Effects of Dietary Bio-Fermented Selenium Supplementation on Growth, Immune Performance, and Intestinal Microflora of Chinese Mitten Crabs, Eriocheir sinensis.

Selenium is a vital trace mineral that is crucial for maintaining regular biological processes in aquatic animals. In this study, a four-week dietary trial was carried out to assess the impact of bio-fermented selenium (Bio-Se) on the growth and immune response of Chinese mitten crabs, Eriocheir sinensis. The crabs were randomly allocated to five dietary treatment groups, each receiving a different dose of Bio-Se. The doses included 0, 0.3, 0.6, 1.5, and 3.0 mg/kg and were accurately measured in basal diet formulations. The results showed the weight gain rate (WGR), specific growth rate (SGR), and survival rate (SR) in the 1.5 mg/kg Bio-Se group were the highest, and 3.0 mg/kg of Bio-Se has an inhibitory effect on the WGR, SGR, and SR. The activities of the immune enzymes, including glutathione peroxidase (GPX), superoxide dismutase (SOD), and acid phosphatase (ACP), of the hepatopancreas were significantly (p < 0.05) increased in the 1.5 mg/kg Bio-Se group, while they decreased (p < 0.05) in the 3.0 mg/kg feeding group compared to the 0 mg/kg feeding group. The concentration of maleic dialdehyde (MDA) exhibited the opposite pattern. Similarly, the mRNA expression levels of antimicrobial peptides (ALF-1, Crus-1, and LYS), ERK, and Relish genes were also observed to be the highest in the 1.5 mg/kg Bio-Se group compared with the other groups. Furthermore, the administration of 1.5 mg/kg of Bio-Se resulted in an increase in the thickness of the intestinal plica and mucosal layer, as well as in alterations in the intestinal microbial profile and bacterial diversity compared to the dose of 0 mg/kg of Bio-Se. Notably, the population of the beneficial bacterial phylum Fusobacteria was increased after crabs were fed the 1.5 mg/kg Bio-Se diet. In conclusion, the oral administration of 1.5 mg/kg of Bio-Se improved the growth efficiency, antioxidant capabilities, immunity, and intestinal health of E. sinensis. Through a broken-line analysis of the WGR against dietary Bio-Se levels, optimal dietary Bio-Se levels were determined to be 1.1 mg/kg. These findings contribute valuable insights to the understanding of crab cultivation and nutrition.

Yorkie negatively regulates the Crustin expression during molting in Chinese mitten crab, Eriocheir sinensis.

Molting is a key biological process of crustaceans, which is mainly regulated by 20-hydroxyecdyone (20E). The molting cycle could be divided into three main stages including pre-molt, post-molt and inter-molt stages. The mechanism of immune regulation during molting process still requires further exploration. Yorkie (Yki) is a pivotal transcription factor in the Hippo signaling pathway, and it plays an essential role in regulating cell growth and immune response. In the present study, a Yki gene was identified from Eriocheir sinensis (designed as EsYki), and the regulatory role of EsYki in controlling the expression of antimicrobial peptide genes throughout the molting process was investigated. The mRNA expression level of EsYki was higher at the pre-molt stage compared to the post-molt stage and inter-molt stage. Following the injection of 20E, there was a notable and consistent rise in the EsYki mRNA expression in haemocytes. The increase was observed from 3 h to 48 h with the maximum level at 12 h. And the phosphorylation of Yki in the haemocytes was also significantly up-regulated at 3 h post 20E injection. Moreover, the levels of EsYki mRNA expression at three molting stages were significantly increased post Aeromonas hydrophila stimulation. The maximum level was detected at post-molt stage following A. hydrophila stimulation, while the lowest level was observed at inter-molt stage. The expression pattern of EsCrus was in contrast to EsCrus. After EsYki mRNA transcripts were inhibited by Yki inhibitor (CA3), the mRNA expression levels of EsCrus1 and EsCrus2 following A. hydrophila stimulation were significantly elevated. Furthermore, the phosphorylation level of NF-κB was also increased following the inhibition of Yki. Collectively, our findings indicated that EsYki could be induced by 20E and has a suppressive effect on the expression of EsCrus via inhibiting NF-κB during molting process. This research contributes to the understanding of the immunological regulation mechanism during molting process in crustaceans.

The involvement of tumor necrosis factor receptor-associated factor 6 in regulating immune response by NF-κB at pre-molt stage of Chinese mitten crab (Eriocheir sinensis).

