Functional properties of ATPIF1 in the orange-spotted grouper (Epinephelus coioides) in response to viral infection.

ATP synthase inhibitory factor 1 (ATPIF1) can activate mitochondrial autophagic pathway and mediates immune response by regulating ATP synthase activity. However, the role of fish ATPIF1 on viral infection is still unknown. In this study, we identified an ATPIF1 homolog (Ec-ATPIF1) from orange-spotted grouper (Epinephelus coioides). Ec-ATPIF1 is mainly expressed in the kidney and liver. The expression of Ec-ATPIF1 was significantly up-regulated after RGNNV stimulation in vitro. Further experiments showed that overexpression of Ec-ATPIF1 inhibited the expression of viral genes (CP and RdRp) and intracellular ATP synthesis. Ec-ATPIF1 overexpression also promoted the expression of mitophagy related genes (PINK1, Parkin, BNIP3, NIX, FUNDC1, LC3), inflammation-related factors (IL-1ß, IL-6, IL-8, IL-10, TNF-α, TLR2) and interferon pathway factors (IRF1, IRF3, IRF7, MX1, ISG15, ISG56, MDA5, TRIF). While the knockdown of Ec-ATPIF1 exhibited the opposite effects on the expression of viral genes and immune-related factors above. These data suggest that Ec-ATPIF1 can impact viral infection by regulating mitophagy, ATP synthesis, the expression of inflammatory factors and interferon pathway factors. These findings will be beneficial to better explore the immune regulatory mechanisms of fish respond to viral infection.

Characterization and functional analysis of SOCS9 from orange-spotted grouper (Epinephelus coioides) during virus infection.

The suppressor of cytokine signaling (SOCS) proteins family have twelve members including eight known mammalian SOCS members (CISH, SOCS1-7) and four new discovery members (SOCS3b, SOCS5b, SOCS8 and SOCS9) that is regarded as a classic feedback inhibitor of cytokine signaling. Although the function of the mammalian SOCS proteins have been well studied, little is known about the roles of SOCS in fish during viral infection. In this study, the molecular characteristics of SOCS9 from orange-spotted grouper (Epinephelus coioides, EcSOCS9) is investigated. The EcSOCS9 protein encoded 543 amino acids with typical SH2 (389-475aa) and SOCS_box (491-527aa), sharing high identities with reported fish SOCS9. EcSOCS9 was expressed in all detected tissues and highly expressed in kidney. After red-spotted grouper nervous necrosis virus (RGNNV) infection, the expression of EcSOCS9 was significantly induced in vitro. Furthermore, EcSOCS9 overexpression enhanced RGNNV replication, promoted virus-induced mitophagy that evidenced by the increased level of LC3-Ⅱ, BCL2, PGAM5 and decreased level of BNIP3 and FUNDC1. Besides, EcSOCS9 overexpression suppressed the expression levels of ATP6, CYB, ND4, ATP level and induced ROS level. The expression levels of interferon (IFN) related factors (IRF1, IRF3, IRF7, P53), inflammatory factors (IL1-ß, IL8, TLR2, TNF-α) and IFN-3, ISRE, NF-κB, AP1 activities were also reduced by overexpressing EcSOCS9. These date suggests that EcSOCS9 impacts RGNNV infection through modulating mitophagy, regulating the expression levels of IFN- related and inflammatory factors, which will expand our understanding of fish immune responses during viral infection.

Effects of Chinese herbal medicines on growth performance, intestinal flora, immunity and serum metabolites of hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatu♂).

