Identification and functional characterization of laminin receptor in the mud crab, Scylla paramamosain, in response to MCDV-1 challenge.

Laminin receptor (LR), which mediating cell adhesion to the extracellular matrix, plays a crucial role in cell signaling and regulatory functions. In the present study, a laminin receptor gene (SpLR) was cloned and characterized from the mud crab (Scylla paramamosain). The full length of SpLR contained an open reading frame (ORF) of 960 bp encoding 319 amino acids, a 5' untranslated region (UTR) of 66 bp and a 3' UTR of 49 bp. The predicted protein comprised two Ribosomal-S2 domains and a 40S-SA-C domain. The mRNA of SpLR was highly expressed in the gill, followed by the hepatopancreas. The expression of SpLR was up-regulated after mud crab dicistrovirus-1(MCDV-1) infection. Knocking down SpLR in vivo by RNA interference significantly down-regulated the expression of the immune genes SpJAK, SpSTAT, SpToll1, SpALF1 and SpALF5. This study shown that the expression level of SpToll1 and SpCAM in SpLR-interfered group significantly increased after MCDV-1 infection. Moreover, silencing of SpLR in vivo decreased the MCDV-1 replication and increased the survival rate of mud crabs after MCDV-1 infection. These findings collectively suggest a pivotal role for SpLR in the mud crab's response to MCDV-1 infection. By influencing the expression of critical innate immune factors and impacting viral replication dynamics, SpLR emerges as a key player in the intricate host-pathogen interaction, providing valuable insights into the molecular mechanisms underlying MCDV-1 pathogenesis in mud crabs.

Transcriptome analysis of Scylla paramamosain hepatopancreas response to mud crab dicistrovirus-1 infection.

Scylla paramamosain, an economically significant crab, is widely cultivated worldwide. In recent years, S. paramamosain has faced a serious threat from viral diseases due to the expansion of culture scale and increased culture density. Among these, mud crab dicistrovirus-1 (MCDV-1) stands out as highly pathogenic, presenting substantial challenges to the healthy development of mud crab aquaculture. Therefore, a comprehensive understanding of the mud crab immune response to MCDV-1 infection is imperative for devising effective disease prevention strategies. In this study, transcriptomic analyses were conducted on the hepatopancreas of mud crabs infected with MCDV-1. The findings revealed a total of 5139 differentially expressed genes (DEGs) between healthy and MCDV-1 infected mud crabs, including 3327 upregulated and 1812 downregulated DEGs. Further analysis showed that mud crabs resist MCDV-1 infection by activating humoral immune-related pathways, including the MAPK signaling pathway, MAPK signaling pathway-fly, and Toll and Imd signaling pathway. In contrast, MCDV-1 infection triggers host metabolic disorders. Several immune-related vitamin metabolism pathways (ascorbate and aldarate metabolism, retinol metabolism, and nicotinate and nicotinamide metabolism) were significantly inhibited, which may create favorable conditions for the virus's self-replication. Notably, endocytosis emerged as significantly upregulated both in GO terms and KEGG pathways, with several viral endocytosis-related pathways showing significant activation. PPI network analysis identified 9 hub genes associated with viral endocytosis within the endocytosis. Subsequent GeneMANIA analysis confirmed the association of these hub genes with viral endocytosis. Both transcriptome data and qPCR analysis revealed a significant upregulation of these hub genes post MCDV-1 infection, suggesting MCDV-1 may use viral endocytosis to enter cells and facilitate replication. This study represents the first comprehensive report on the transcriptomic profile of mud crab hepatopancreas response to MCDV-1 infection. Future investigations should focus on elucidating the mechanisms through which MCDV-1 enters cells via endocytosis, as this may holds critical implications for the development of vaccine targets.

The role of Nrf2 signaling pathway in the mud crab (Scylla paramamosain) in response to Vibrio parahaemolyticus infection.

