins are important functional components that are embedded in the shell, which play important roles in shell formation. The proteome of the oyster shell had been determined based on the oyster genome sequence by our group and gives the chance for further deep study in this area. The classical model of shell formation posits that the shell proteins are mantle-secreted. But, in this study, we further analyzed the shell proteome data in combination with organ transcriptome data and we found that the shell proteins may be produced by multiple organs though the mantle is still the most important organ for shell formation. To identify the transport pathways of these shell proteins not in classical model of shell formation, we conducted a shell damage experiment and we determined the shell-related gene set to identify the possible transport pathways from multiple organs to the shell formation front. We also found that there may exist a remodeling mechanism in the process of shell formation. Based on these results along with some published results, we proposed a new immature model, which will help us think about the mechanism of shell formation in a different way.

THE IMPACT OF Aeromonas salmonicida INFECTION ON INNATE IMMUNE PARAMETERS OF ATLANTIC SALMON Salmo salar L

Enzyme activities and gene expression of a number of innate immune parameters in the serum, mucus and skin of Atlantic salmon (Salmo salar) were investigated after challenge with a pathogenic strain of Aeromonas salmonicida (A. salmonicida). Fish were injected in the dorsal muscle with either 100 μl bacterium solution, about 3.05 × 10(7) CFU/ml A. salmonicida, or 100 μl 0.9% NaCl (as control group) and tissue samples were collected at days 0, 2, 4 and 6 post-injection. Lysozyme (LSZ) and alkaline phosphatase (AKP) activities in serum, mucus and skin, and LSZ and AKP mRNA expression in skin of the challenged fish were higher than those of the control at most of the experimental time, with significant differences at several time points (P < 0.05), indicating the involvement of LSZ and AKP in the innate immunity of Atlantic salmon to A. salmonicida. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities in mucus and skin, along with the SOD, POD and CAT mRNA expression in skin significantly decreased at day 4 and 6, indicating the decreased antioxidant capacity of the challenged fish. Glutamate pyruvate transaminase (GPT) and glutamic oxalacetic transaminase (GOT) activities in serum, mucus and skin of the challenged group were all higher than those of the control after the injection, and at several time points significant differences were found between the two groups, suggesting organs of fish were impaired after the pathogen infection. The changes of the GPT and GOT activities could be used as potential biomarkers for the impairment of physiological functions caused by the pathogen infection. Identified biomarkers of the immune responses will contribute to the early-warning system of the disease. So this study will not only provide a theoretical basis for vaccine development, but also provide basic data for the establishment of early warning systems for diseases caused by A. salmonicida in Atlantic salmon rearing.

Effect of dietary β-glucan on growth, survival and regulation of immune processes in rainbow trout (Oncorhynchus mykiss) infected by Aeromonas salmonicida

ABSTRACT The present study evaluated the effects of dietary &bgr;‐glucan (0, 0.05%, 0.1%, and 0.2%) on growth performance after 42 days of feeding. Thereafter, rainbow trout (Oncorhynchus mykiss) were infected with Aeromonas salmonicida, and survival rates as well as the regulating processes of stress‐ and immune‐related factors were analyzed. In general, higher dietary &bgr;‐glucan levels obviously improved specific growth rate (SGR), weight gain (WG) and feed efficiency (FE) (P ≤ 0.05). Survival rates in &bgr;‐glucan groups increased significantly compared with the control group after A. salmonicida infection (P ≤ 0.05). Serum total superoxide dimutase (T‐SOD), peroxidase (POD) as well as catalase (CAT) activities, and their mRNA expressions in the head kidney of fish in the &bgr;‐glucan groups generally increased to higher levels after infection, and more quickly, compared with in the control group. Serum lysozyme (LSZ) and its expression in the head kidney in &bgr;‐glucan groups reached a higher peak earlier than in the control group. Serum glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels in the &bgr;‐glucan groups were significantly lower than in the control group (P ≤ 0.05). The peak of heat shock protein 70 (HSP70) expression in the 0.2% &bgr;‐glucan group was higher and occurred earlier than in other groups (P ≤ 0.05). These results confirm that 0.1% and 0.2% dietary &bgr;‐glucan are beneficial for promoting growth in rainbow trout and enhancing resistance against A. salmonicida. Furthermore, &bgr;‐glucan could play an important role in regulating stress‐ and immune‐related factors in rainbow trout to more quickly fight against bacterial infection. HIGHLIGHTSDietary &bgr;‐glucan in dosage of 0.1% and 0.2% can enhance growth performance of rainbow trout.&bgr;‐glucan from 0.05% to 0.2% is able to significantly enhance resistance to A. salmonicida in rainbow trout.It can stimulate immune factors more quickly to higher levels to fight against bacteria.In this paper, the optimal level of dietary &bgr;‐glucan concentration for rainbow trout is 0.2%.

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.