The immunomodulatory roles of Chinese herbal Medicine (CHM) in aquatic animals have been well-recorded. However, how CHM impacts the intestinal microbiota and serum metabolism is not fully understood. In this study, the effects of different additive levels of CHM on the growth performance, immunity, intestinal flora and serum metabolism of hybrid grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatu) were investigated. The addition of 0.5%, 1.0%, 1.5% and 2.0% Chinese herbal medicine compound to feed could significantly improve the weight gain rate (WGR), specific growth rate (SGR) and survival rate (SR) of grouper, reduced feed coefficient, while had no significant difference on morphometric parameter. The most significant improvement for the parameters above was observed in 1.5% group. Different addition levels of CHM could also significantly enhance the activities of ACP, AKP, SOD, CAT and LZM in serum. Accordingly, the supplementation of CHM significantly induced up-regulation of immune genes such as IL-8, IL-1ß, TNF-α, Nrf2, Lzm in the liver, spleen and head kidney of grouper, improved the resistance of grouper to V. harveyi as well. The intestinal flora analysis showed that at the phylum level, the main dominant species of intestinal microorganisms were Firmicutes, Proteobacteria, Bacteroidota, Actinobacteriota, Gemmatimonadota, Desulfobacterota, Fusobacteriota and Myxococcota. At the genus level, the high abundance was Lactobacillus, Streptococcus, Bacteroides, Escherichia, Romboutsia, Sphingomonas and Muribaculaceae. The abundance of probiotics (such as Lachnospiraceae, Lactobacillaceae, Streptococcaceae, etc) in CHM-supplement groups were higher (highest in 1.5% group) compared with control group. Moreover, a total of 11 common differential metabolic pathways were screened by LC-MS metabolism analysis of serum, they were Neuroactive ligand-receptor interaction, Purine metabolism, Linoleic acid, Glycerophospholipid metabolism, Taurine and hypotaurine metabolism, Arginine and proline metabolism, ABC transporters, Aminoacyl-tRNA biosynthesis, Arachidonic acid metabolism, Drug metabolism-cytochrome P450, alpha-Linolenic acid metabolism. Also, three common differential metabolites (PI(20:4(5Z,8Z,11Z,14Z)/18:1(11Z)), PC(20:3(8Z,11Z,14Z)/22:1(13Z)), PC(22:0/20:4(5Z,8Z,11Z,14Z)) associated with intestinal health, growth and disease resistance was found. These data will contributes to a comprehensive understand for the regulatory roles of CHM on fish, which is also beneficial for the disease control and sustainable development of aquaculture.

Characterization of S100A12 from nile tilapia (Oreochromis niloticus) and its roles on inflammatory responses.

S100A12 is a member of S100 proteins family that induces pro-inflammatory response via ligating with the receptor for advanced glycation end products (RAGE) and subsequent activation of intracellular signal transduction pathways. But information about fish S100A12 remain largely unclear. In this study, the S100A12 homolog (On-S100A12) was identified from Nile tilapia (Oreochromis niloticus). On-S100A12 was mainly expressed in liver and intestine. After Streptococcus agalactiae infection in vivo, S100A12 significantly increased in brain, intestine, liver and head kidney, suggesting S100A12 might played roles in immune response. The further in vitro experiments found that recombinant protein of S100A12 (rOn-S100A12) upregulated the expression of IL1-ß, TLR2, TNF-α and inhibited the expression of IL-10, indicating On-S100A12 promoted inflammatory response and activation of M1 macrophages. The present data lay a foundation to further explore the roles of fish S100 during immune defense and will also be beneficial for better understanding of fish immune-regulatory network.

Functional characterization of BAG3 in orange-spotted grouper (Epinephelus coioides) during viral infection.

Bcl-2-associated athanogene 3 (BAG3) is a cochaperone protein that interacts with Bcl-2 and mediate cell death. However, little is known about the roles of fish BAG3 during viral infection. In this study, we characterized a BAG3 homolog from orange-spotted grouper (Epinephelus coioides) (EcBAG3) and investigated its roles during viral infection. The EcBAG3 protein encoded 579 amino acids with typical WW, PXXP and BAG domains, which shared high identities with reported fish BAG3. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBAG3 was highly expressed in brain and heart. And the expression of EcBAG3 was significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) stimulation in vitro. EcBAG3 overexpression could promoted the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)), which was enhanced by co-transfection with Hsp70 and Hsp22. Also, EcBAG3 overexpression up-regulated the expression of LC3-Ⅱ and down-regulated the expression of Bax and BNIP3, the IFN- (IRF1, IRF3, IRF7, IFP35, Mx1) or inflammation-related (IL-1ß and TNFα) factors, as well as decreased the activities of NF-κB, ISRE and IFN-3. While knockdown of EcBAG3 decreased the transcripts of RGNNV CP gene and RdRp gene. Further studies showed that EcBAG3 knockdown impaired the expression level of autophagy factor LC3-Ⅱ, and promoted the expression level of Bax and BNIP3, inflammatory factors and interferon factors. These data indicate that EcBAG3 can affect viral infection through modulating virus-induced cell death, regulating the expression of IFN- and inflammation-related factors, which will be helpful to further explore the immune response of fish during viral infection.