The transcription factor Nrf2 plays vital roles in detoxification and antioxidant enzymes against oxidative stress. However, the function of Nrf2 in crustaceans is not well studied. In this study, a novel Nrf2 gene from the mud crab (Sp-Nrf2) was identified. It was encoded 245 amino acids. Sp-Nrf2 expression was ubiquitously expressed in all tested tissues, with the highest expression level in the gill. Sp-Nrf2 protein was mainly located in the nucleus. The expression levels of Sp-Nrf2, and antioxidant-related genes (HO-1 and NQO-1) were induced after Vibrio parahaemolyticus infection, indicating that Nrf2 signaling pathway was involved in the responses to bacterial infection. Over-expression of Sp-Nrf2 could improve cell viability after H2O2 exposure, indicating that Sp-Nrf2 might relieve oxidative stress. Silencing of Sp-Nrf2 in vivo decreased HO-1 and NQO-1 expression. Moreover, knocking down Sp-Nrf2 in vivo can increase malondialdehyde content and the mortality of mud crabs after V. parahaemolyticus infection. Our results indicated that Nrf2 signaling pathway played a significant role in immune response against bacterial infection.

Essential role of the HSC70 in the mud crab Scylla paramamosain in response to Vibrio parahaemolyticus infection.

Heat shock proteins play an important role in host defense, and modulate immune responses against pathogen infection. In this study, a novel HSC70 from the mud crab (designated as SpHSC70) was cloned and characterized. The full length of SpHSC70 contained a 58 bp 5'untranslated region (UTR), an open reading frame (ORF) of 2,046 bp and a 3'UTR of 341 bp. The SpHSC70 protein included the conserved DnaK motif. The mRNA of SpHSC70 was highly expressed in the hemocytes, heart and hepatopancreas, and lowly expressed in the intestine. The subcellular localization results indicated that SpHSC70 was localized in both the cytoplasm and the nucleus. Moreover, SpHSC70 was significantly responsive to bacterial challenge. RNA interference experiment was designed to investigate the roles of SpHSC70 in response to bacterial challenge. V. parahaemolyticus infection induced the expression levels of SpPO, SpHSP70, SpSOD and SpCAT. Knocking down SpHSC70 in vivo can decrease the expression of these genes after V. parahaemolyticus infection. These results suggested that SpHSC70 could play a vital role in defense against V. parahaemolyticus infection via activating the immune response and antioxidant defense signaling pathways in the mud crab.

Identification and functional characterization of activating transcription factor 6 (ATF6) from the mud crab (Scylla paramamosain) in response to hydrogen peroxide and bacterial challenge.

Activating transcription factor 6 (ATF6) is critical for regulation of unfolded protein response (UPR), which is involved in the endoplasmic reticulum (ER) proteostasis maintenance and cellular redox regulation. In the present study, a ATF6 gene from the mud crab (designated as Sp-ATF6) has been cloned and identified. The open reading frame of Sp-ATF6 was 1917 bp, encoding a protein of 638 amino acids. The deduced amino acid sequences of Sp-ATF6 contained a typical basic leucine zipper (BZIP domain). Sp-ATF6 was widely expressed in all tested tissues, with the highest expression levels in the hemocytes and the lowest in the muscle. Subcellular localization showed that Sp-ATF6 was expressed in both nucleus and cytoplasm of S2 cells. The expression level of Sp-ATF6 was induced by hydrogen peroxide and V. parahaemolyticus challenge, indicating that the ATF6 pathway was activated in response to ER stress. In order to know more about the regulation mechanism of the Sp-ATF6, RNA interference experiment was investigated. Knocking down Sp-ATF6 in vivo can decrease the expression of antioxidant-related genes (CAT and SOD) and heat shock proteins (HSP90 and HSP70) after V. parahaemolyticus infection. All these results suggested that Sp-ATF6 played a crucial role in the defense against environmental stress and pathogen infection in crustaceans.

Essential role of the Cytochrome P450 2 (CYP2) in the mud crab Scylla paramamosain antioxidant defense and immune responses.

