USP46 promotes the interferon antiviral signaling in black carp by deubiquitinating TBK1.

Ubiquitin-specific peptidase 46 (USP46) functions as a deubiquitinating enzyme, facilitating the removal of ubiquitin molecules attached to substrate proteins and playing a critical role in cancer and neurodegenerative diseases. However, its function in innate antiviral immunity is unknown. In this study we cloned and identified bcUSP46, a homolog of USP46 from black carp. We discovered that overexpression of bcUSP46 enhanced the transcription of interferon (IFN) promoters and increased the expression of IFN, PKR, and Mx1. In addition, bcUSP46 knockdown significantly inhibited the expression of ISG genes, as well as the antiviral activity of the host cells. Interestingly, when bcUSP46 was co-expressed with the RLR factors, it significantly enhanced the activity of the IFN promoter mediated by these factors, especially TANK-binding kinase 1 (TBK1). The subsequent co-immunoprecipitation (co-IP) and immunofluorescence (IF) assay confirmed the association between bcUSP46 and bcTBK1. Noteworthily, co-expression of bcUSP46 with bcTBK1 led to an elevation of bcTBK1 protein level. Further analysis revealed that bcUSP46 stabilized bcTBK1 by eliminating the K48-linked ubiquitination of bcTBK1. Overall, our findings highlight the unique role of USP46 in modulating TBK1/IFN signaling and enrich our knowledge of the function of deubiquitination in regulating innate immunity in vertebrates.

Grass carp IL-20 binds to IL-20R2 but induces STAT3 phosphorylation via IL-20R1.

IL-20 is a pleiotropic cytokine that belongs to the IL-10 family and has a variety of biological functions in tissue homeostasis and regulation of host immune defenses. It signals through a heterodimeric receptor composed of a subunit with a long intracellular domain (R1 type receptor) and a subunit with a short intracellular domain (R2 type receptor). In this study, the R1 type receptor (CiIL-20R1/CRFB8) and the R2 type receptor (CiIL-20R2/CRFB16) were identified in grass carp Ctenopharyngodon idella. Expression analysis revealed that IL-20R2 was highly expressed in the gills and skin in healthy fish. Infection with Flavobacterium columnare resulted in the downregulation of both receptors in the gill at 48 and 72 h, whilst infection with grass carp reovirus induced their expression in the head kidney and spleen at 72 h. In the primary head kidney leucocytes, the expression levels of IL-20R1 and IL-20R2 were decreased after stimulation with 250 ng/mL IL-1ß but not affected by IFN-γ. Co-immunoprecipitation analysis showed that CiIL-20R2/CRFB16 but not CiIL-20R1/CRFB8 bound to CiIL-20L. Furthermore, it was shown that CiIL-20R1/CRFB8 was responsible for activating the phosphorylation of STAT3, whilst CiIL-20R2/CRFB16 was not involved. Structural modeling analysis showed that key residues involved in the interaction between IL-20 and receptors were highly conserved between grass carp and humans, suggesting that the signal transduction and functions of IL-20/IL-20R axis are evolutionarily conserved.

Expression and functional characterization of three ß-defensins in grass carp (Ctenopharyngodon idella).

ß-defensins (BDs) are a group of cysteine-rich cationic antimicrobial peptides and play important roles in the first line of defense against infection. In this study, the expression and antibacterial activities of three grass carp (Ctenopharyngodon idella) (Ci) ß-defensin (BD) peptides were comparatively investigated. Expression analysis reveals that CiBD1-3 were constitutively expressed in tissues, with the highest expression detected in the skin. The CiBD-1 transcripts were more abundant than CiBD-2 and CiBD-3. In the primary head kidney leukocytes, CiBDs were induced by PHA, LPS, poly(I:C) and cytokines such as IL-1ß and IFN-γ. In vivo challenge of fish with Aeromonas hydrophila resulted in the up-regulation of CiBDs in the head kidney and hindgut. To determine the biological activities, recombinant CiBD proteins were produced in the HEK293-F cells and purified for the minimum inhibitory concentration assay. It was found that all three recombinant CiBD proteins were effective to inhibit the growth of Gram-negative fish bacterial pathogens including Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare and Klebsiella pneumoniae and Gram-positive Staphylococcus aureus. CiBD-2 and CiBD-3 were more effective than CiBD-1. Our results demonstrate that all the three CiBDs have broad antibacterial activity against fish bacterial pathogens.