Molting is a crucial biological process of crustaceans. Crustaceans go through three separate stages throughout their molting process, including pre-molt, post-molt and inter-molt. However, the exact mechanism of immunological modulation during molting remains unclear. Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been extensively documented to participate in immune defense. In the present study, a TRAF6 gene with two TRAF-type zinc finger domains was identified from Eriocheir sinensis (designed as EsTRAF6), and its role in regulating immune response during molting process was explored. The mRNA expression level of EsTRAF6 at pre-molt stage was higher than that at post-molt stage and inter-molt stage. After Aeromonas hydrophila stimulation, the expression levels of EsTRAF6, EsRelish and anti-lipopolysaccharide factors (ALFs) genes exhibited a considerable increase at three molting stages. Subsequently, the expression patterns of EsTRAF6 and EsRelish in response to the treatment with 20-hydroxyecdysone (20E) were examined. The mRNA expression of EsTRAF6 and EsRelish were significantly increased at 12 h after 20E injection. Additionally, the protein expression level of TRAF6 was also up-regulated in 20E group compared to control group. Furthermore, the role of EsTRAF6 in regulating the anti- ALFs expression at pre-molt stage post A. hydrophila stimulation was investigated. Following the inhibition of the EsTRAF6 transcript using RNAi or the injection of inhibitor (TMBPS), there was a notable decrease of the EsALF1, EsALF2 and EsALF3 transcripts. Moreover, a significant reduction in the phosphorylation level of NF-κB at pre-molt stage was observed after A. hydrophila stimulation in TRAF6-inhibited crabs. Collectively, our results suggest that EsTRAF6 could be induced by 20E and promoted the EsALFs expression by activating NF-κB at pre-molt stage, which provides a novel insight into the research of immune regulatory mechanism during the process of molting of crustaceans.

The interaction between 20-hydroxyecdysone and AMPK through PI3K activation in Chinese mitten crab, Eriocheir sinensis.

In crustaceans, the steroid hormone 20-hydroxyecdysone (20E) initiates molting, and the molting process is also regulated by energy metabolism. AMPK is an energy sensor and plays a critical role in systemic energy balance. Here, the regulatory mechanism in the interaction between 20E and AMPK was investigated in Chinese mitten crab, Eriocheir sinensis. The results showed that the 20E concentration and the mRNA expression levels of 20E receptors in hepatopancreas were down-regulated post AMPK activator (AICAR) treatment, and were up-regulated after AMPK inhibitor (Compound C) injection in crabs. Besides, the molt-inhibiting hormone (MIH) gene expression in eyestalk showed the opposite patterns in response to the AICAR and Compound C treatment, respectively. Further investigation found that there was a significant reduction in 20E concentration post PI3K inhibitor (LY294002) treatment, and the phosphorylation level of PI3K was increased in hepatopancreas after AMPK inhibitor injection. On the other hand, the positive regulation of PI3K-mediated activation of AMPK was also observed, the phosphorylation levels of AMPKα, AMPKß and PI3K in hepatopancreas were significantly increased post 20E injection. In addition, the phosphorylation levels of AMPKα and AMPKß induced by 20E were decreased after the injection of PI3K inhibitor. Taken together, these results suggest that the regulatory cross-talk between 20E and AMPK is likely to act through PI3K pathway in E. sinensis, which appeared to be helpful for a better understanding in molting regulation.

IGF2BP2 promotes glycolysis and hepatocellular carcinoma stemness by stabilizing CDC45 mRNA via m6A modification.

A growing number of studies have shown the prognostic importance of Cell division cycle protein 45 (CDC45) in hepatocellular carcinoma (HCC). This study aims to investigate the biological function and mechanism of CDC45 in HCC. The differential expression and prognostic significance of CDC45 in HCC and normal tissues were analyzed by bioinformatics. CDC45 was knocked down and the biological effects of CDC45 in HCC in vitro and in vivo were measured. Subsequently, using RNA m6A colorimetry and Methylated RNA Immunoprecipitation (MeRIP), the levels of m6A modification of total RNA and CDC45 were evaluated in cells. RIP was applied to establish that CDC45 and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) interact. A test using actinomycin D was performed to gauge the stability of the CDC45 mRNA. Furthermore, the regulatory role of IGF2BP2 on CDC45 expression in HCC progression was explored by overexpressing IGF2BP2. High expression of CDC45 was correlated with poor prognosis in HCC patients. Knocking down CDC45 inhibited HCC cell proliferation, migration, invasion, EMT, stemness, and glycolysis, and promoted apoptosis, which was verified through in vitro experiments. Additionally, IGF2BP2 was highly expressed in HCC cells, and it was found to interact with CDC45. Knocking down IGF2BP2 resulted in reduced stability of CDC45 mRNA. Moreover, overexpression of IGF2BP2 promoted HCC cell proliferation, migration, invasion, EMT, stemness, and glycolysis, while inhibiting apoptosis, which was reversed by knocking down CDC45. In general, IGF2BP2 promoted HCC glycolysis and stemness by stabilizing CDC45 mRNA via m6A modification. [Figure: see text].

