Roles of ß-catenin in innate immune process and regulating intestinal flora in Qi river crucian carp (Carassius auratus).

In mammals, ß-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of ß-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). ß-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, ß-catenin increased in gill, intestine, liver and kidney, indicating that ß-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the ß-catenin pathway using FH535, an inhibitor of Wnt/ß-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3, defb3, LYZ-C, INF-γ), upregulation of inflammatory factors (NF-κB, TNF-α, IL-1, IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that ß-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp.

Effect of dietary koumine on the immune and antioxidant status of carp (Cyprinus carpio) after Aeromonas hydrophila infection.

Koumine (KM) has anxiolytic, anti-inflammatory and growth-promoting effects in pigs and sheep. Based on the growth-promoting and immunological effects of koumine, the present study was conducted on Cyprinus carpio (C. carpio) with four KM concentrations: 0 mg/kg, 0.2 mg/kg, 2 mg/kg, and 20 mg/kg for 10 weeks, followed by a 1-week Aeromonas hydrophila (A. hydrophila) infection experiment. The effect of KM on the immunity of A. hydrophila infected carp was analyzed by histopathology, biochemical assay, and qRT-PCR to assess the feasibility of KM in aquaculture. The results showed that the presence of KM alleviated pathogen damage to carp tissues. At 2 mg/kg and 20 mg/kg concentrations of KM successively and significantly elevated (p < 0.05) the SOD activities in the intestinal tract, hepatopancreas and kidney of carp. The expression levels of hepatopancreatic antioxidant genes Nrf2 and IGF-1 were significantly up-regulated in the same group (p < 0.05), while the expression levels of immune genes IL-8 and IL-10 were down-regulated. In summary, KM at concentrations of 2 mg/kg and 20 mg/kg could regulate the expression of antioxidant and immune genes in various tissues in an orderly and rapid manner, and significantly improve the antioxidant and immune abilities of carp, which is conducive to the improvement of the resilience of carp.

The Yin Yang 1 of Penaeus vannamei regulates transcription of the small subunit hemocyanin gene during Vibrio parahaemolyticus infection.

Hemocyanin is a respiratory protein, it is also a multifunctional immune molecule that plays a vital role against pathogen invasion in shrimp. However, the regulation of hemocyanin gene expression in shrimp hemocytes and the mechanisms involved during pathogen infection remains unclear. Here, we used DNA pull-down followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the Yin Yang 1 transcription factor homolog in Penaeus vannamei (PvYY1) as a key factor that modulates transcription of the small subunit hemocyanin gene of P. vannamei (PvHMCs) in hemocytes during Vibrio parahaemolyticus AHPND (VPAHPND) infection. Bioinformatics analysis revealed that the core promoter region of PvHMCs contains two YY1 motifs. Mutational and oligoprecipitation analyses confirmed that PvYY1 could bind to the YY1 motifs in the PvHMCs core promoter region, while truncation of PvYY1 revealed that the N-terminal domain of PvYY1 is essential for the transactivation of PvHMCs core promoter. Besides, the REPO domain of PvYY1 could repress the activity of the PvHMCs core promoter. Overexpression of PvYY1 significantly activates the promoter activity of PvHMCs core promoter, while PvYY1 knockdown significantly decreases the expression level of PvHMCs in shrimp hemocytes and survival rate of shrimp upon infection with VPAHPND. Our present study provides new insights into the transcriptional regulation of PvHMCs by PvYY1 in shrimp hemocytes during bacteria (VPAHPND) infection.

Transcriptomic analysis of the antioxidant responses and immunomodulatory effects of dietary melatonin in red swamp crayfish (Procambarus clarkii).

This study aimed to investigate the effects of dietary melatonin (MT) levels on the antioxidant capacity, immunomodulatory, and transcriptional regulation of red swamp crayfish. Six experimental diets with different levels of MT (0, 22.5, 41.2, 82.7, 165.1, and 329.2 mg/kg diet) were fed to juvenile crayfish for 60 d. The transcriptome data of the control group and the group supplemented with dietary MT at 165.1 mg/kg were obtained using RNA-seq. In total, 3653 differentially expressed genes (2082 up-regulated and 1571 down-regulated) were identified. Pathways and genes related to antioxidant immune and growth performance were verified by qRT-PCR. The total hemocyte count, phagocytosis rate, and respiratory burst were significantly increased in the MT (165.1 mg/kg) group compared to the control group. Analysis of antioxidant immune-related enzymes in the hepatopancreas demonstrated that dietary MT (165.1 mg/kg) significantly increased activities of catalase, superoxide dismutase, glutathione reductase, and glutathione peroxidase and significantly decreased aspartate aminotransferase and alanine aminotransferase activity. At the transcriptional level, dietary MT up-regulated expression levels of genes associated with antioxidant immune and development, which included toll-like receptors, Crustin, C-type lectin, and so on. To conclude, MT could be used as a supplement in crayfish feed to increase immunity and antioxidant capacity and according to the broken line regression, the ideal MT concentration was the 159.02 mg/kg. Overall, this study demonstrates the role of melatonin in the antioxidant responses and immunomodulatory of Procambarus clarkii, laying the foundation for the development of melatonin as a feed additive in the aquaculture of this species.