Conservation of structural and interactional features of CD226 and Necl5 molecules from Nile tilapia (Oreochromis niloticus).

CD226 interacts with its ligand Necl5 as a costimulatory signal. In this study, we cloned a CD226 from Nile tilapia (Oreochromis niloticus, named OnCD226) and a Necl5 (named OnNecl5). The open reading frame of OnCD226 was 1071 bp, encoding a protein of 356 amino acids. Sequence alignment analysis indicated that OnCD226 contained two Ig-like domains in ectodomain. The open reading frame of OnNecl5 was 1155 bp, encoding a protein of 384 amino acids, and there are three lg-like domains in the extracellular domain. In healthy tilapia, OnCD226 was distributed in all tested tissues and relatively higher in the brain, while OnNecl5 was relatively higher in the skin. After Streptococcus agalactiae infection, OnCD226 has the same up-regulated expression pattern as OnNecl5 in different tissues. After HKLs stimulation with S. agalactiae and Poly I:C, respectively. OnCD226 was significantly up-regulated (0.01 < p < 0.05) at 12 h and extremely significant up-regulation was observed (p < 0.01) at 48 h and 96 h, the peak was observed at 96 h after stimulation by S. agalactiae. After stimulation by Poly I:C, OnCD226 expression was extremely significant (p < 0.01) at 72 h and 96 h, the peak was observed at 96 h. After stimulation by Keyhole limpet hemocyanin (KLH), a classical T cell-dependent antigen, the expression of OnCD226 was significantly up-regulated in blood, head kidney, spleen, and thymus. Moreover, when compared with the first challenge, the gene expression of OnCD226 which response to the second challenge was up-regulated earlier. Subcellular co-localization studies showed that OnCD226 and OnNecl5 were distributed mainly in the cytomembrane. Yeast two-hybrid results, indicated a strong interaction between OnCD226 and OnNecl5. These results suggested that OnCD226 plays an important role during pathogens infection, and the interaction between CD226 and Necl5 is conserved in Nile tilapia.

Characterization and functional analysis of a galectin-related protein B from Nile tilapia involved in the immune response to bacterial infection.

Galectin-related protein is a kind of lectin without canonical activity that regulates cell adhesion and cell growth. In this study, a novel galectin-related protein B (OnGRPB) was identified from Nile tilapia (Oreochromis niloticus). The open reading frame of OnGRPB was 438 bp and encoded a peptide of 145 amino acids. The deduced protein sequence of OnGRPB possessed a conserved carbohydrate recognition domain (CRD) with partial sugar binding sites (N-R, V-N and W-E) and shared high identities with other fish GRPB proteins. The qRT-PCR analysis found that OnGRPB was widely distributed in various tissues and monocyte/macrophages (Mo/MΦ) of healthy tilapia. After Streptococcus agalactiae infection, OnGRPB transcripts were significantly up-regulated in liver, spleen, head kidney and Mo/MΦ. The recombinant OnGRPB protein (rOnGRPB) had the binding activity and agglutination ability to bacteria. Also, rOnGRPB could modulate antibacterial activity and inflammatory factor expression of Mo/MΦ. These data collectively indicate that OnGRPB plays roles in the immune response of Nile tilapia against bacterial infection.

Single-cell RNA-seq reveals different subsets of non-specific cytotoxic cells in teleost.

Non-specific cytotoxic cells (NCC) are important cytotoxic leukocytes in teleost immune system. However, the NCC subsets have not been clarified. Thus, we create a comprehensive cell map of ~24,062 head kidney-derived leukocytes from Nile tilapia post poly I:C stimulation using single-cell RNA sequencing (scRNA-seq). Based on cell heterogeneity and known markers, the cells were classified into four cell types including B cell, T cell, NCC and monocytes/macrophages (Mo/MΦ). In the meantime, the regulatory network of NCC population was predicted by WGCNA and four hub genes (Stbd1, VWF, PGP, and GRN) and one transcription factor (Hvcn1) were identified. To further study the differentiation of NCC, four subsets including memory-like NCC, mature NCC, immature NCC, and pre-NCC were revealed in NCC population for the first time. Our data will provide new insight into the biology of NCC and enable more accurate functional and developmental analysis of NCC in immune system of lower vertebrates.