A circadian clock protein cryptochrome inhibits the expression of inflammatory cytokines in Chinese mitten crab (Eriocheir sinensis).

Cryptochrome (Cry), as important flavoprotein, plays a key role in regulating the innate immune response, such as the release of inflammatory cytokines. In the present study, a cryptochrome homologue (EsCry) was identified from Chinese mitten crab Eriocheir sinensis, which contained a typical DNA photolyase domain, a FAD binding domain. The transcripts of EsCry were highly expressed at 11:00, and lowest at 3:00 within one day, while those of Interleukin enhancer binding factor (EsILF), Lipopolysaccharide-induced TNF-alpha factor (EsLITAF), Tumor necrosis factor (EsTNF) and Interleukin-16 (EsIL-16) showed a rhythm expression pattern contrary to EsCry. After EsCry was knocked down by dsEsCry injection, mRNA transcripts of Timeless (EsTim), Cycle (EsCyc), Circadian locomotor output cycles kaput (EsClock), Period (EsPer), and EsLITAF, EsTNF, EsILF, EsIL-16, as well as phosphorylation level of Dorsal significantly up-regulated. The transcripts of EsLITAF, EsTNF, EsILF, and EsIL-16 in EsCry-RNAi crabs significantly down-regulated after injection of NF-κB inhibitor. The interactions of EsCyc and EsCry, EsCyc and Dorsal were observed in vitro. These results indicated that EsCry negatively regulated the expression of the cytokine TNF and IL-16 via inhibiting their transcription factor LITAF and ILF through NF-κB signaling pathway, which provide evidences to better understand the circadian regulation mechanism of cytokine production in crabs.

A neural cell adhesion molecule from oyster Crassostrea gigas: Molecular identification and immune functional characterization.

Neural cell adhesion molecules (NCAMs) are large cell-surface glycoproteins playing important roles in cell-cell and cell-extracellular matrix interactions in nervous system. Recent study identified a homologue of NCAM (CgNCAM) from the Pacific oyster Crassostrea gigas. Its ORF was of 2634 bp which encodes a protein (877 amino acids) consisting of five immunoglobulin domains and two fibronectin type III domains. CgNCAM transcripts were broadly distributed in oyster tissues especially in mantle, labial palp and haemolymph. CgNCAM showed up-regulated expression in haemocytes of oysters after Vibrio splendidus and Staphylococcus aureus stimulation. The recombinant CgNCAM protein (rCgNCAM) was able to bind manose, lipopolysaccharide and glucan, as well as different microbes including Gram-negative bacteria and fungi. rCgNCAM displayed bacterial agglutination and hemagglutination activity. CgNCAM improved the phagocytosis of haemocytes towards V. splendidus by regulating the expression of CgIntegrin, CgRho J and CgMAPKK. Moreover, CgNCAM was involved in the extracellular trap establishment of haemocytes after V. splendidus stimulation. The results collectively indicated that CgNCAM acted as a recognition receptor executing multiple immune functions to recognize and eliminate invading microorganisms in innate immunity of oysters.

Molecular characterization of a cation-dependent mannose-6-phosphate receptor gene in Crassostrea hongkongensis and its responsiveness in Vibrio alginolyticus infection.

The cation-dependent mannose-6-phosphate receptor (CD-M6PR) is a P-type lectin that plays a crucial role in lysosomal enzyme transport, bacterial resistance, and viral entry. In this study, we cloned and analyzed the ORF of the CD-M6PR gene from Crassostrea hongkongensis and named it ChCD-M6PR. We analyzed the nucleotide and amino acid sequence of ChCD-M6PR, its tissue expression pattern and immune response to Vibrio alginolyticus. Our results showed that the ORF of ChCD-M6PR was 801 bp long and encoded a protein of 266 amino acids with a signal peptide at the N-terminus, as well as Man-6-P_recep, ATG27 and transmembrane structural domains. Phylogenetic analysis indicated that Crassostrea hongkongensis shared the highest similarity with Crassostrea gigas in the terms of CD-M6PR. The ChCD-M6PR gene was found to be expressed in various tissues, with the highest expression observed in the hepatopancreas and the lowest in the hemocytes by the fluorescence quantitative PCR. Furthermore, the expression of ChCD-M6PR gene was significantly up-regulated for a short time in response to Vibrio alginolyticus infection in the gill and hemocytes, while it was down-regulated in the gonads. The expression patterns of ChCD-M6PR also varied in the other tissues. The 96 h cumulative mortality rate of Crassostrea hongkongensis infected with Vibrio alginolyticus after knockdown the ChCD-M6PR gene was significantly higher. Overall, our findings suggests that ChCD-M6PR plays a crucial role in the immune response of Crassostrea hongkongensis to Vibrio alginolyticus infection, and its tissue-specific expression patterns may be indicatitive of varied immune responses across tissues.