The regulatory function of GATA3 on immune response in Japanese flounder (Paralichthys olivaceus).

GATA3 belongs to the GATA family, and it could interact with the target gene promoter. It has been reported to play a central role in regulating lymphocyte differentiation. In this study, the GATA3 cDNA sequence was identified by a homologous clone and the RACE technology from Japanese flounder (Paralichthys olivaceus). The full-length of the GATA3 cDNA sequence was 2904 bp, including 1332 bp open reading frame (ORF), 265 bp 5 '-untranslated region (5' UTR), and 1308 bp 3 '-UTR, encoding 443 amino acids. GATA3 protein sequence was conserved in vertebrates and invertebrates, including two zinc finger domains. qRT-PCR showed that the expression of GATA3 was high in the gill, kidney, and spleen. Expression of GATA3 slowly increased at the earlier stages and culminated at the late gastrula and somatic stages. Immunohistochemistry (IHC) results showed that the GATA3 protein was expressed in lymphocyte cells, undifferentiated basal and pillar cells of the gills, as well as lymphocyte cells and melanin macrophages of the kidney. The expression of GATA3 was significantly regulated in tissues and different types of lymphocytes after stimulation with Edwardsiella tarda. Dual-luciferase reporter assay indicated that the GATA3 protein could directly interact with promoters of target genes involved in the immune response. These findings suggested that GATA3 plays a major role in regulating the immune response. This study provided a theoretical basis for the immune response mechanism of teleost and a useful reference for later research on fish immunology.

Effects of dietary supplementation of Gracilaria lemaneiformis-derived sulfated polysaccharides on the growth, antioxidant capacity, and innate immunity of rabbitfish (Siganus canaliculatus).

The dietary supplementation of red seaweed-derived polysaccharides has been shown to be beneficial to fish and shellfish aquaculture. However, the function of red seaweed (Gracilaria lemaneiformis)-extracted polysaccharide (GLP) on the health status of rabbitfish (Siganus canaliculatus) is still unknown. This study explored the influences of GLP on growth performance, antioxidant activity, and immunity of rabbitfish. Herein, the fish were fed commercial pelleted feed incorporated with the diverse amount of GLP: 0 (control), 0.10 (GLP0.10), and 0.15 g kg-1 (GLP0.15) for 60 days. The results demonstrated that dietary GLP0.15 significantly elevated FBW and WG, while feed utilization efficiency improved (reduced feed conversion ratio and increased protein efficiency ratio) upon GLP0.10 treatment, regarding the control (P < 0.05). Also, dietary administration of GLP0.15 suggestively improved the serum acid phosphatase and lysozyme activity as well as hepatic total antioxidant capacity, catalase, and superoxide dismutase activity. In contrast, GLP0.15decreased the serum alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and malonaldehyde activity when compared to the control (P<0.05). Moreover, the lipase (36.08 and 16.46 U/mgprot in GLP0.10 and GLP0.15, respectively) and amylase (0.43 and 0.23 U/mgprot in GLP0.10 and GLP0.15, respectively) activity recorded the maximum values than the control (8.61 and 0.13 U/mgprot, respectively).Further, the intestinal morphometry was developed (such as increased villus length, width, and area) in the fish fed with a GLP-supplemented diet compared to the control. The KEGG pathway analysis unveiled that several differentially expressed genes (DEGs) in control vs. GLP0.10 and control vs. GLP0.15 were associated with metabolic or immune-associated pathways like antigen processing and presentation, phagosome, complement and coagulation cascades, and platelet activation. The DEGs, namely C3, f5, fgb, MHC1, and cfb, were evaluated in control vs. GLP0.10 and C3 and MHC1 in control vs. GLP0.15, suggesting their possible contributions to GLP-regulated immunity. Additionally, the cumulative mortality of rabbitfish after the Vibrio parahaemolyticus challenge was lower in both GLP0.10 (8.88%) and GLP0.15 (11.11%) than in control (33.33%) (P<0.05). Thus, these findings direct the potential use of GLP as an immunostimulant and growth promoter in rabbitfish aquaculture.