Construction and evaluation of Vibrio alginolyticus ΔclpP mutant, as a safe live attenuated vibriosis vaccine.

Vibrio alginolyticus is a common and serious pathogen threatening the progress of coastal aquaculture. ClpP protease has been proved to be closely associated with biofilm formation, stress tolerance, autolysis and virulence in several pathogens. Hence, targeting ClpP may be a potentially viable, attractive option for the preparation of vaccine in preventing vibriosis. In this study, an in-frame deleted mutant strain (ΔclpP) was constructed by allelic exchange mutagenesis to investigate physiological role of clpP in pathogenicity of V. alginolyticus and evaluate its potential as a live attenuated vaccine. The results exhibited that ΔclpP showed no differences in external morphology, growth, swarming motility and ECPase activity. However, ΔclpP represented an increment in biofilm formation, and a decrement in adherence to CIK cells. In addition, virulence of ΔclpP was examined in pearl gentian grouper and was found to be seriously attenuated. ΔclpP induced high antibody titers and provided a valid protection with a relative percent survival value of 83.8% without histopathologic abnormality. Our results indicated ΔclpP showed a great potential to be a live attenuated vaccine.

Biological characterisation, expression and functional analysis of non-specific cytotoxic cell receptor protein 1 in Nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cell receptor protein 1 (NCCRP-1) plays a role in recognition of target cell and activation of non-specific cytotoxic cell (NCC). In this study, the full length of Nile tilapia NCCRP-1 (On-NCCRP-1) was cloned. cDNA is composed of 1045 bp with a 90 bp of 5'-Untranslated Regions (UTR), 702 bp open reading frame (ORF) and 253 bp 3'-UTR, encoding 233 amino acids (GenBank accession no: MF162296). The On-NCCRP-1 genomic sequence is 4471 bp in length and contains six exons and five introns. On-NCCRP-1 possesses some inherent conservative domains, such as proline-rich motifs, antigen recognition site, and F-box-related domain. Subcellular localisation and Western blot analysis indicated that On-NCCRP-1 is located in the cell membrane. The transcript of On-NCCRP-1 was detected in all the examined tissues of healthy Nile tilapia by using qRT-PCR, with the highest expression levels in the liver. Following Streptococcus agalactiae challenged in vivo, the On-NCCRP-1 expression was up-regulated significantly in brain, intestines, head kidney and spleen. In the in vitro analysis, the On-NCCRP-1 expression in NCCs was up-regulated significantly from 8 h to 12 h after LPS challenge, and up-regulated significantly at 12 h after challenged with polyI:C. After NCCs were challenged with inactivated S. agalactiae, the On-NCCRP-1 expression was down-regulated significantly after 24 h. NF-кB pathway was strongly activated by the over-expression of On-NCCRP-1 in HEK-293T cells. These results indicate that On-NCCRP-1, as a membrane surface receptor of NCCs, may play an important role in immune response to pathogenic infection in Nile tilapia.

Molecular characterization and expression of CD48 in Nile tilapia (Oreochromis niloticus) in response to different stimulus.