Antibiotic-induced alternations in gut microflora are associated with the suppression of immune-related pathways in grass carp (Ctenopharyngodon idellus).

Gut microbiota play a vital role in fish health homeostasis. Antibiotics are known to alter microbial community composition and diversity; however, the substantial effects of antibiotics upon the gut microbiome with respect to immune-related pathways in healthy fish remain unclear. Accordingly, here we explored the impact of two antibiotics on the intestinal health, immune response, microbiome dynamics, and transcriptome profiles of grass carp. A two-week feeding trial was carried out in which the basal diet was complemented with enrofloxacin (10 mg/kg) or florfenicol (10 mg/kg). The results showed that: (1) Enrofloxacin and florfenicol both induced intestinal oxidative stress and reduced the digestive enzyme activity of grass carp. (2) High-throughput sequencing of 16S rDNA revealed that enrofloxacin but not the florfenicol treatment influenced gut microbiota diversity in grass carp by shifting α/ß-diversity with more abundant pathogens detected. (3) Transcriptome profiling demonstrated that florfenicol down-regulated the immune-related pathways of grass carp, and the network analysis revealed that IgA was negatively correlated with certain pathogens, such as Shewanella and Aeromonas. (4) Antibiotic-induced alternations of gut core microbes were revealed via immune-related transcripts, as were lower mRNA expression levels of mucosal-related genes. (5) Apoptosis and histopathological changes were detected in the enrofloxacin- and florfenicol-treated groups compared with the control group. Overall, administering antibiotics will promote oxidative stress, cause intestinal flora dysbiosis, inhibit the mucosal immune system, and induce apoptosis in grass carp.

Herbivorous Juvenile Grass Carp (Ctenopharyngodon idella) Fed with Genetically Modified MON 810 and DAS-59122 Maize Varieties Containing Cry Toxins: Intestinal Histological, Developmental, and Immunological Investigations.

Feeding experiments with juvenile grass carp (Ctenopharyngodon idella) fed with genetically modified maize MON 810 or DAS-59122 dried leaf biomass were carried out with 1-, 3- and 6-month exposures. Dosages of 3-7 µg/fish/day Cry1Ab or 18-55 µg/fish/day Cry34Ab1 toxin did not cause mortality. No difference occurred in body or abdominal sac weights. No differences appeared in levels of inorganic phosphate, calcium, fructosamine, bile acids, triglycerides, cholesterol, and alanine and aspartame aminotransferases. DAS-59122 did not alter blood parameters tested after 3 months of feeding. MON 810 slightly decreased serum albumin levels compared to the control, only in one group. Tapeworm (Bothriocephalus acheilognathi) infection changed the levels of inorganic phosphate and calcium. Cry34Ab1 toxin appeared in blood (12.6 ± 1.9 ng/mL), but not in the muscle. It was detected in B. acheilognathi. Cry1Ab was hardly detectable in certain samples near the limit of detection. Degradation of Cry toxins was extremely quick in the fish gastrointestinal tract. After 6 months of feeding, only mild indications in certain serum parameters were observed: MON 810 slightly increased the level of apoptotic cells in the blood and reduced the number of thrombocytes in one group; DAS-59122 mildly increased the number of granulocytes compared to the near-isogenic line.

Bactericidal efficacy of non-thermal plasma activation against Aeromonas hydrophila and immunological responses of koi (Cyprinus carpio haematopterus).

In the aquaculture industry, an efficient and safe water purification system is important to prevent mass mortality by virulent pathogens. As extensive use of traditional methods (e.g.: povidone-iodine, ozone, ultraviolet irradiation, formalin, and chlorine dioxide) have adverse effects on cultured fish, an appropriate and alternative water purification method is vital for the sustainability of the industry. Non-thermal plasma technology has been successfully used for various biomedical purposes (e.g: food sterilization, medical device disinfection, wound healing, cancer therapy, etc.) and has great potential to be used as a sterilizing system. However, few studies have been conducted on its usefulness in the aquaculture industry. In this study, we investigated the bactericidal efficacy of plasma-activated water induced by non-thermal plasma and its histopathological as well as immunological adverse effects on koi. A highly virulent Aeromonas hydrophila SNU HS7, which caused massive mortality of koi, was used for this study. Non-thermal plasma was applied for 10 min to the fish tanks with 1.2 × 109 CFU/mL SNU HS7 using PLMB-20 system to confirm the sterilization efficacy and to observe the survival and immunological reaction of koi for 14 days. As a result, gross pathological, histopathological, and immunological investigations did not reveal any significant adverse effects in fish as compared to the control groups. To the best of our knowledge, this is the first study showing that non-thermal plasma can be used for sterilization of rearing water without giving significant physiological damage to the fish, even under the assumption of extreme situations. As plasma can effectively sterilize not only bacteria but also other unknown pathogens, the results of this study are showing a promising future in purifying water in aquaculture practice.