Peroxiredoxin-4 supplementation modulates the immune response, shapes the intestinal microbiome, and enhances AHPND resistance in Penaeus vannamei.

Peroxiredoxin-4 from Penaeus vannamei (LvPrx4) is considered a damage-associated molecular pattern (DAMP) that can activate the expression of immune-related genes through the Toll pathway. We previously demonstrated that the recombinant LvPrx4 (rLvPrx4) can enhance shrimp resistance against Vibrio parahaemolyticus, causing acute hepatopancreatic necrosis disease (VPAHPND), which causes great production losses in shrimp farming. Herein, we showed that the rLvPrx4 had a thermal tolerance of around 60 °C and that the ionic strength had no noticeable effect on its activity. We discovered that feeding a diet containing rLvPrx4 to shrimp for three weeks increased the expression of the immune-related genes LvPEN4 and LvVago5. Furthermore, pre-treatment with rLvPrx4 feeding could significantly prolong shrimp survival following the VPAHPND challenge. The shrimp intestinal microbiome was then characterized using PCR amplification of the 16S rRNA gene and Illumina sequencing. Three weeks of rLvPrx4 supplementation altered the bacterial community structure (beta diversity) and revealed the induction of differentially abundant families, including Cryomorphaceae, Flavobacteriaceae, Pirellulaceae, Rhodobacteraceae, and Verrucomicrobiaceae, in the rLvPrx4 group. Metagenomic predictions indicated that some amino acid metabolism pathways, such as arginine and proline metabolism, and genetic information processing were significantly elevated in the rLvPrx4 group compared to the control group. This study is the first to describe the potential use of rLvPrx4 supplementation to enhance shrimp resistance to VPAHPND and alter the composition of a beneficial bacterial community in shrimp, making rLvPrx4 a promising feed supplement as an alternative to antibiotics for controlling VPAHPND infection in shrimp aquaculture.

Characterization of hepcidin gene and protection of recombinant hepcidin supplemented in feed against Aeromonas hydrophila infection in Yellow River carp (Cyprinus carpio haematopterus).

Hepcidin is a small peptide of defensins with antibacterial activity, and plays an important role in innate immunity against pathogenic microorganisms, which can also participate in the regulation of iron metabolism. The hepcidin gene in Yellow River carp (Cyprinus carpio haematopterus) (CcHep) was cloned and identified. The total length of CcHep cDNA was 480 bp, containing an open reading frame (ORF) that encoded 91 amino acids (aa), which contained a 24-aa signal peptide, a 42-aa propeptide, and a 25-aa mature peptide. The mature peptide had a typical RX (K/R) R motif and eight conserved cysteine residues forming four pairs of disulfide bonds. Homology and phylogenetic tree analysis showed that CcHep had the closest relationship with that of crucian carp. The expression levels of hepcidin mRNA in healthy and Aeromonas hydrophila stimulated fish were measured by real-time fluorescence quantitative PCR. The results showed that CcHep mRNA was expressed in different tissues of healthy fish with the highest relative expression level in liver, followed by kidney and intestine, and the lowest expression level was observed in heart. The hepcidin gene was extremely significantly up-regulated in head kidney, intestine, liver, skin, spleen, and gill at 6 h and 12 h after A. hydrophila infection. Furthermore, the immunoregulation effect of dietary recombinant protein was evaluated. The recombinant hepcidin protein (rCcHep) was successfully expressed by Pichia pastoris X-33 and showed strong antibacterial activity against A. hydrophila, Escherichia coli, Vibrio anguillarum and Bacillus subtilis in vitro. In order to evaluate the preventive effect of rCcHep, fish were fed with basal diet or diet supplemented with different doses of rCcHep, and then challenged with A. hydrophila. The results showed that immune genes were up-regulated to varying degrees, and feed additive groups exhibited a significantly improved up-regulation expressions of Lysozyme, Toll-like receptor 5 (TLR 5), Major histocompatibility complex classⅡ (MHCⅡ), while inhibited up-regulation expressions of Interleukin 1ß (IL-1ß), Interleukin 8 (IL-8), and Tumor necrosis factor α (TNF-α) in liver and spleen compared to the control. Meanwhile, the relative immune protection rate in 120 mg/kg feed additive group was 28%, and the bacterial clearance rate in tissues of this group was higher than that of the control. Collectively, these results indicated that rCcHep had antibacterial activity and showed an immune protection effect against A. hydrophila, and could be considered as a dietary supplement to apply in aquaculture.

Effect of Clostridium butyricum on intestinal microbiota and resistance to Vibrio alginolyticus of Penaeus vannamei.