The involvement of hypoxia inducible factor-1α on the proportion of three types of haemocytes in Chinese mitten crab under hypoxia stress.

Hypoxia triggers diverse cell physiological processes, and the hypoxia inducible factors (HIFs) are a family of heterodimeric transcription factors that function as master regulators to respond to hypoxia in different cells. However, the knowledge about the hypoxic responses especially cell alteration mediated by HIFs under hypoxia stress is still limited in crustaceans. In the present study, a hypoxia-inducible factor-1α (HIF-1α) gene was identified (designed as EsHIF-1α). The relative mRNA expression level of EsHIF-1α was highest in hyalinocytes and lowest in granulocytes among three types of haemocytes in crabs. Hypoxia could significantly increase the EsHIF-1α protein expression level in haemocytes. Meanwhile, the proportion of hyalinocytes began to increase from 3 h post hypoxia treatment, and reached the highest level at 24 h. However, the opposite variation in proportion of granulocytes was observed under hypoxia stress. Further investigation showed that the inhibition of EsHIF-1α induced by KC7F2 (HIF-1α inhibitor) could lead to the significant decrease in the proportion of hyalinocytes under hypoxia stress, and also resulted in an increase of granulocytes proportion. While, after EsHIF-1α was activated by IOX4 (HIF-1α activator), the proportion of hyalinocytes was significantly up-regulated and the proportion of granulocytes was significantly down-regulated under post hypoxia treatment. These results collectively suggested that EsHIF-1α was involved in the regulation of proportion of three types of haemocytes induced by hypoxia stress, which provided vital insight into the understanding of the crosstalk between hypoxia and cell development in invertebrates.

ERK is involved in the regulation of CpG ODN 2395 on the expression levels of anti-lipopolysaccharide factors in Chinese mitten crab, Eriocheir sinensis.

CpG oligodeoxynucleotides (ODN), as an effective adjuvant or immunopotentiator, activate the immune system and induce various immune responses. Recently, it has also been reported that high dose of CpG ODN can lead to immunosuppression. However, the underlying mechanism of CpG ODN-mediated immune response remains largely unknown in invertebrates. In the present study, the role of ERK in regulating expression levels of anti-lipopolysaccharide factors (ALFs) induced by different doses of CpG ODN 2395 was analyzed in Chinese mitten crab, Eriocheir sinensis. The mRNA expression levels of EsALFs (EsALF1, EsALF2 and EsALF3) and EsERK in haemocytes were observed to increase from 6 h to 48 h post low doses of CpG ODN 2395 (0.5 µg and 2.5 µg) stimulation, while they were suppressed after high dose of CpG ODN 2395 (12.5 µg) injection. Meanwhile, the phosphorylation levels of ERK in haemocytes were significantly promoted after low doses of CpG ODN 2395 injection, and a reduce level of ERK phosphorylation was observed after high dose of CpG ODN 2395 injection. Further investigation showed that the expression levels of EsALFs induced by CpG ODN 2395 were markedly down-regulated after knocking down the expression of EsERK. Similarly, the EsALFs mRNA expression were also inhibited post different doses of CpG ODN 2395 stimulation in PD98059 (ERK inhibitor) injection crabs. These results collectively suggest that ERK is involved in regulating the expression level of EsALFs induced by different dose of CpG ODN 2395 in Chinese mitten crab, which contribute to the understanding of the regulation of CpG ODN involving in immune response in crustaceans.

Spontaneous hepatic rupture during late pregnancy in a patient with solitary necrotic nodule of the liver: A case report.