Phylogeny of the HO family in cyprinus carpio and the response of the HO-1 gene to adding Bacillus coagulans in feed under Cd2+ stress.

The heavy metal cadmium (Cd2+) is an environmental pollutant that poses serious health hazards. Due to the increasing contamination of aquatic systems with Cd2+, the increased accumulation of Cd2+ in fish has become a food safety and public health concern. Heme oxygenase (HO) is an important antioxidant enzyme that plays a key role in defending the body against oxidative damage, but little research has been done in common carp. In this study, 6 HO genes were identified in the common carp genome database. Comparative genomics analysis showed considerable expansion of the HO genes and verified the four-round whole genome duplication (WGD) event in common carp. Phylogenetic analysis revealed that all HO genes of common carp were clustered into orthologous groups, indicating high conservation during evolution. In addition, the tissue distribution results showed that most HO genes had extensive tissue distribution and showed tissue-specific expression patterns. Exposure to 0.5 mg/L Cd2+ significantly reduced the expression of TGF-ß and IL-10 in common carp, which may indicate that Cd2+ exposure can destroy the physical barrier function of the intestine, inhibit intestinal immune defense and induce intestinal inflammation. To find a suitable concentration of Bacillus coagulans that could activate HO-1 genes and the immunity of the organism, we investigated the changes in HO-1 gene expression levels in the intestinal tract of common carp under Cd2+ stress at 30 days and 60 days by adding different concentrations of B. coagulans to the feed. Compared with the Cd2+ stress group without supplementation, the expression levels of the HO-1 gene in the gut of three different concentrations of B. coagulans were almost increased. And B. coagulans with L2 concentrations had better activation effect on the HO-1 gene. Similarly, compared to the Cd2+ stressed group, adding B. coagulans to the diet can almost cause the early upregulation of IL-10 and TGF-ß genes. Therefore, the addition of appropriate concentrations of B. coagulans may be a good way to activate HO-1, IL-10, and TGF-ß genes, reduce oxidative damage, and encourage the immune.

Effects of four different adjuvants separately combined with Aeromonas veronii inactivated vaccine on haematoimmunological state, enzymatic activity, inflammatory response and disease resistance in crucian carp.

The purpose of the current study was to explore the immunomodulatory effects of different adjuvants combined with inactivated vaccines under Aeromonas veronii TH0426 infection in crucian carp. This study explored the best conditions for A. veronii as an inactivated vaccine, and included an animal safety test. Furthermore, we expressed the flagellin FlaA of the A. veronii TH0426 strain for use as an adjuvant supplemented in the diet. Crucian carp were fed 12 different experimental diets for 35 days, including the administration of 10 different adjuvants and inactivated vaccine combinations (50% aluminum hydroxide gel and inactivated vaccine combination, and inactivated vaccine with 20%, 30%, or 50% glucan, astragalus polysaccharide or flagellin), inactivated vaccine alone, and PBS control without adjuvant and inactivated vaccine. After the 42 day feeding trials, the fish were challenged with A. veronii TH0426, and the survival rate over 14 days was recorded. In addition, flagellin FlaA can be expressed normally in large amounts. All experimental groups produced higher levels of IgM serum titres than the control group in the different feeding cycles. Moreover, the activity of serum ACP, AKP, SOD, and LZM, and the expression of inflammatory factors were significantly increased in the experimental groups compared with the control group. The results of qRT-PCR analysis showed that the transcription levels of the IL-10, IL-1ß, IFN-γ and TNF-α genes in heart, liver, spleen and kidney tissues were significantly enhanced by adjuvant treatment, indicating that the addition of adjuvants can significantly promote the body's inflammatory response. In addition, the phagocytic activity of leukocytes in each adjuvant treated group was significantly enhanced compared to that in the groups without adjuvant. After the A. veronii challenge, the survival rate of all adjuvant-treated groups was significantly higher than that of the control group, and the 50% flagellin adjuvant group had the highest rate of 78.37%. Overall, our findings strongly indicate that adjuvants not only significantly improve the body's immunity, but also exhibit a strong anti-infection ability. Importantly, this work provides a new perspective for the prevention and control of aquaculture diseases.