In order to evaluate the effect of Clostridium butyricum (C. butyricum) feeding on intestinal microorganisms and protection against infection by Vibrio alginolyticus (V. alginolyticus) in Penaeus vannamei (P. vannamei). We set up two groups, CG30 (fed normal feed) and CB30 (fed feed supplemented with C. butyricum), for the 30d C. butyricum feeding test, and four groups, CG (CG30 group injected with PBS), CB (CB30 group injected with PBS), VACG (CG30 group injected with V. alginolyticus), and VACB (CB30 group injected with V. alginolyticus), for the 24 h infection test. The protective effect of C. butyricum against acute V. alginolyticus infection in P. vannamei was explained in terms of survival, histopathology, changes in enzyme activity, transcriptome analysis, and immune-related genes. We found that feeding C. butyricum significantly altered intestinal microbial populations' abundance and significantly reduced Vibrio spp. In the V. alginolyticus stress test, C. butyricum improved the survival rate and alleviated pathological changes in hepatopancreatic tissues, alleviated the reduction of superoxide dismutase (SOD) and phenoloxidase (PO) activity caused by infection, and increased the lysozyme content in P. vannamei. VACB group compared with the VACG group, 1730 up-regulated differentially expressed genes (DEGs) and 2029 down-regulated DEGs were screened. Quantitative real-time PCR (qRT-PCR) showed that dietary supplementation with C. butyricum suppressed the upregulation of alkaline phosphatase (AKP) transcription factors and the downregulation of prophenoloxidase (proPO), alpha-2-macroglobulin (A2M), and anti-lipopolysaccharide factor (ALF) induced by V. alginolyticus infection. In conclusion, feed supplementation with C. butyricum changed P. vannamei's population ratio of intestinal microorganisms. Moreover, C. butyricum has the potential to act as an inhibitor of V. alginolyticus infection and enhance the resistance of P. vannamei to V. alginolyticus infection.

Animal models of Soft Tissue Sarcoma for alternative anticancer therapy studies: characterization of the A-72 Canine Cell Line.

Canine Soft Tissue Sarcoma (STS) cell line A-72 has been largely employed for antiviral and antiproliferative studies. However, there are few information on their characteristics. Our aim was to evaluate A-72 expression level of genes and proteins involved in the innate immune response and cell cycle, their ability to respond to infective stressors and their possible use as a cellular model for anti-cancer studies in human and animal medicine. For this purpose, we evaluated the basal expression of immune-related, cell cycle and DNA repair genes on this cell line and tumoral tissues. A-72 ability to respond to a wild-type strain of Salmonella typhimurium was assessed. S. typhimurium showed ability to penetrate A-72 causing pro-inflammatory response accompanied by a decrease of cell viability. IL10 and IL18 genes were not expressed in A-72 while CXCL8, NOS2, CXCR4 and PTEN were highly expressed in all samples and TP53 was slightly expressed, as shown in human STS. Our results outline the ability of A-72 to respond to a bacterial agent by modifying the expression of important genes involved in innate immune response and provide a useful model for in vitro evaluation of new therapeutic approaches that could be translated into the human oncology.

The enhancement effect of low-dose dietary lipopolysaccharide on the growth and immunity of Litopenaeus vannamei, and transcriptome analysis.

In this study, the effects of dietary lipopolysaccharide (LPS) on Litopenaeus vannamei were investigated to determine whether LPS could play a role as a potential immunostimulant in shrimp. L. vannamei with an initial body weight of 0.30 ± 0.02 g were fed a diet containing LPS at doses of 0, 0.2, 1, 5, 25 or 125 mg kg-1 for eight weeks (groups LPS0, LPS0.2, LPS1, LPS5, LPS25 and LPS125, respectively). After eight weeks of feeding, the growth performance, immunity and transcriptome response of L. vannamei were analysed. Only dietary LPS at 0.2 and 1 mg kg-1 resulted in a significant increase in the growth of L. vannamei (P < 0.05). According to the weight gain rate (WGR) and specific growth rate (SGR), the optimum dietary LPS level was 2.462 and 2.455 mg kg-1, respectively. When compared with the control group, the survival rate (SR) of L. vannamei in the LPS0.2 group was significantly increased after white spot syndrome virus (WSSV) infection and the SR of L. vannamei in the LPS1 group was significantly increased after Vibrio parahaemolyticus infection (both P < 0.05). Compared with the LPS0 group, immune enzyme activity in the serum of L. vannamei could be significantly increased and the content of maleic dialdehyde (MDA) significantly decreased by dietary LPS. Transcriptome analysis of the haemocytes of L. vannamei identified 399 up-regulated differentially expressed genes (DEGs) and 5000 down-regulated DEGs in the LPS0.2 compared to the control group. Most of the DEGs were significantly enriched in the following pathways: phosphatidylinositol signalling, Wnt signalling, Jak-STAT signalling and inositol phosphate metabolism. In conclusion, this study revealed that diets supplemented with low-dose LPS had positive effects on the growth and immunity of L. vannamei.