CD48 is a glycosylphosphatidylinositol-anchored protein involved in lymphocyte adhesion, activation, and costimulation. In this study, the CD48 gene of tilapia (Oreochromis niloticus, named On-CD48), was cloned from the head kidney of tilapia. The coding sequences is 654 bp and encoding 217 amino acids. The deduced amino acid sequence of On-CD48 with an estimated molecular weight of 24.4 kDa and a theoretical pI of 5.03. Amino acid alignment indicated that it had two immunoglobulin-like domain conserved region. In healthy tilapia, the On-CD48 could be detected in all the examined tissues and the highest expression level in the spleen. The expression of On-CD48 in the spleen and head kidney was decreased after immunized by formalin-inactivated Streptococcus agalactiae, and the peak was observed in the spleen at 24 h and appeared again at 96 h, and in the head kidney gradual decline before 48 h then gradually increased to the original level. qPCR analysis of inactivated S. agalactiae, LPS and Poly I:C stimulated at the whole lymphocyte level showed that the stimulation of the Poly I:C was more sensitive. Prokaryotic expression results showed that efficient expression of On-CD48 protein could be realized after induced with 0.5 mmol L-1 IPTG in E. coli BL21 (DE3) for 10 h at 18 °C. The result of subcellular localization showed that On-CD48 were evenly distributed in the whole cell of HEK-293T. Western Blot confirmed that the molecular weight of the recombinant On-CD48 was about 21 kDa, consistent with the predicted result. The results of this study will lay a strong foundation for the further study of On-CD48 molecular function in tilapia.

The in vivo roles of galectin-2 from Nile tilapia (Oreochromis niloticus) in immune response against bacterial infection.

Our previous study has recorded that the recombinant protein of Nile tilapia (Oreochromis niloticus) galectin-2 (rOnGal-2) can enhance immune response against Streptococcus agalactiae (S.agalactiae) infection in vitro. In this study, we further explored the effects of OnGal-2 in immune response against bacterial infection in vivo. The administration of rOnGal-2 could improve serum antibacterial activity (ALKP, ACP, and LZM) and antioxidant capacity (CAT, POD, and SOD). After S. agalactiae infection, rOnGal-2 injection could reduce bacterial burden and decrease tissue damage in head kidney, spleen, and liver of tilapia. Also, rOnGal-2 regulated the inflammatory-related genes expression including IL-6, IL-8 and IL-10 during bacterial infection. Furthermore, rOnGal-2 administration could increase the relative percentage survival of tilapia infected with S.agalactiae. Taken together, our results indicate that OnGal-2 can protect fish from bacterial infection through reducing bacterial burden, impairing tissue damage and modulating anti-bacterial immune response, which also can be applied as a potential vaccine adjuvant in O.niloticus culture.

Superoxide dismutase B (sodB), an important virulence factor of Vibrio alginolyticus, contributes to antioxidative stress and its potential application for live attenuated vaccine.

Vibrio alginolyticus is an opportunistic and halophilic Gram-negative pathogen in limiting the development of aquatic industry and affecting human health. SODs are oxidative enzymes that play a critical role in oxidative defense. In this study, an in-frame deleted mutant strain (ΔsodB) was constructed by allelic exchange mutagenesis to investigate physiological role of sodB in pathogenicity of V. alginolyticus. The results exhibited that ΔsodB showed no differences in growth compared with wild-type strain HY9901 (WT), but led to increasing in biofilm formation, ECPase activity and sensitivity to hydrogen peroxide, decreasing in swarming motility, adherence to CIK cells, SOD activity and virulence. In addition, ΔsodB induced a high antibody titer and provided a valid protection with a relative percent survival value of 86.5% without inducing clinical symptoms after challenging with WT. These results suggest that sodB is important for normal physiological function, oxidation resistance and virulence in V. alginolyticus, and ΔsodB may be considered as an effective live attenuated vaccine against V. alginolyticus.

Characterization of a tandem-repeat galectin-9 from Nile tilapia (Oreochromis niloticus) involved in the immune response against bacterial infection.

Galectin-9 is a -galactoside-binding lectin which could modulate a variety of biological functions including recognition, aggregation and clearance of pathogen. In this study, a galectin-9 homologue (OnGal-9) was identified from Nile tilapia (Oreochromis niloticus) and its expression model and biological effects on bacterial infection were analyzed. The open reading frame of OnGal-9 sequence was 975 bp encoding 324 amino acids. It shares 45%-92% identities with other galectin-9 proteins. The deduced mature peptide of OnGal-9 possesses two conserved carbohydrate recognition domain (CRD) that connected with a linker peptide. Expression analysis indicated that OnGal-9 was distributed in all the tested tissues of healthy tilapia. The OnGal-9 expression was significantly up-regulated in spleen, head kidney, and intestine after challenged by Streptococcus agalactiae. Meanwhile, the recombinant OnGal-9 (rOnGal-9) protein displayed strong binding and agglutination activity toward both Streptococcus agalactiae and Aeromonas hydrophila. Moreover, rOnGal-9 could promote phagocytosis of macrophages. Taken together, the results here indicate that OnGal-9 might be involved in the immune response of Nile tilapia against bacterial infection.