Cytochrome P450 (CYPs) enzymes are one of the critical detoxification enzymes, playing a key role in antioxidant defense. However, the information of CYPs cDNA sequences and their functions are lacked in crustaceans. In this study, a novel full-length of CYP2 from the mud crab (designated as Sp-CYP2) was cloned and characterized. The coding sequence of Sp-CYP2 was 1479 bp in length and encoded a protein containing 492 amino acids. The amino acid sequence of Sp-CYP2 comprised a conserved heme binding site and chemical substrate binding site. Quantitative real-time PCR analysis revealed that Sp-CYP2 was ubiquitously expressed in various tissues, and it was highest in the heart followed by the hepatopancreas. Subcellular localization showed that Sp-CYP2 was prominently located in the cytoplasm and nucleus. The expression of Sp-CYP2 was induced by Vibrio parahaemolyticus infection and ammonia exposure. During ammonia exposure, ammonia exposure can induce oxidative stress and cause severely tissue damage. Knocking down Sp-CYP2 in vivo can increase malondialdehyde content and the mortality of mud crabs after ammonia exposure. All these results suggested that Sp-CYP2 played a crucial role in the defense against environmental stress and pathogen infection in crustaceans.

Identification and functional characterization of glutaredoxin 5 from the mud crab (Scylla paramamosain) in response to cadmium and bacterial challenge.

Glutaredoxin (Grx) is a class molecule oxidoreductase, which plays a key role in maintaining redox homeostasis and regulating cell survival pathways. However, the expression pattern and function of Grx remain unknown in the mud crab (Scylla paramamosain). In the present study, a novel full-length of Grx 5 from the mud crab (designated as Sp-Grx 5) was cloned and characterized. The open reading frame of Sp-Grx 5 was 441 bp, which encoded a putative protein of 146 amino acids. The amino acid sequence of Sp-Grx 5 contained a typical C-G-F-S redox active motif and several GSH binding sites. Sp-Grx 5 widely existed in all tested tissues with a high-level expression in hepatopancreas. Subcellular localization showed that Sp-Grx 5 was located in the cytoplasm and nucleus. The expression of Sp-Grx 5 was significantly up-regulated after Vibrio parahaemolyticus infection and cadmium exposure, suggesting that Sp-Grx 5 was involved in innate immunity and detoxification. Furthermore, overexpression of Sp-Grx 5 could improve cells viability after H2O2 exposure. All these results indicated that Sp-Grx 5 played important roles in the redox homeostasis and innate immune response in crustaceans.

Identification of co-chaperone Cdc37 in Penaeus monodon: coordination with Hsp90 can reduce cadmium stress-induced lipid peroxidation.

Cell division cycle 37 (Cdc37) is an important cytoplasmic phosphoprotein, which usually functions as a complex with heat shock protein 90 (Hsp90), to effectively reduce the damage caused by heavy metals, such as cadmium (Cd), in aquatic animals. The high toxicity of Cd in aquatic systems generally has a deleterious effect on healthy farming of shrimps. In the present study, a novel Cdc37 gene from Penaeus monodon was identified and designated as PmCdc37. Following exposure to Cd stress, the expression levels of PmCdc37 were upregulated at the transcriptional level in both the hepatopancreas and hemolymph. RNA interference and recombinant protein injection experiments were carried out to determine the function of PmCdc37 in P. monodon following Cd exposure. To clarify the correlations between PmCdc37 and PmHsp90, the respective recombinant proteins were expressed in vitro, and the ATPase activity of PmHsp90, with or without PmCdc37, was assessed. Moreover, a pull-down assay was conducted to detect the correlation between PmCdc37 and PmHsp90. After analyzing the expression patterns of PmHsp90 following Cd challenge, whether PmHsp90 can promote the ability of PmCdc37 to resist Cd stress or not was investigated. The results showed that formation of a PmHsp90/PmCdc37 complex protected shrimp against Cd stress-induced damage. Moreover, we also confirmed that PmSOD is involved in Cd stress, and that the PmHsp90/PmCdc37 complex can regulate SOD enzymatic activity. PmSOD was involved in decreasing the MDA content in shrimp hemolymph caused by Cd stress. We concluded that during exposure to Cd, the PmHsp90/PmCdc37 complex increases SOD enzyme activity, and in turn decreases the MDA content, thereby protecting shrimp against the damage caused by Cd stress. The present studies contribute to understanding the molecular mechanism underlying resistance to Cd stress in shrimp.