Grass carp MAP3K4 participates in the intestinal immune response to bacterial challenge.

Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) is a multifunctional mediator of the conserved MAPK signaling pathway that plays essential roles in the regulation of immune responses in mammals. However, the function of teleost MAP3K4s in innate immunity, especially in the intestinal immune system, is still poorly understood. In the current study, we identified a fish MAP3K4 homolog (CiMAP3K4) in Ctenopharyngodon idella as well as its immune function in intestine following bacterial infection in vivo and in vitro. The open reading frame (ORF) of CiMAP3K4 encodes putative peptide of 1544 amino acids containing a predicted serine/threonine protein kinase (S_TKc) domain with high identity with other fish MAP3K4s. Phylogenetic analysis revealed the CiMAP3K4 belonged to the fish cluster and showed the closest relationship to Pimephales promelas. Quantitative real-time PCR (qRT-PCR) analysis revealed that CiMAP3K4 transcripts were widely distributed in all tested tissues, especially with high expression in the muscle and intestine of healthy grass carp. In vitro, CiMAP3K4 gene expression was upregulated by bacterial PAMPs (lipolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) and muramyl dipeptide (MDP)) and pathogens (Aeromonas hydrophila and Aeromonas veronii) in primary intestinal cells. In vivo, the mRNA expression levels of CiMAP3K4 in the intestine were significantly induced by bacterial MDP challenge in a time-dependent manner; however, this effect could be inhibited by the bioactive dipeptides ß-alanyl-l-histidine (carnosine) and alanyl-glutamine (Ala-Gln). Moreover, CiMAP3K4 was located primarily in the cytoplasm, and its overexpression increased the transcriptional activity of AP-1 in HEK293T cells. Collectively, these results suggested that CiMAP3K4 might play an important role in the intestinal immune response to bacterial infections, which paves the way for a better understanding of the intestinal immune system of grass carp.

Evaluation of the effects of Astragalus polysaccharides as immunostimulants on the immune response of crucian carp and against SVCV in vitro and in vivo.

This experiment was conducted to evaluate the immunomodulatory effect and antiviral activity of Astragalus polysaccharides (APS) in crucian carp and epithelioma papulosum cyprinid (EPC) cells. Two diets containing 0 and 2 g/kg, APS were fed crucian carp for 56 days. The results showed that supplementation with APS significantly upregulated the immune-related indices including the levels of IgM, the activities of LZM, AKP and ACP, and the contents of C3 and C4. At the same time, compared with the CK group, adding APS to the feed significantly upregulated the expression of IL-8, IL-10, IL-1ß, IFN-α, IFN-γ, MyD88, TGF-ß and TNF-α in the spleen, kidney, liver and intestine of crucian carp. In addition, when the crucian carp were injected with SVCV, the survival rates of fish in the APS group and the control group were 48.87% and 13.76%, respectively. These results indicated that dietary APS could improve the resistance of crucian carp against SVCV infection. APS also significantly decreased viral titer and inhibited apoptosis induced by SVCV in EPC cells. These results indicated that APS could stimulate the immune response of crucian carp and improve the abilities of crucian carp and EPC cells to resist SVCV infection.

Stress responses of the intestinal digestion, antioxidant status, microbiota and non-specific immunity in Songpu mirror carp (Cyprinus carpio L.) under starvation.

Songpu mirror carp, Cyprinus carpio L., is a new variety of common carp that has become an economically important freshwater fish in China. However, it remains unknown how its metabolism is regulated under starvation. Here, we investigated how intestinal digestion, antioxidant status, microbiota and immune activities were affected under starvation stress. The feeding regimes were designed as follows: ST0 comprised fish allowed to feed continuously; ST1 comprised fish starved for 1 week; ST2 comprised fish starved for 2 weeks; ST3 comprised fish starved for 3 weeks; ST4 comprised fish starved for 4 weeks. Our results showed a significant decrease in the level of intestinal amylase, lipase, and protease activities in the group ST4 (P < 0.05). Compared with the control group, intestinal antioxidant enzyme activities were significantly increased during short-term starvation. The gene expression levels of interleukin 1ß (IL-1ß), interleukin 8 (IL-8) and tumor necrosis factor-alpha (TNF-α) were elevated in the groups ST3 and ST4. We also detected the reduction in the expression levels of interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß2) compared with those of the group ST0. Notably, the gut microbial composition was dominated by Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. The relative abundance of the dominant microbial phyla changed significantly under starvation stress. Taken together, our results suggest that starvation can induce the change of intestinal digestion, non-specific immunity and microbiota in Songpu mirror carp, and provide new insights into its habitat selection and adaptation to environmental changes.