Characterization of Galectin-2 from Nile tilapia (Oreochromis niloticus) involved in the immune response to bacterial infection.

galectin-2 plays important roles in innate and adaptive immunity. In this study, galectin-2 (OnGal-2) was identified from Nile tilapia (Oreochromis niloticus). Its tissue distribution and expression patterns following bacterial infection were also investigated. OnGal-2 is widely distributed in various tissues of healthy tilapia. After Streptococcus agalactiae challenge, OnGal-2 expressions were significantly up-regulated in all tested tissues. Meanwhile, the recombinant OnGal-2 (rOnGal-2) protein showed strong agglutinating activities against both Gram-negative bacteria and Gram-positive bacteria. Moreover, rOnGal-2 could promote phagocytosis of macrophages. Taken together, the present study indicated that OnGal-2 might play roles in the immune responses of Nile tilapia against bacterial pathogens.

An IL-6 gene in humphead snapper (Lutjanus sanguineus): Identification, expression analysis and its adjuvant effects on Vibrio harveyi OmpW DNA vaccine.

Interleukin 6 (IL-6) is a pleiotropic cytokine that plays important role in mediating the innate and adaptive immune responses against pathogen infection. In this study, an IL-6 homolog (Ls-IL6) was identified and characterized from humphead snapper, Lutjanus sanguineus. The full-length cDNA of Ls-IL6 was 1066 bp, containing an open reading frame (ORF) of 639 bp encoding 212 amino acids, 5' untranslated region(UTR) of 63 bp and 3' UTR of 605 bp. The predicted Ls-IL6 protein had typical motif of IL-6 family and shared high identities to teleost IL-6s. Ls-IL6 extensively expressed in various tissues, and the highest expression of Ls-IL6 was detected in head kidney, spleen and thymus. In vivo, the transcript levels of Ls-IL6 were significantly up-regulated in response to Vibrio harveyi infection. Moreover, the DNA plasmid containing the OmpW of V. harveyi together with the gene encoding Ls-IL6 were successfully constructed and administered to fish, the protective efficacy of Ls-IL6 was investigated. Compared with the pcDNA-OmpW group, the level of specific antibodies against V. harveyi increased in pcDNA-IL6-OmpW injected group. After V. harveyi infection, the pcDNA-IL6-OmpW vaccinated fish showed higher relative percent survival (76%) than the relative survival of fish immunized with pcDNA-OmpW (60%). These results indicated that Ls-IL6 was involved in immune response against V. harveyi infection and could be applied as a promising adjuvant for DNA vaccines against V. harveyi.

Functional characterization of galectin-3 from Nile tilapia (Oreochromis niloticus) and its regulatory role on monocytes/macrophages.

Galectin-3 is a kind of ß-galactoside-binding lectin involved in host defense against pathogen infection. However, the immune functions of fish galectin-3 remain poorly understood. In this study, the roles of a fish galectin-3 (OnGal-3) from Nile tilapia (Oreochromis niloticus) on the binding activity on bacterial pathogens or PAMPs, the agglutinating activity on bacterial pathogens and the regulatory effects on monocytes/macrophages activity were investigated. After in vitro challenge of Streptococcus agalactiae and Aeromonas hydrophila, OnGal-3 expressions were significantly up-regulated in monocytes/macrophages. In addition, recombinant OnGal-3(rOnGal-3) protein showed strong binding activity on bacterial pathogens or PAMPs. Also, rOnGal-3 agglutinated Gram-positive and Gram-negative bacteria. Moreover, rOnGal-3 could induce the inflammatory factors expressions in monocytes/macrophages and enhance phagocytosis and respiratory burst activity of monocytes/macrophages. These results suggest that fish galectin-3 participates in anti-bacterial immune response through recognizing pathogens and modulating monocytes/macrophages activity.

Traditional Chinese Medicine Enhances Growth, Immune Response, and Resistance to Streptococcus agalactiae in Nile Tilapia.