Multiple functions of thymosin ß4 in the pearl oyster Pinctada fucata suggest its multiple potential roles in artificial pearl culture.

Thymosin ß4 is a multifunctional protein in vertebrates that participates in physiological processes, such as wound healing, immune response, cell proliferation and migration. We assessed the multifarious roles of this small peptide in Pinctada fucata, an oyster commonly used in pearl culture in China. Our results showed that when P. fucata was challenged by bacterial pathogens or LPS, the relative expression level of Pfthymosin ß4 mRNA was significantly up-regulated, suggesting its involvement in immune response of the animal. Recombinant Pfthymosin ß4 (rPfthymosin ß4) was produced and showed in vitro different antibacterial activities against different pathogenic bacteria; the inhibitory effect of rPfthymosin ß4 on bacterial growth was relatively stronger in the broth culture than agar culture. The overexpression of Pfthymosin ß4 in Escherichia coli BL21(DE3) cells could improve their resistance to Cu2+, Zn2+, Cd2+, and H2O2, suggesting that Pfthymosin ß4 is likely involved with antioxidant. rPfthymosin ß4 also significantly promoted the proliferation and migration of mouse aortic vascular smooth muscle cells as indicated by MTT assay and cell scratch assay, respectively. In addition, chemically synthesized or recombinant Pfthymosin ß4 could transiently increase the circulating total hemocytes counts but down-regulated by RNAi in P. fucata. Taking together above results and previous studies suggested that Pfthymosin ß4 is potentially able to promote wound healing through enhancing antibacterial activity and antioxidant capacity, promotion of cell proliferation and migration, and increase of circulating hemocytes in P. fucata due to nucleus implantation injury. Thus, the future of recombinant Pfthymosin ß4 should be promising in the culture of pearls in P. fucata.

Novel 2-Cys Peroxiredoxin gene confers biotic and abiotic stress resistance in Penaeus monodon.

Peroxiredoxins (Prxs) are crucial antioxidant proteins that protect against biotic and abiotic stresses in many organisms, ranging from bacteria to mammals. In the present work, a novel 2-Cys Peroxiredoxin gene (PmPrxn), which contains a 153 bp 5'-terminal untranslated region (5'-UTR), a 636 bp open reading frame encoding a protein with 211 amino acids, and an 898 bp 3'-UTR, was successfully identified and characterized in the black tiger shrimp, Penaeus monodon. Tissue-specific expression analysis revealed that the PmPrxn mRNA was ubiquitously expressed and was comparatively highly expressed in the hepatopancreas. To explore the immunity-related and anti-stress roles of PmPrxn, the gills and hepatopancreas were chosen as target tissues in P. monodon and challenged with Vibrio harveyi, Streptococcus agalactiae, and toxic environmental stressors. The results indicate that PmPrxn might play a vital role in response to biotic and abiotic stresses. Furthermore, the antimicrobial and heavy metal toxicity stress-resistance properties of PmPrxn were evaluated and investigated in vitro using a prokaryotic expression system. These results provide useful information that will help further understand the functional mechanisms of PmPrxn in the defense against bacterial pathogens and environmental acute stresses in shrimp.

Molecular characterization and functional analysis of TRAF6 in the spotted sea bass (Lateolabrax maculatus).