Background: Spontaneous hepatic rupture (SHR) during pregnancy is a rare and life-threatening event, which usually occurs together with preeclampsia, eclampsia, HELLP syndrome, or liver tumors. However, SHR resulting from solitary necrotic nodule of the liver (SNNL) is extremely rare. Case presentation: We report the case of a 32-year-old pregnant woman who presented at 33 weeks of gestation with abdominal pain and emesis. Transabdominal ultrasound and magnetic resonance imaging revealed massive hemoperitoneum and lesions in the left lobe of the liver. An emergency cesarean section was performed and the hepatic rupture was managed surgically via left lateral lobectomy. The postprocedural course was uneventful. The premature baby successfully survived, and the patient was discharged 8 days after admission without complications. Histological examination revealed a diagnosis of SNNL, which resulted in the hepatic hematoma and SHR. Conclusion: To our knowledge, this is the first case of SHR resulting from SNNL during late pregnancy. Multidisciplinary collaboration and surgical management are important cornerstones for improving the perinatal outcomes when SHR is suspected in a pregnant patient.

The Elevated Expressions of Anti-lipopolysaccharide Factors After Priming Stimulation Confer Lastingly Humoral Protection in Crab Eriocheir sinensis.

Evidence of immune memory in invertebrates (immune priming) has accumulated in various organisms, and both cellular and humoral immune reactions are speculated to be involved in immune priming. However, there is a lack of understanding of the molecular mechanisms involved. In the present study, the protective effect of primed haemolymph was further validated by the increased survival rate of naïve crabs receiving a transfusion of primed haemolymph. By proteomic analysis, there were 474 proteins identified from the primed haemolymph, and most of them were functionally annotated in transport and metabolism classes. A total of 70 proteins were found to be differentially expressed in haemolymph at 12 hours and 7 days after priming stimulation with Aeromonas hydrophila, among which anti-lipopolysaccharide factor 1 (EsALF-1) and 3 (EsALF-3) were identified as the most significant (p < 0.05). After being challenged with A. hydrophila, EsALF-1 and EsALF-3 were highly expressed at both mRNA (in haemocytes) and protein (in haemolymph) levels compared with blank crabs, and the mRNA expressions of components in the EsTLR1-EsMyd88-EsPelle-EsALF pathway also increased significantly (p < 0.05). The EsALF-3 and EsMyd88 were even significantly higher expressed in response to the second A. hydrophila challenge, but their expressions all decreased (p < 0.05) when EsTLR1 was knocked down by RNAi. After the naïve crabs received an injection with the recombinant protein of EsALF-1 (rEsALF-1) or EsALF-3 (rEsALF-3), their survival rate increased significantly (p < 0.05) upon A. hydrophila stimulation. In contrast, the survival rate of the primed crabs reduced significantly (p < 0.05) after they received an injection with the antibody of EsALF-1 or EsALF-3. The enhanced expressions of EsALF-1 and EsALF-3 after A. hydrophilap riming stimulation could sustain for four weeks. All the results suggested that the EsTLR1-mediated productions of EsALF-1 and EsALF-3 in haemolymph played an indispensable role in the month-long humoral immune protection induced by A. hydrophila, which provides solid evidence of immune priming in crabs and a valuable reference for further understanding immune memory in invertebrates.

A CD63 Homolog Specially Recruited to the Fungi-Contained Phagosomes Is Involved in the Cellular Immune Response of Oyster Crassostrea gigas.

Cluster of differentiation 63 (CD63), a four-transmembrane glycoprotein in the subfamily of tetraspanin, has been widely recognized as a gateway from the infection of foreign invaders to the immune defense of hosts. Its role in Pacific oyster Crassostrea gigas is, however, yet to be discovered. This work makes contributions by identifying CgCD63H, a CD63 homolog with four transmembrane domains and one conservative CCG motif, and establishing its role as a receptor that participates in immune recognition and hemocyte phagocytosis. The presence of CgCD63H messenger RNA (mRNA) in hepatopancreas, labial palps, gill, and hemocytes is confirmed. The expression level of mRNA in hemocytes is found significantly (p < 0.01) upregulated after the injection of Vibrio splendidus. CgCD63H protein, typically distributed over the plasma membrane of oyster hemocytes, is recruited to the Yarrowia lipolytica-containing phagosomes after the stimulation of Y. lipolytica. The recombinant CgCD63H protein expresses binding capacity to glucan (GLU), peptidoglycan (PGN), and lipopolysaccharide (LPS) in the presence of lyophilized hemolymph. The phagocytic rate of hemocytes toward V. splendidus and Y. lipolytica is significantly inhibited (p < 0.01) after incubation with anti-CgCD63H antibody. Our work further suggests that CgCD63H functions as a receptor involved in the immune recognition and hemocyte phagocytosis against invading pathogen, which can be a marker candidate for the hemocyte typing in C. gigas.