Transcriptome analysis of the immunomodulatory effects of Salvia miltiorrhiza polysaccharide on hemocyte immune response in Procambarus clarkii.

Natural plant polysaccharide as immune modulator is considered an effective strategy for healthy aquaculture to reduce medicine treatment. Salvia miltiorrhiza polysaccharides (SMP) had applications to regulate immune activity and enhance antioxidant in vertebrates, but the potential function has been rarely reported in crustaceans. In this study, the immunological effects of SMP on hemocytes of Procambarus clarkii were analyzed. Results showed that total superoxide dismutase (T-SOD), phenoloxidase (PO) activity and respiratory burst were up-regulated after SMP treatment. After high-throughput sequencing, 2170 differentially expressed genes (DEGs) including 1294 up-regulated and 876 down-regulated genes were identified. KEGG function enrichment analysis indicated that DEGs are involved in crustaceans cellular immune-related signaling pathways, including lysosome, phagosome and endocytosis. Transcriptome mining and qRT-PCR showed that SMP up-regulated humoral immunity factors gene expression. Diets supplemented with 0.8% SMP significantly up-regulated the total number of hemocytes (THC), T-SOD and PO activity, improved the survival of crayfish after Citrobacter freundii infection. This study suggested that SMP could improve the cellular and humoral immunity of P. clarkii. Furthermore, this finding supplied a molecular foundation for further comprehending the immunopotentiator effects of plant polysaccharides in crustaceans.

Substituting fish meal with castor meal in diets of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂): Effects on growth performance, immune response, antioxidant and digestive enzyme activities, gut morphology, and inflammatory-related gene expression.

The present study was conducted to investigate the effects of replacing fishmeal (FM) with castormeal (CM) on the growth performance, immune response, antioxidant and digestive enzyme activities, intestinal morphology, and expression of inflammatory-related genes in juvenile hybrid grouper (Epinephelus fuscoguttatus♀ ×E. lanceolatus♂). Six iso-nitrogenous (50% crude protein) and iso-lipidic (10% crude lipid) diets were formulated; namely, a reference diet (FM) containing 50% FM and five experimental diets (4% (CM4), 8% (CM8), 12% (CM12), 16% (CM16), and 20% (CM20)) in which FM protein was substituted with CM at varying levels to feed fish (initial weight: 9.12 ± 0.01 g) for 8 weeks. The results showed that the final weight, weight gain rate, and specific growth rate were highest in the FM, CM4, and CM8 groups, whereas the feed conversion ratio, hepatosomatic and viscerosomatic indexes were significantly enhanced in the CM4 group in comparison to the others. The CM4 and CM12 groups were observed to show the highest intestinal length index values compared to the other groups, with the CM20 revealing the worst growth performance. The serum total protein content first increased (P < 0.05) in the CM4 group and decreased (P < 0.05) afterward. Nonetheless, a decreasing significant (P < 0.05) cholesterol and triglyceride contents were witnessed with the increasing replacement of FM with CM. Compared to the control group, a significant increase (P < 0.05) in the activities of serum and liver immunoglobulin-M, superoxide dismutase, glutathione peroxidase, total antioxidant capacity, and complement-3 (except serum activity for CM12 group); liver lysozyme; intestinal amylase, and lipase, was witnessed in the CM groups. However, the serum lysozyme activity was highest (P < 0.05) in the CM4 group and lowest in the CM20 group. While the least serum malondialdehyde contents were observed in the CM4 group, that of the liver malondialdehyde was least witnessed in the FM, CM4, CM8, CM12, and CM16 groups as compared to the CM20. The intestinal histological examination revealed a significantly decreasing trend for villi height and villi width with increasing replacement levels. However, the muscle thickness, crypt depth, and type II mucus cells first increased upto 4% replacement level and later decreased. The increasing of dietary replacement levels significantly up-regulated pro-inflammatory (il-1ß, tnf-α, myd88, ifn-γ, tlr-22, and il-12p40) and down-regulated anti-inflammatory (il-10, tgf-ß, mhc-iiß) and anti-bacterial peptide (epinecidin and hepcidin) mRNA levels in the intestine. The mRNA levels of il-6 was up-regulated firstly upto 4 and 8% replacement levels, and later down-regulated with increasing replacement. These results suggested that, although higher dietary CM replacement enhances the immune, antioxidant and digestive enzymes, it aggravates intestinal inflammation. Replacing 4 and 8% of FM with CM could enhance the growth performance of fish.