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial adapter protein in the toll-like receptor signaling pathway that triggers downstream molecules involved in innate immunity. Although TRAF6 has been well studied in mammals, the molecular information and function of TRAF6 in fish is still limited. Here, we identified and analyzed a TRAF6 homolog (LmTRAF6) from the spotted sea bass (Lateolabrax maculatus). Similar to its counterparts in mammals and other fish species, LmTRAF6 shares the domain topology containing one N-terminal RING, two TRAF-type zinc fingers, a coiled-coil region and a C-terminal MATH domain. Despite a sequence similarity of 60% with mammalian TRAF6s, LmTRAF6 shares higher similarities with teleost homologs (~68%-93%). The coding region of LmTRAF6 gene contains seven exons and six introns, which is consistent to the genetic organization in grouper and rock bream, but not in zebrafish, common carp and tetrapods (the sixth intron was lost resulting in a combined exon). Quantitative real-time polymerase chain reaction analysis revealed that LmTRAF6 transcripts were ubiquitously expressed in all tested tissues and upregulated after Vibrio. harveyi and S. agalactiae infection. LmTRAF6 could assist HEK293T cells to survive by inhibiting apoptosis under both V. harveyi and S. agalactiae stimulation. Intracellular localization showed that LmTRAF6 was localized mainly in the cytoplasm. Overexpression of wild-type (WT) LmTRAF6 and the truncated form of △MATH increased the ability of NF-κB in HEK293T cells, whereas truncations, including the △RING and △coiled-coil domain, did not significantly activate NF-κB, indicating that the RING finger and coiled-coil domain play crucial roles in downstream signal transduction. In addition, overexpression of LmTRAF6-WT significantly increased the activation of NF-κB in HEK293T cells under V. harveyi and S. agalactiae stimulation. These results suggest that LmTRAF6 activates NF-κB and plays a potential role in the immune defense system against bacterial infection.

Molecular characterization and functional analysis of Akirin from black tiger shrimp (Penaeus monodon).

Akirin, which are members of the NF-κB signaling pathway, play critical roles in regulating the expression of antimicrobial peptides. In the present study, the Akirin gene from Penaeus monodon was identified from a transcriptome database and designated as PmAkirin. The complete sequence of the PmAkirin cDNA was 1508 bp, encoding a protein of 213 amino acids, and it showed 99% amino acid identity to the Litopenaeus vannamei Akirin. Two predicted nuclear localization signals (NLSs) were found, and the amino acid sequence alignments showed that PmAkirin was highly conserved at the N-terminus and C-terminus. PmAkirin expression was found to be the highest in the hemolymph, followed by the heart, gill, stomach, hepatopancreas, intestine, and muscle. When challenged with Vibrio parahaemolyticus infection, the PmAkirin mRNA and three antimicrobial peptides (AMPs: PmALF2, PmALF3, and PmCrus4) were upregulated. However, another five AMPs (PmALF6, PmCrus1, PmPEN3a, PmPEN3b, and PmPEN5) were downregulated by V. parahaemolyticus infection. Silencing PmAkirin by dsRNA significantly decreased the expression of the eight AMPs, which lead to an increase in the blood concentration of V. parahaemolyticus and higher mortality in the shrimp. In contrast, the overexpression of PmAkirin significantly increased the expression of the eight AMPs, which led to a reduction in the blood concentration of V. parahaemolyticus and promoted the survival of the shrimp. Taken together, we concluded that PmAkirin plays an important role in regulating the expression of AMPs in black tiger shrimp to defend against V. parahaemolyticus infection.

The acute stresses role of the atypical 2-cys peroxiredoxin PmPrx5 in black tiger shrimp (Penaeus monodon) from biological immunity and environmental toxicity stress.