The involvement of ecdysone and ecdysone receptor in regulating the expression of antimicrobial peptides in Chinese mitten crab, Eriocheir sinensis.

The ecdysone, 20-hydroxyecdysone (20E) and ecdysone receptor (EcR), are regarded as the key regulators of development, metamorphosis, and growth in arthropods. In the present study, the role of 20E and EsEcR in regulating the expression of antimicrobial peptides (AMPs) was investigated in Chinese mitten crab, Eriocheir sinensis. The concentration of 20E in plasma was significantly (p < 0.05) up-regulated from 3 h to 12 h after lipopolysaccharide (LPS) stimulation. The mRNA expression level of EsEcR-4 in hemocytes was significantly (p < 0.01) up-regulated from 6 h to 24 h after LPS stimulation, while no significant changes of EsEcR-2 and EsEcR-3 transcripts were observed. After 20E injection, EsEcR-4 expression level was significantly increased from 12 h to 48 h with the highest level at 24 h (4.34-fold compared to the control group, p < 0.01), and the mRNA expression levels of AMPs (EsALF-2, EsLYZ and EsCrus) in hemocytes were significantly increased from 6 h to 24 h with the peak level of 2.93-fold (p < 0.01), 2.33-fold (p < 0.01) and 2.75-fold (p < 0.01) at 12 h, respectively. After EsEcR-4 expression was interfered with specific dsRNA, a significant reduction of EsALF-2 (0.56-fold compared to the control group, p < 0.01), EsLYZ (0.27-fold, p < 0.01) and EsCrus (0.41-fold, p < 0.01) mRNA expression level was observed in dsEsEcR-4+LPS group at 12 h post LPS stimulation. Moreover, the mRNA expression levels of EsDorsal and EsJNK in hemocytes were significantly (p < 0.05) increased from 6 h to 24 h post 20E injection, and the phosphorylation of Dorsal and JNK in the hemocytes were significantly (p < 0.01) up-regulated at 3 h post 20E injection, while that in dsEsEcR-4+LPS group were significantly decreased after LPS stimulation compared to dsEsEGFP+LPS group. Taken together, these results suggested that 20E and EsEcR-4 play important roles in regulating the expression level of AMPs in the immune responses of E. sinensis by regulating the mRNA expression level and phosphorylation of Dorsal and JNK.

The involvement of a regucalcin in suppressing hemocyte apoptosis in Pacific oyster Crassostrea gigas.

Regucalcin (RGN), also known as senescence marker protein-30 (SMP30), plays a vital role in the regulation of Ca2+ homeostasis. In the present study, a regucalcin (designated as CgRGN) was identified from Pacific oyster Crassostrea gigas. The complete cDNA sequence of CgRGN was of 1059 bp, containing an open reading frame of 933 bp which encoded a protein of 310 amino acids. The deduced amino acid sequence of CgRGN shared similarity with other RGNs from the genome of C. gigas as well as other species. The mRNA transcripts of CgRGN were universally detected in all tested tissues, with higher level in hepatopancreas, labial palp, and gills. The relative expression level of CgRGN in hemocytes was significantly up-regulated (p < 0.05) at 3, 12, 72, and 96 h after the stimulation of lipopolysaccharide (LPS). After CgRGN expression was interfered by specific CgRGN-dsRNA, the hemocytes apoptosis rate increased dramatically at 12 h post LPS stimulation (1.56 fold, p < 0.01), compared to the control group. The caspase-3 activity in hemocytes and NO concentration in hemolymph increased significantly (p < 0.05) in dsCgRGN injection oysters. These results collectively indicated that CgRGN could suppress LPS-induced apoptosis and be involved in the immune response of oysters.

Transcriptomic profile of oyster Crassostrea gigas hemocyte after short-term cadmium exposure and bacteria stimulation.