Effect of dietary Ficus carica polysaccharides on the growth performance, innate immune response and survival of crucian carp against Aeromonas hydrophila infection.

Ficus carica polysaccharides (FCPS), one of the most effective and important compo-nents in Ficus carica L., had been considered to be a beneficial immunostimulant and may be used in immunotherapy for animals and human. However, studies were little about the effect of FCPS used as immunomodulatory and the suitable dosage in fish. The present study investigated the effect of four different dietary levels of FCPS (0.1%, 0.2%, 0.4%, 0.8%) on the growth performance, innate immune responses and survival of crucian carp against Aeromonas hydrophila infection. The results showed that compared with control group, dietary FCPS had positive effects the growth performance (final weight, feed conversion ratio and survival rate) of crucian carp. FCPS induced significant higher (p < 0.05) leukocyte phagocytosis activity, serum bactericidal activity, lysozyme activity, com-plement C3, SOD activity and total protein level in the serum of crucian carp. Moreover, innate immune response of fish in FCPS groups increased first and then decreased with increasing dietary FCPS from 0.1% to 0.8%, and reached up to the peak in 0.4% dietary FCPS groups. Besides, the cumulative mortalities in FCPS groups were remarkably lower than that of control group when challenged with A. hydrophila, the relative percent survivals were 22.67%, 55.56%, 62.22% and 17.78% in 0.1% group, 0.2% group, 0.4% group and 0.8% group, respectively. These results suggested that dietary FCPS could improve the growth performance, innate immune response and disease resistance against A. hydrophila in fish, and the suitable dietary dose of FCPS was 0.4% in crucian carp.

Blood cell characterization and transcriptome analysis reveal distinct immune response and host resistance of different ploidy cyprinid fish following Aeromonas hydrophila infection.

Aeromonas hydrophila can pose a great threat to survival of freshwater fish. In this study, A. hydrophila infection could decrease blood cell numbers, promote blood cell damage as well as alter the levels of alkaline phosphatase (ALP), lysozyme (LZM), aspartate aminotransferase (AST), total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) in immune-related tissues of red crucian carp (RCC, 2 N = 100) and triploid cyprinid fish (3 N fish, 3 N = 150). In addition, the significant alternation of antioxidant status was observed in PBMCs isolated from RCC and 3 N following LPS stimulation. The core differential expression genes (DEGs) involved in apoptosis, immunity, inflammation and cellular signals were co-expressed differentially in RCC and 3 N following A. hydrophila challenge. NOD-like receptor (NLR) signals appeared to play a critical role in A. hydrophila-infected fish. DEGs of NLR signals in RCCah vs RCCctl were enriched in caspase-1-dependent Interleukin-1ß (IL-1ß) secretion, interferon (IFN) signals as well as cytokine activation, while DEGs of NLR signals in 3Nah vs 3Nctl were enriched in caspase-1-dependent IL-1ß secretion and antibacterial autophagy. These results highlighted the differential signal regulation of different ploidy cyprinid fish to cope with bacterial infection.

A novel ferritin L (FerL) in hybrid crucian carp could participate in host defense against Aeromonas hydrophila infection and diminish inflammatory signals.

FerL, a multifunctional iron-storage polypeptide, not only exhibited a regulatory role in iron metabolism, but also participated in the regulation of fish immunity. In this study, ORF sequence of WR-FerL was 522 bp, encoding 173 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-FerL was detected in spleen. A. hydrophila challenge and LPS stimulation could sharply enhance WR-FerL mRNA expression in tissues and fish cells, respectively. Purified WR-FerL fusion peptide exhibited in vitro binding activity to A. hydrophila and endotoxin, limited bacterial dissemination to tissues as well as attenuated A. hydrophila-induced production of pro-inflammatory cytokines. Moreover, WR-FerL overexpression could abrogate NF-κB and TNFα promoter activity in fish cells. These results indicated that WR-FerL could play an important role in host defense against A. hydrophila infection.