As a unique atypical 2-Cys Peroxiredoxin (Prx) of the Prx-like superfamily, Peroxiredoxin5 (Prx5) possesses special properties, such as its enzymatic mechanism, wide subcellular distribution and high affinity for peroxides and peroxynitrite. Prx5 plays a crucial role in oxidative stress, immune responses, cell apoptosis, proliferation, differentiation, intracellular signaling, the modulation of gene expression, ecdysis, etc. In this paper, we obtained a full-length Prx5 cDNA sequence (designated PmPrx5) from black tiger shrimp (P. monodon). The full-length PmPrx5 cDNA sequence was 1686 bp containing a 5' untranslated region (UTR) of 76 bp with two nucleotide sequences (AAA), a 3' UTR of 1040 bp with a poly (A) tail and two canonical polyadenylation signal sequences (AATAAA), and an open reading frame of 570 bp encoding 189 amino acid residues with a predicted molecular mass of 20 kDa and a theoretical isoelectric point of 6.29. Phylogenetic trees and multiple sequence alignment showed that the PmPrx5 had strong homology with Prx5 proteins from other species, such as similarity with Palaemon carinicauda (69%) and Macrobrachium rosenbergii (69%), containing the highly conserved functional domain. PmPrx5 mRNA was ubiquitously detected in all tested tissues. After P. monodon was exposed to pathogenic bacteria, osmotic pressure, acidity and alkalinity and the heavy metal, the mRNA expression of PmPrx5 in the gills and hepatopancreas was significantly enhanced (P < 0.01) because of the immune response and declined with heavy metal copper and cadmium challenges as time progressed. The recombinant PmPrx5 protein purified in E. coli (DE3) was further confirmed to exhibit antioxidant activity and antibacterial properties to a certain extent using a bacterial growth inhibition test in both liquid and solid cultures in vitro. E. coli transformed with pRSET-PmPrx5 were dramatically protected in response to metal toxicity stress. Thus, PmPrx5 may be developed as a potential therapeutic drug against pathogenic bacteria and as a biomarker for pollutant levels. This work offers useful clues to further explore the functional mechanism of Prx5 in marine shrimp immunity.

TLR3 gene in Japanese sea perch (Lateolabrax japonicus): Molecular cloning, characterization and expression analysis after bacterial infection.

Toll-like receptors (TLRs) play important roles in fish innate immune and are involved in the defense process of bacteria invasion. In the present study, the full-length cDNA of TLR3 from the sea perch, Lateolabrax japonicus, was cloned and characterized. The full length of LjTLR3 cDNA was 3265 bp including an open reading frame of 2679 bp encoding a peptide of 922 amino acids. Tissues distribution analysis indicated that LjTLR3 showed a tissue-specific variation with high expression in spleen, head-kidney and liver. In order to investigate LjTLR3 functions against bacteria infection, the expression patterns of LjTLR3 after Vibrio harveyi and Streptococcus agalactiae challenge were detected by qRT-PCR, and the results showed that LjTLR3 was significant up-regulated after both bacteria stimulation in head-kidney, spleen and liver in a time-depended manner. Furthermore, the results by in situ hybridization experiments showed that positive signals of LjTLR3 mRNA in infected spleen and head-kidney were more numerous than that in the control group. In addition, intracellular localization revealed that LjTLR3 is distributed in the cytoplasm. In summary, these findings suggest that LjTLR3 was involved in the immune process under bacteria infection. This study would benefit to further clarify the roles of fish TLRs in the immune process and contribute to further study on enhancing disease resistance of L. japonicus.

Transcriptome analysis of the immune reaction of the pearl oyster Pinctada fucata to xenograft from Pinctada maxima.

The pearl oyster Pinctada maxima exhibits great difficulty to culture pearls through nuclear insertion with an allograft, but it is easy for P. fucata to culture pearls after allografting. If P. fucata could be used as a surrogate mother to culture P. maxima pearls, it would benefit the pearl culture industry of P. maxima. However, this is blocked by the immune rejection of P. fucata against P. maxima mantle grafts. In this study, the immune responses of P. fucata hemocyte to allograft and xenograft were investigated after transplantation by transcriptome analysis. In total, 107.93 Gb clean reads were produced and assembled using the reference genome of P. fucata. Gene Ontology Term enrichment and KEGG enrichment analyses indicated that apoptosis, hippo signaling pathway, oxidation-reduction, MAPK signaling pathway, ribosome, protein processing in endoplasmic reticulum, purine metabolism, NF-kappa B signaling pathway, oxidative phosphorylation, Ras signaling pathway, and ubiquitin mediated proteolysis were involved in response to transplantation. Many genes related to oxidation-reduction reactions, the MAPK signaling pathway, and apoptosis were identified by comparison of the allograft group and the xenograft group at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h post-transplantation. Among them, the expression levels of NADH dehydrogenase, succinate dehydrogenase and other dehydrogenases were increased significantly in the xenograft groups compared with allograft groups at 0 h post transplantation, indicating that a respiratory burst of neutrophils occurred immediately after xenograft transplantation. Additionally, HSP70 was highly expressed from 0 h to 96 h in the xenograft groups, indicating an oyster immune response to the xenograft. The genes enriched in the ribosome and hippo-signaling pathways were also identified, and expression patterns of these DEGs were different as compared between transplantation and control groups. Finally, altered expression levels of 10 randomly selected immune-related DEGs were confirmed by quantitative real-time PCR. These results indicated that oxidation-reduction is likely the key factor responsible for immune rejection to transplantation. The findings should provide some new insight into the molecular mechanism of immune rejection of the host against xenograft, and thus benefit to development of immunosuppressive reagents to facilitate effective xenograft pearling.