Oyster Crassostrea gigas, is considered as a useful environmental indicator since it is widely distributed along the intertidal zone whereby it tends to accumulate cadmium and is always exposed to various pathogen agents. However, its molecular responses to both cadmium and pathogen stimulation remain unclear. In the present study, transcriptome data of hemocytes from oysters were analyzed to reveal specific molecular responses of oyster to cadmium or cadmium/bacteria stimulation. A total of 21591, 22872 and 20107 genes were detected in the BLANK, Cd24h and Cd/Bac24h group, respectively. Among them, there were 685 differentially expressed genes collected in the comparison of Cd24h versus BLANK. GO analysis of these genes found that sixteen terms into the Molecular Function category displayed transporter activities, and were all over-enrichment by cadmium exposure, whereas twelve terms into Biological Process category involved mainly in metabolic process of the various cellular components and two terms into Cellular Component category were all under-enrichment. The 330 immune responsive genes were shared by two gene lists of CdBac24h versus BLANK and CdBac24h versus Cd24h, and seven out of thirty terms in GO analysis were related to the immune process. Further annotation of these genes from the KEGG database revealed fourteen pathways, including two nervous system related pathways, arachidonic acid pathway, four immune pathways, MAPK cascade and other four cell signaling pathways, and two energy related pathways. Twenty-two differentially expressed genes were identified to responsive to both cadmium exposure and bacteria stimulation, but in different expression patterns, suggesting that bilateral responsive genes, such as alkaline phosphatase and sodium and chloride-dependent glycine transporter gene, could be candidate biomarkers for early warning of cadmium pollution. The present results collectively indicated that a profound neuro-endocrine-immune regulatory network was activated in response to cadmium and bacteria stimulation in oyster C. gigas, and the expression pattern of some cadmium responsive genes may be either reversed or strengthened by bacteria stimulation. The results provide knowledge on the transcriptomic response profile of oyster after short-term cadmium exposure and bacteria stimulation, which would be useful for future studies on stress response mechanism of mollusc, and some cadmium-bacteria responsive genes may be explored as potential biomarkers for monitoring marine pollution.

The involvement of TLR signaling and anti-bacterial effectors in enhanced immune protection of oysters after Vibrio splendidus pre-exposure.

The phenomena of enhanced protection of innate immunity responding to a pre-exposed pathogen have been reported in invertebrates. The underpinning molecular basis and mechanism for the enhanced immune protection are still missing. In order to explore the possible molecular basis for enhanced immune protection in molluscs, the transcriptomic analysis of oysters Crassostrea gigas hemocytes after twice stimulation of Vibrio splendidus were conducted, and a total of 403 M clean reads and 34254 differentially expressed genes (DEGs) were collected. There were 2964 common DEGs up-regulated in hemocytes after both the first and second immune stimulation, which were mostly enriched in metabolic processes and immune related pathways, such as endocytosis, MAPK signaling pathway and TLR signal pathway. Moreover, 187 and 55 DEGs were higher expressed at resting (0 h after stimulation) and activating state (12 h after stimulation) of the second immune response than that of the first response, respectively, mainly including immune recognition receptor scavenger receptor 2, signal molecule MAPK2, immune regulator IL17-d, apoptosis inhibitor IAP and effector cathepsin. More importantly, 13 DEGs were long-lastingly higher expressed at both the resting and activating state within the second immune response than that of the first, including TLR signal molecule MyD88, anti-virulent tissue inhibitor of metalloproteinase, anti-bacterial proline-rich transmembrane protein, which might play indispensable roles in enhanced immune protection against V. splendidus re-infection. The expression patterns of TLR signals (CgTLR6 and CgMyD88) and effector molecules (CgTIMP and CgPRTP) were further validated by RT-PCR, which were consistent to transcriptomic results. All the results provided an overall molecular basis of enhanced immune protection for hemocytes defensing against the second stimulation of V. splendidus in oyster, which would be valuable for understanding the protection mechanisms of pre-exposure in invertebrates.

The sensing pattern and antitoxic response of Crassostrea gigas against extracellular products of Vibrio splendidus.

Serious juvenile oyster disease induced by pathogenic Vibrio splendidus has resulted in tremendous economic loss, but the molecular mechanisms underlying this killing mechanism remain unclear. The resistance of adult oyster to V. splendidus or its virulence factors might provide a possible access to cognize the interaction between pathogen and host. In the present study, the extracellular products (ECP) from less virulent V. splendidus JZ6 were injected into adult Pacific oyster Crassostrea gigas, and the cellular and humoral immune response induced by ECP were investigated. The phagocytosis rate of hemocytes was significantly up-regulated (30.57%) at 6 h after ECP injection compared with that (21%) of control groups. And significantly high level of ROS production was also observed from 3 h to 12 h in ECP-injected oysters, concomitant with increased apoptosis rate of hemocytes (16.4% in ECP-injected group, p < 0.01) compared with control group (6.7%). By RT-PCR analysis, the expression level of antioxidant CgSOD in hemocytes significantly increased to 6.41-fold of that in control groups (p < 0.01) at 12 h post ECP injection. The expression levels of anti-toxic metalloprotease inhibitors CgTIMP629 and CgTIMP628 were also significantly up-regulated at the early (3-6 h) and late (6-24 h) stage of immune response, respectively. Moreover, after the ECP were incubated with serum proteins isolated from the ECP-injected oysters in vitro, the metalloprotease activity of ECP significantly declined by 21.39%, and less degraded serum proteins were detected by SDS-PAGE. When the primarily cultured hemocytes were stimulated with heat-inactivated ECP or fragments derived from ECP-degraded serum proteins, the expressions of CgTIMP629 (13.64 and 7.03-fold of that in saline group, respectively, p < 0.01) and CgTIMP628 (5.07 and 6.08-fold of that in saline group, respectively, p < 0.01) in hemocytes were all significantly induced. All the results indicated that the adult oysters could launch phagocytosis, antioxidant and anti-toxic response to resist the virulence of ECP, possibly by sensing heterologous ECP and ECP-induced endogenous alarm signals. These results provided a possible clue for the resistance mechanism of adult oysters towards the ECP of less virulent V. splendidus, which might be valuable for exploring strategies for the control of oyster disease.