Traditional Chinese herb formulas in diet enhance the non-specific immune responses of yellow catfish (Pelteobagrus fulvidraco) and resistance against Aeromonas hydrophila.

The effects of a traditional Chinese herbal mixture (TCHM) composed of Glycyrrhiza uralensis, Astragalus membranaceus, Rheum palmatum, Catsia tora and Lonicera japonica on immune response and disease resistance of yellow catfish (Pelteobagrus fulvidraco) were studied. Fish were fed diets containing 0% (control), 1.0%, 3.0% or 5.0% TCHM (w/w) for 28 d. Immune parameters including cytokine genes interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and Immunoglobulin M (IgM), acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), catalase (CAT), superoxide dismutase (SOD) and immunoglobulin M (IgM) were measured during the test period. After 28 d of feeding, fish were infected with Aeromonas hydrophila, and mortality was recorded. The TCHM-supplementation diet stimulated ACP, AKP, LZM, CAT, SOD, and IgM activity in serum and induced IL-1ß, TNF-α, and IgM mRNA expression in the spleen. All TCHM groups showed reduced mortality after A. hydrophila infection compared to the control group. These results suggest that the TCHM-supplemented diet can improve fish immunity and disease resistance against A. hydrophila.

The involvement of an interferon-induced protein 44-like (CgIFI44L) in the antiviral immune response of Crassostrea gigas.

Interferon-stimulated genes (ISGs) encoding proteins are the essential executors of interferon (IFN) mediated antiviral defense. In the present study, an ISG member, interferon-induced protein 44-like (IFI44L) gene (designed as CgIFI44L-1) was identified from the Pacific oyster Crassostrea gigas. The ORF of CgIFI44L-1 cDNA was of 1437 bp encoding a polypeptide of 479 amino acids with a TLDc domain and an MMR_HSR1 domain. The mRNA transcripts of CgIFI44L-1 were detected in all the tested tissues with highest level in haemocytes, which was 15.78-fold of that in gonad (p < 0.001). Among the haemocytes, the CgIFI44L-1 protein was detected to be highly expressed in granulocytes with dominant distribution in cytoplasm. The mRNA expression level of CgIFI44L-1 in haemocytes was significantly induced by poly (I:C) stimulation, and the expression level peaked at 24 h, which was 24.24-fold (p < 0.0001) of that in control group. After the treatment with the recombinant protein of an oyster IFN-like protein (rCgIFNLP), the mRNA expression level of CgIFI44L-1 was significantly enhanced at 6 h, 12 h and 24 h, which was 2.67-fold (p < 0.001), 5.44-fold (p < 0.001) and 5.16-fold (p < 0.001) of that in control group, respectively. When the expressions of CgSTAT and CgIFNLP were knocked down by RNA interference (RNAi), the mRNA transcripts of CgIFI44L-1 were significantly down-regulated after poly (I:C) stimulation, which was 0.09-fold (p < 0.001) and 0.06-fold (p < 0.001) of those in EGFP group, respectively. These results suggested that CgIFI44L-1 was a conserved ISG in oyster, which was regulated by CgIFNLP and CgSTAT, and involved in the oyster antiviral immune response.

Effects of recombinant anti-lipopolysaccharide factor expressed by Pichia pastoris on the growth performance, immune response and disease resistance of Litopenaeusvannamei.

The codon-optimized anti-lipopolysaccharide factor (ALF) sequence was introduced into pPICZαA vector and transformed into Pichia pastoris GS115. The recombinant ALF yeast supernatant (rALF-mix) was freeze-dried and evaluated as a feed additive for Litopenaeus vannamei. It was found by antibacterial activity test in vitro that the rALF-mix had antibacterial activity under different pH and temperature conditions. The 0, 0.00375%, 0.0075%, 0.015%, 0.03% and 0.06% of rALF-mix were added respectively to make the six experimental diets. After a 10-week feeding trial with shrimps (2.36 ± 0.02 g), it was found that the weight gain rate (WGR) and protein efficiency ratio (PER) of shrimp in the groups with 0.0075%, 0.015% and 0.03% of dietary rALF-mix supplementation were significantly higher than those in the control group (P < 0.05). Dietary rALF-mix supplementation significantly increased the total haemocyte count, respiratory burst, phagocytic activity, total anti-oxidative capacity (T-AOC), phenol oxidase activity, nitric oxide synthase activity, lysozyme (LYZ) activity, serum antibacterial capacity in the hemolymph and the T-AOC, LYZ in the hepatopancreas of shrimps (P < 0.05). The malondialdehyde contents in hemolymph and hepatopancreas were significantly decreased (P < 0.05). Meanwhile, the expression levels of toll, immune deficiency, heat shock protein 70, crustin and lipopolysaccharide-ß-glucan binding protein in the gill of shrimps were significantly increased (P < 0.05). After the challenge test, it was showed that dietary rALF-mix supplementation significantly improved the resistance of L. vannamei to Vibrio parahaemolyticus (P < 0.05). In conclusion, the rALF-mix can be used as a functional feed additive to improve the growth, immunity and disease resistance of shrimp. Based on the quadratic regression analysis for WGR, the optimal supplemental level of rALF-mix in diet for shrimp was estimated to be 0.02813%.