Serum immune response of pearl oyster Pinctada fucata to xenografts and allografts.

The mantle piece from the donor pearl oyster would be rejected by the immune system of recipient oyster in pearl culture practice, especially in the case that the donor and receptor are different species. Thus, investigation of the immune response of recipient oyster to grafted mantle pieces, particularly to xenografts, is of importance in creating xenograft transplantation technology for pearl culture industry. The humoral immune responses of P. fucata to allograft (mantle piece of P. fucata) and xenografts (mantle pieces of P. maxima and P. margaritifera, respectively) were studied in this paper. The oysters receiving no transplantations were served as the control group. The serum was collected from recipient P. fucata at 1 d, 2 d, 3 d, 4 d, 5 d, 7 d, 9 d, 11 d, 13 d, and 15 d, respectively after transplantation, and the serum antibacterial activity, lysozyme activity (LZM), alkaline phosphatase (AKP), acid phosphatase (ACP), total antioxidant capacity (TAC), and agglutination to rabbit red blood cells were investigated. The result indicated that serum of both the experimental groups and the control group can agglutinate rabbit red blood cells, with variation between groups and between time points, respectively. The antibacterial activity in the experimental group was significantly higher than that in the control group at 2-4 d, but lower at 5-11 d and returned back to normal at 15 d, with significant differences among experimental groups (P < 0.05). The LZM in the experimental group was significantly higher than that in the control group at 3-7 d, with significant differences in bacteriolytic activity among various groups (P < 0.05). Both the ACP and AKP activity levels in the experimental groups were higher than those in the control group at 2-9 d, with significant differences among various groups at 3-9 d (P < 0.05). The TAC level in the experimental groups was higher than that in the control group at 1-7 d, with significant differences among various groups at 4-7 d (P < 0.05). The above results indicated that all of the humoral immune factors investigated showed immune responses to both allografts and xenografts, with no specific to any of them. Thus, it is necessary to further screen immune rejection factors specific to xenografts, including cellular immune components.

High carotenoids content can enhance resistance of selected Pinctada fucata families to high temperature stress.

Carotenoids are a class of natural antioxidants widely found in aquatic, and they have significant effects on the growth, survival, and immunity of these organisms. To investigate the mechanisms of carotenoids in high temperature resistance, we observed the immune response of selected pearl oyster Pinctada fucata (Akoya pearl oyster) families with different carotenoids contents to high temperature stress. The results indicated that the survival rate (SR) of P. fucata decreased significantly with increase in temperature from 26 °C to 34 °C and with the decrease of total carotenoids content (TCC); when the TCC was higher, the SR tended to be higher. TCC and total antioxidant capacity (TAC) decreased significantly at 30 °C with increasing stress time. Correlation analysis indicated that TAC was positively and linearly correlated with TCC, and SR was S-type correlated with TCC and TAC. Immune analysis indicated that levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in selected families (with higher TCC) under temperature stress (at 30 °C) were generally significantly lower than in the control group (with lowest TCC) and from 0 to 96 h, the levels of each of these substances varied significantly. Levels of SOD, CAT, and MDA within each family first rose from 0 to 3 h, then decreased to their lowest point after 24 h, and then rose again to their highest levels at 96 h. When TCC was higher, the levels of SOD, CAT, and MDA tended to be lower. These findings indicated that carotenoids play an important role in improving survival rates of P. fucata under high temperature stress by enhancing animals' antioxidant system, and could serve as an index for breeding stress-resistant lines in selective breeding practices.