A novel nuclear factor Akirin regulating the expression of antimicrobial peptides in Chinese mitten crab Eriocheir sinensis.

Akirin, a recently discovered nuclear factor, participates in regulating various processes, including cell proliferation and differentiation, embryonic development, and immunity. In the present study, a novel Akirin was identified from Chinese mitten crab Eriocheir sinensis (designated as EsAkirin), and its primary functions in regulating antimicrobial peptides were explored. The open reading frame of EsAkirin was of 615 bp, encoding a polypeptide of 204 amino acid residues. The deduced amino acid sequence of EsAkirin shared high similarities ranging from 44.1% to 89.2% with other Akirins. In the phylogenetic tree, EsAkirin was firstly clustered with Akirins from shrimp and then assigned into the invertebrate branch. The mRNA transcripts of EsAkirin were constitutively expressed in all the tested tissues, with the highest expression level (5.07-fold compared to the stomach, p < 0.01) in hepatopancreas. The mRNA expression of EsAkirin in hemocytes was significantly increased at 6 h, and reached the maximum level at 24 h post stimulations with either lipopolysaccharide (LPS) (5.04-fold, p < 0.01) or Aeromonas hydrophila (3.10-fold, p < 0.01). After the injection of EsAkirin-dsRNA, the mRNA expressions of EsALF2, EsLYZ, EsCrus2 and EsDWD1 were significantly decreased (p < 0.01) upon LPS stimulation. EsAkirin protein was prominently distributed in the nucleus of E. sinensis hemocytes after LPS and A. hydrophila stimulations. The relative luciferase reporter system analysis revealed that the activity of nuclear factor-κB was significantly up-regulated (2.64-fold, p < 0.01) in human embryonic kidney (HEK293T) cells after the over-expression of EsAkirin. Collectively, these results suggested that EsAkirin might play an important role in the immune responses of E. sinensis by regulating the expression of antimicrobial peptides.

An inhibitor of apoptosis protein (EsIAP1) from Chinese mitten crab Eriocheir sinensis regulates apoptosis through inhibiting the activity of EsCaspase-3/7-1.

Inhibitor of apoptosis proteins (IAPs) maintain the balance between cell proliferation and cell death by inhibiting caspase activities and mediating immune responses. In the present study, a homolog of IAP (designated as EsIAP1) was identified from Chinese mitten crab Eriocheir sinensis. EsIAP1 consisted of 451 amino acids containing two baculoviral IAP repeat (BIR) domains with the conserved Cx2 Cx6 Wx3 Dx5 Hx6 C motifs. EsIAP1 mRNA was expressed in various tissues and its expression level in hemocytes increased significantly (p < 0.01) at 12-48 h after lipopolysaccharide stimulation. In the hemocytes, EsIAP1 protein was mainly distributed in the cytoplasm. The hydrolytic activity of recombinant EsCaspase-3/7-1 against the substrate Ac-DEVD-pNA decreased after incubation with rEsIAP1. Moreover, rEsIAP1 could directly combine with rEsCaspase-3/7-1 in vitro. After EsIAP1 was interfered by dsRNA, the mRNA expression and the hydrolytic activity of EsCaspase-3/7-1 increased significantly, which was 2.26-fold (p < 0.05) and 1.71-fold (p < 0.05) compared to that in the dsGFP group, respectively. These results collectively demonstrated that EsIAP1 might play an important role in apoptosis pathway by regulating the activity of EsCaspase-3/7-1 in E. sinensis.