Molecular characterization of the evolutionary conserved signaling intermediate in Toll pathways (ECSIT) of soiny mullet (Liza haematocheila).

Mammalian evolutionary conserved signaling intermediate in Toll pathways (ECSIT) is an important intracellular protein that involves in innate immunity, embryogenesis, and assembly or stability of the mitochondrial complex I. In the present study, the ECSIT was characterized in soiny mullet (Liza haematocheila). The full-length cDNA of mullet ECSIT was 1860 bp, encoding 449 amino acids. Mullet ECSIT shared 60.4%∼78.2% sequence identities with its teleost counterparts. Two conserved protein domains, ECSIT domain and C-terminal domain, were found in mullet ECSIT. Realtime qPCR analysis revealed that mullet ECSIT was distributed in all examined tissues with high expressions in spleen, head kidney (HK) and gill. Further analysis showed that mullet ECSIT in spleen was up-regulated from 6 h to 48 h after Streptococcus dysgalactiae infection. In addition, the co-immunoprecipitation (co-IP) assay confirmed that mullet ECSIT could interact with tumor necrosis factor receptor-associated factor 6 (TRAF6). Molecular docking revealed that the polar interaction and hydrophobic interaction play crucial roles in the forming of ECSIT-TRAF6 complex. The resides of mullet ECSIT that involved in the interaction between ECSIT and TRAF6 were Arg107, Glu113, Phe114, Glu124, Lys120 and Lys121, which mainly located in the ECSIT domain. Our results demonstrated that mullet ECSIT involved in the immune defense against bacterial and regulation of TLRs signaling pathway by interaction with TRAF6. To the best of our knowledge, this is the first report on ECSIT of soiny mullet, which deepen the understanding of ECSIT and its functions in the immune response of teleosts.

The positive effects of dietary inositol on juvenile hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu) fed high-lipid diets: Growthperformance, antioxidant capacity and immunity.

The objective of the present research was to assess the influence of inositol supplementation on growth performance, histological morphology of liver, immunity and expression of immune-related genes in juvenile hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu). Hybrid grouper (initial weight 6.76 ± 0.34 g) were fed isonitrogenous and isolipidic diets (16%) with various inositol levels of 0.17 g/kg (J1, the control group), 0.62 g/kg (J2), 1.03 g/kg (J3), 1.78 g/kg (J4), 3.43 g/kg (J5), 6.59 g/kg (J6), respectively. The growth experiment lasted for 8 weeks. The results indicated that dietary inositol had a significant promoting effect on final mean body weight of the J5 and J6 groups and specific growth rate (SGR) of the J3, J4, J5 and J6 groups (P < 0.05). In the serum, superoxide dismutase (SOD) of the J4 group became significantly active compared with that of the control group (P < 0.05), while aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (AKP) activities in the inositol-treated groups showed distinctly decreased compared with those of the control group (P < 0.05). In the liver, dietary inositol could significantly increase the activities of SOD, catalase (CAT), lysozyme (LYZ) and the contents of total antioxidative capacity (T-AOC) and immunoglobulin M (IgM) (P < 0.05), and distinctly reduce the content of malondialdehyde (MDA) as well as reactive oxygen species (ROS) (P < 0.05). Compared with the control group, the damaged histological morphology of the liver was relieved and even returned to normal after an inositol increase (0.4-3.2 g/kg). In the liver, the remarkable up-regulation of SOD, CAT, glutathione peroxidase (GPX), heat shock protein70 (HSP70) and heat shock protein90 (HSP90) expression levels were stimulated by supply of inositol, while interleukin 6 (IL6), interleukin 8 (IL8) and transforming growth factor ß (TGF-ß) expression levels were down-regulated by supply of inositol. In head kidney, the mRNA of toll-like receptor 22 (TLR22), myeloid differentiation factor 88 (MyD88) and interleukin 1ß (IL1ß) expression levels were significantly down-regulated (P < 0.05), which could further lead to remarkable down-regulation of IL6 and tumor necrosis factor α (TNF-α) expression (P < 0.05). These results indicated that high-lipid diets with supply of inositol promoted growth, increased the antioxidant capacity, and suppressed the inflammation of the liver and head kidney by inhibiting the expression of pro-inflammation factors (IL6, IL8, TGF-ß and TNF-α). In conclusion, these results indicated that dietary inositol promoted growth, improved antioxidant capacity and immunity of hybrid grouper fed high-lipid diets. Based on SGR, broken-line regression analysis showed that 1.66 g/kg inositol supply was recommended in high-lipid diets of juvenile grouper.