Herbal supplements are suitable for improving fish health and combating diseases in fish culture. However, the mechanism of action of many herbal mixtures remains unclear. This study was conducted to evaluate the effects of traditional Chinese medicine (TCM; a mix of Astragalus membranaceus, Angelica sinensis, and Crataegus hupehensis at a ratio of 1:1:1 on a weight basis) on growth, immune response, and disease resistance in Nile tilapia Oreochromis niloticus. Experimental fish (mean ± SE weight = 57 ± 1 g) were divided into two groups: a control group and a TCM (10 g/kg) group. After 4 weeks of the experimental trial, a significant increase in weight gain and specific growth rate and a lower feed conversion ratio were observed in fish fed a TCM-supplemented diet compared with control fish. Similarly, the immune response of Nile Tilapia in the TCM group showed enhanced lysozyme, superoxide dismutase, catalase, and immunoglobulin levels compared with the control fish. In comparison with the control, fish fed TCM showed significant up-regulation of ß-defensin, lysozyme, heat shock protein 70, superoxide dismutase, and catalase genes in the intestine and head-kidney tissues. After a Streptococcus agalactiae challenge, survival of Nile Tilapia in the TCM group was 70% compared with 35% in the control. These results indicate that the TCM mixture in this study can elevate the immune response and disease resistance of Nile Tilapia.

Conservation of structural and interactional features of CD28 and CD80/86 molecules from Nile tilapia (Oreochromis niloticus).

Interaction of CD28 with CD80 or CD86 molecules provides a costimulatory signals required in T cell activation. In this study, we cloned and analyzed a CD28 gene (On-CD28) and a CD80/86 gene (On-CD80/86) from Nile tilapia (Oreochromis niloticus). Sequence analysis revealed the typical characteristics of On-CD28 protein; for instance, the proline-based motif (117TYPPPL122) is essential in binding of CD28 to CD80/86 ligands. Moreover, an extracellular Ig domain was found in On-CD80/86; this domain is responsible in binding of CD28 to CD80/86 receptors. Subcellular localization analysis showed that both On-CD28 and On-CD80/86 were distributed predominantly in the cytomembrane. Yeast two-hybrid assay showed that On-CD28 directly interacted with On-CD80/86. On-CD28 and On-CD80/86 transcripts were detected in all the examined tissues of healthy Nile tilapia, and the highest expression levels of On-CD28 and On-CD80/86 were detected in the brain and heart, respectively. Following a bacterial challenge using Streptococcus agalactiae in vivo, On-CD28 and On-CD80/86 were upregulated in head kidney, spleen, intestines, and brain. However, they showed different expression profiles in response to stimulation with inactivated S. agalactiae in vitro. These findings indicated that the interaction of On-CD28 with On-CD80/86 provides a costimulatory signals that possibly play an important role in T cell activation during S. agalactiae infection.

Biological characterization, expression, and functional analysis of tumor necrosis factor receptor-associated factor 6 in Nile tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is a pivotal economic fish that has been plagued by Streptococcus agalactiae infections for many years. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial adaptor molecule of the interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily, which can trigger downstream signaling cascades involved in innate immunity. In this study, the full-length cDNA of TRAF6 was cloned from O. niloticus (named On-TRAF6), which has an open reading frame of 1716 bp, and encode a polypeptide of 571 amino acids. The predicted amino acid sequence of On-TRAF6 contained the characteristic motifs of TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, and a MATH (meprin and TRAF homology) domain. Multiple sequence alignment revealed that On-TRAF6 shares a relatively high level of identity with those of other fishes (64-98%). In healthy tilapia, mRNA expression of On-TRAF6 could be detected in all the examined tissues and the highest expression occurred in the spleen. Moreover, we found that On-TRAF6 was involved immune response of Nile tilapia following the stimulation with Streptococcus agalactiae and polyinosinic: polycytidylic acid (Poly I:C) when determined by using qPCR. The result of subcellular localization showed that On-TRAF6 distributed in the cytoplasm, and over-expression of On-TRAF6 could strongly activated NF-кB pathway in HEK293T cells. These findings indicated that On-TRAF6 may play an important role in the immune response to intracellular bacteria in Nile tilapia.