Proteomic analysis in kidneys of Atlantic salmon infected with Aeromonas salmonicida by iTRAQ

Abstract Aeromonas salmonicida is a major etiologic agent which induces furunculosis and is globally harmful in salmonid and turbot cultures, especially in Atlantic salmon (Salmo salar) farming. In order to improve knowledge of its poorly understood pathogenesis, we utilized high‐throughput proteomics to display differentially expressed proteins in the kidney of Atlantic salmon challenged with high and low infection dose of A. salmonicida at 7 and 14 days. In quantitative proteomic assays, isobaric tags for relative and absolute quantitation (iTRAQ) combined with 2D LC‐MS/MS is emerging as a powerful methodology in the search for disease‐specific targets and biomarkers. In this study, 4009 distinct proteins (unused ≥ 1.3, which is a confidence ≥ 95%) were identified in three two‐dimensional LC/MS/MS analyses. Then we chose 140 proteins (fold change ratio ≥ 1.5 and P < 0.01) combined with protein–protein interaction analysis to ultimately obtain 39 proteins in network which could be considered as potential biomarkers for Atlantic salmon immune responses. Nine significant differentially expressed proteins were consistent with those at the proteomic level used to validate genes at the transcriptomic level by qPCR. Collectively, these data was first reported using an iTRAQ approach to provide additional elements for consideration in the pathophysiology of A. salmonicida and pave the way to resolve the influence of this disease in Atlantic salmon. Graphical abstract Figure. No Caption available. HighlightsIt’s first time by using iTRAQ to identify diferentially expressed proteins in the kidney of Atlantic salmon.A total of 4009 proteins were identified by the international standardized gene functional classification system of GO.2323 proteins were sub‐categorized into 24 COG classifications.2552 proteins were sub‐categorized into 264 KEGG classifications.39 significant differential expressed proteins were grouped into 13 major categories by protein‐protein network analysis.The results of our work provide a list of new protein targets that could be used to further explore disease mechanisms of A. salmonicida.

Characterization of the Mollusc RIG-I/MAVS Pathway Reveals an Archaic Antiviral Signalling Framework in Invertebrates

Despite the mitochondrial antiviral signalling protein (MAVS)-dependent RIG-I-like receptor (RLR) signalling pathway in the cytosol plays an indispensable role in the antiviral immunity of the host, surprising little is known in invertebrates. Here we characterized the major members of RLR pathway and investigated their signal transduction a Molluscs. We show that genes involved in RLR pathway were significantly induced during virus challenge, including CgRIG-I-1, CgMAVS, CgTRAF6 (TNF receptor-associated factor 6), and CgIRFs (interferon regulatory factors. Similar to human RIG-I, oyster RIG-I-1 could bind poly(I:C) directly in vitro and interact with oyster MAVS via its caspase activation and recruitment domains. We also show that transmembrane domain-dependent self-association of CgMAVS may be crucial for its signalling and that CgMAVS can recruit the downstream signalling molecule, TRAF6, which can subsequently activate NF-κB signal pathway. Moreover, oyster IRFs appeared to function downstream of CgMAVS and were able to activate the interferon β promoter and interferon stimulated response elements in mammalian cells. These results establish invertebrate MAVS-dependent RLR signalling for the first time and would be helpful for deciphering the antiviral mechanisms of invertebrates and understanding the development of the vertebrate RLR network.

Molecular characterization and functional analysis of tumor necrosis factor receptor-associated factor 2 in the Pacific oyster

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are a family of crucial adaptors, playing vital roles in mediating signal transduction in immune signaling pathways, including RIG-I-like receptor (RLR) signaling pathway. In the present study, a new TRAF family member (CgTRAF2) was identified in the Pacific oyster, Crassostrea gigas. Comparison and phylogenetic analysis revealed that CgTRAF2 could be a new member of the invertebrate TRAF2 family. Quantitative real-time PCR revealed that CgTRAF2 mRNA was highly expressed in the digestive gland, gills, and hemocytes, and it was significantly up-regulated after Vibrio alginolyticus and ostreid herpesvirus 1 (OsHV-1) challenge. The CgTRAF2 mRNA expression profile in different developmental stages of oyster larvae suggested that CgTRAF2 could function in early larval development. CgTRAF2 mRNA expression pattern, after the silence of CgMAVS (Mitochondrial Antiviral Signaling) -like, indicated that CgTRAF2 might function downstream of CgMAVS-like. Moreover, the subcellular localization analysis revealed that CgTRAF2 was localized in cytoplasm, and it may play predominately important roles in signal transduction. Collectively, these results demonstrated that CgTRAF2 might play important roles in the innate immunity and larval development of the Pacific oyster.

Involvement of LuxS in Aeromonas salmonicida metabolism, virulence and infection in Atlantic salmon (Salmo salar L)

ABSTRACT Quorum sensing is a bacterial density dependent communication system, which regarded to regulate co‐operative behaviors of community and mediated by extracellular signal molecules named autoinducers (AI). Among various signals, autoinducer‐2 (AI‐2) is believed to be the messengers inter species and produced by LuxS. For Aeromonas salmonicida (A. salmonicida), an opportunistic pathogen to many cold‐water teleost, little information has been known about the function of AI‐2 and LuxS. Therefore, our aim was to preliminarily clarify the function of LuxS in A. salmonicida. The consequences demonstrated that wild type A. salmonicida exhibited AI‐2 activity and luxS defective mutant strain fail to produce AI‐2 signals. Furthermore, it was suggested that luxS deficiency could impact bacterial morphology, surface properties and virulence dramatically. Challenge experiment showed a tendency that immune factors expressed earlier when Atlantic salmon was infected with &Dgr;luxS strain. Overall, we hypothesis that AI‐2 quorum sensing could regulate the expression of A‐layer protein coding gene vapA, and then influence bacterial survival ability when suffered from attack of the host immune system. Though additional studies are warranted, our study will supply a new thinking to control the damage caused by A. salmonicida. HIGHLIGHTSAI‐2 activity exists in A. salmonicida and correlates to the expression of luxS.LuxS plays a dual role in cellular metabolism and virulent factors expression.LuxS deficiency affects bacterial survival ability during its infection to Atlantic salmon.