Effects of Zinc Adaptation on Histological Morphology, Antioxidant Responses, and Expression of Immune-Related Genes of Grass Carp (Ctenopharyngodon idella).

The study was conducted to evaluate the effect of zinc adaptation on histological morphology and antioxidant and immune responses of grass carp(Ctenopharyngodon idella). A total of 180 young grass carp (20.0 ± 2.0 g) was equally distributed into 9 groups, and triplicate groups were subjected to 0 µg/L Zn2+ (control group), 200 µg/L Zn2+, and 300 µg/L Zn2+ solution for 42 days, respectively. The results indicated that the liver and gill have obvious pathological changes after long-term adaptation to zinc except the intestine; the zinc adaptation can positively influence intestinal morphology. The activities of GPX (glutathione peroxidase activity), SOD (superoxide dismutase), and CAT (Catalase) were significantly increased in zinc treatment groups (P < 0.05). The genes expression levels of CuZnSOD (copper zinc superoxide dismutase), CAT, Hsp70 (heat shock protein-70), IL-1b (interleukin-1-b), and TGF-ß1 (transforming growth factor-ß1) were upregulated in the gill and intestine of grass carp following waterborne adaptation to zinc solution for 42 days (P < 0.05). In conclusion, zinc adaptation has different effects on organs of grass carp and may reduce the inflammatory response of the body's gills and intestines by improving the body's antioxidant and anti-stress defense capabilities.

Dietary plant pigment on blood-digestive physiology, antioxidant-immune response, and inflammatory gene transcriptional regulation in spotted snakehead (Channa punctata) infected with Pseudomonas aeruginosa.

The current study addressed to investigate the effect of lycopene (LYC) on blood physiology, digestive-antioxidant enzyme activity, specific-nonspecific immune response, and inflammatory gene transcriptional regulation (cytokines, heat shock proteins, vitellogenins) in spotted snakehead (Channa punctata) against Pseudomonas aeruginosa. In unchallenged and challenged fish treated with 200 mg LYC enriched diet the growth performance and digestive-antioxidant enzymes increased after 30 days, whereas with inclusion of 100 or 400 mg LYC in the diets, the increase manifested on or after 45 days. No mortality in fish treated with any LYC diet against P. aeruginosa was revealed. In the unchallenged and challenged fish the phagocytic (PC) activity in head kidney (HK) and spleen were significantly enhanced when fed the control diet or other LYC diets, whereas the respiratory burst (RB) activity and nitric oxide (NO) production significantly increased when fed the 200 mg diet for 45 and 60 days. Similarly, the lysozyme (Lyz) activity in the HK and spleen, and total Ig content in serum were significantly higher in both groups fed the 200 mg LYC diet for 15, 45, and 60 days. Heat shock protein (Hsp 70) was significantly improved in the uninfected group fed the 200 mg LYC diet for 45 and 60 days, but Hsp27 did not significantly change among the experimental groups at any time points. TNF-α and IL-6 mRNA pro-inflammatory cytokine expression significantly increased in both groups fed the 200 mg LYC diet after 45 and 60 days, while the IL-12 mRNA expression was moderate in both groups fed the same diet for 60 days. The IL-10 did not significant mRNA expression between groups at any sampling. The iNOS and NF-κB mRNA expression was pointedly high in both groups fed the 200 mg LYC diet on day 45 and 60. Vitellogenin A (VgA) mRNA was significantly higher in the uninfected fish fed the 100 and 200 mg LYC diets for 45 and 60 days, but VgB did not reveal significant difference between the treatment groups at any time points. The present results suggest that supplementation of LYC at 200 mg significantly modulate the blood physiology, digestive-antioxidant enzymes, specific-nonspecific immune parameters, and cytokines, Hsp, and vitellogenins in spotted snakehead against P. aeruginosa.