Differentially expressed immune-related genes in hemocytes of the pearl oyster Pinctada fucata against allograft identified by transcriptome analysis.

The pearl oyster Pinctada fucata is commonly cultured for marine pearls in China. To culture pearls, a mantle piece from a donor pearl oyster is grafted with a nucleus into a receptor. This transplanted mantle piece may be rejected by the immune system of the recipient oyster, thus reducing the success of transplantation. However, there have been limited studies about the oyster's immune defense against allograft. In this study, hemocyte transcriptome analysis was performed to detect the immune responses to allograft in P. fucata at 0 h and 48 h after a transplant. The sequencing reaction produced 92.5 million reads that were mapped against the reference genome sequences of P. fucata. The Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to identify all immune-related differentially expressed genes (DEGs). Compared with patterns at 0 h, a total of 798 DEGs were identified, including 410 up-regulated and 388 down-regulated genes at 48 h. The expression levels of interleukin receptor and toll-like receptor in hemocytes were increased significantly 48 h post-transplant, indicating that the oyster immune response was induced. Finally, altered levels of 18 randomly selected immune-related DEGs were confirmed by quantitative real-time PCR (qRT-PCR). Our results provide the basis for further analysis of the immune rejection of allotransplantation.

Expression profiles and interaction suggest TBK1 can be regulated by Nrdp1 in response to immune stimulation in large yellow croaker Larimichthys crocea.

TBK1 has been extensively studied in mammals because of its important roles as a molecular bridge, linking the TLRs (TLR3 and TLR4) and RLRs signals to activate transcriptional factors IRF3 and IRF7 for IFN-I production. However, the information on molecular and functional characteristics of TBK1 in teleosts is limited. In this study, the molecular characterization and immune response of TBK1 in Larimichthys crocea (named as LcTBK1) as well as its interaction with Nrdp1 were investigated. Sequence analysis demonstrated that LcTBK1 included four functional motifs, the N-terminal protein kinase domain and ATP-binding site, middle ULD and C-terminal coiled-coil domain. The tissue expression profiles indicated that LcTBK1 gene was constitutively expressed in the twelve tissues examined, with high expression in brain. Temporal expression analysis showed that LcTBK1 mRNA was obviously increased in the liver after injection of LPS, Poly I:C and inactive Vibrio parahaemolyticus, however, declined at some time points in spleen and head-kidney. Furthermore, we found that LcTBK1 can interact with LcNrdp1, an E3 ubiquitin ligase that involved in immune response to Cryptocaryon irritans infection in L. crocea. The qPCR showed that LcNrdp1 was also significantly up-regulated in liver, down-regualted at some time points in spleen and head-kidney after LPS, Poly I:C and inactive V. parahaemolyticus injection, although the expression patterns of the two genes after the three treatments were different in change magnitude and up-regulation timespan. These results suggested that LcTBK1 was involved in L. crocea defense against the pathogen infection and can be regulated by Nrdp1 in PPRs signaling pathway of fishes.

The complete mitochondrial genome of bamboo shrimp Atyopsis moluccensis (Atyidae, Decapoda).

Atyopsis moluccensis, belonging to the family Atyidae, is one of the popular species in aquarium industry. Here, we sequenced the mitochondrial genome of A. moluccensis. The mitogenome of A. moluccensis is 15,933 bp in length, consisting 22 transfer RNAs, 13 protein-coding genes (PCGs), and two ribosomal RNAs. The composition of A. moluccensis mitogenome is 33.77% for A, 13.81% for G, 28.74% for T, and 23.68% for C. The A + T content of the heavy-strand was 62.51%. Except ND5, most of the PCGs had ATN as the start codon. Only COX2 and ND4 were stopped by incomplete stop codon. The phylogenetic relationship was reconstructed with 16 shrimp from six genera of family Atyidae, which revealed that A. moluccensis and A. gabonensis clustered together and species of the same genus were grouped together in a clade. The data are beneficial in understanding the evolution and phylogenetic relationships of Atyidae shrimp.