Effect of Pandanus tectorius extract as food additive on oxidative stress, immune status, and disease resistance in Cyprinus carpio.

The present study investigated the effect of Pandanus tectorius (PT) extract on Cyprinus carpio growth, antioxidant capacity, immunological and oxidative stress, immune-related gene expression, and resistance against pathogen challenge. Fish (average weight: 12.18 ± 0.32 g) were fed with diets containing various concentration (g kg-1) of PT extract: 0 g (basal diet), 5 g [PT5], 10 g [PT10], 20 g [PT20], and 30 g [PT30] for 8 weeks. Our results revealed an increase (p < 0.05) in the final weight gain only in the PT20 (63.12 ± 1.4 g) group. Activity of liver antioxidant parameters such as catalase (CAT) (27.67 ± 1.12 U mg protein-1), superoxide dismutase (SOD) (13.17 ± 0.428 U mg protein-1), and glutathione peroxidase (GPx) (47.14 ± 1.47 U mg protein-1) was highest in the PT20 group. Among the serum immunological parameters examined, lysozyme (37.45 ± 0.67 U mL-1), alternative complement pathway (133.22 ± 1.92 U mL-1), and phagocytic activities (25.68 ± 0.93%) were high (p < 0.05) in PT20, whereas there was no significant effect on serum immunoglobulin or total protein levels, compared to the control. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and malondialdehyde (MDA) levels were found to be lower (p < 0.05) in the PT20 and PT30 groups than those in the control. However, myeloperoxidase (MPO) levels were significantly higher (p < 0.05) in the PT20 and PT30 groups. Gene expression analysis revealed that mRNA expressions of antioxidant genes (SOD, CAT, nuclear factor erythroid 2-related factor 2) and anti-inflammatory cytokine (IL-10) genes were significantly upregulated in the PT10 - PT30 groups, except for the SOD gene in PT10. Conversely, the signalling molecule NF-κBp65 was downregulated in PT20 and PT30. Expression of Toll-like receptor 22 (TLR22) and hepcidin was significantly upregulated in PT20 and PT30, respectively. Fish in the PT20 group exhibited highest relative post-challenge survival (70.37%) against Aeromonas hydrophila challenge. The results of the present study suggest that dietary supplementation of P. tectorius extract at 20 g kg-1 can significantly improve weight gain, serum antioxidant parameters, strengthen immunity, and increase the disease resistance of C. carpio. Therefore, P. tectorius extract could be exploited for its use as a food additive in aquaculture.

Remarkable positive effects of figwort (Scrophularia striata) on improving growth performance, and immunohematological parameters of fish.

This study was conducted to investigate the effect of figwort on the growth and immunohematological parameters of common carp (14.20 ± 0.53 g). Four experimental diets were developed to feed fish for eight weeks: control, Figw10 (10 g/kg figwort), Figw20 (20 g/kg figwort), and Figw30 (30 g/kg figwort). The results showed that fish fed dietary Figw10 gained more weight (38.25 g) than control (P < 0.05). Regarding immunohematological parameters, fish fed dietary Figw30 had a higher level of white blood cells (31.2 103/mm3), hematocrit (35.82%), blood performance (14.63), total protein (1.96 g/dL), albumin (0.79 g/dL), globulin (1.17 g/dL), lymphocyte (70.53%), monocyte (3.03%), alternative hemolytic complement activity (ACH50) (147.76 u/mL), lysozyme (62.19 u/mL), and bactericidal activities (135.24) than the control group (P < 0.05). After 14 days of the challenge with Aeromonas hydrophila, the Figw30 treatment had the highest survival ratio (61.76%) compared to the control with 26.46%. Further, after the challenge, fish fed dietary Figw30 had a higher value of immunoglobulin M (42.00 µg/mL), antibody titer (19.23), complement component 3 (296.39 µg/mL), and complement component 4 (97.91 µg/mL) when compared with those fed control diet (P < 0.05). In conclusion, the optimum dosage for providing the best immune response was 30 g/kg in diet.

Effect of phosphorus on growth performance, intestinal tight junctions, Nrf2 signaling pathway and immune response of juvenile mirror carp (Cyprinus carpio) fed different α-ketoglutarate levels.

Previous research has shown that dietary α-ketoglutarate (AKG) supplementation can promote growth performance, phosphorus metabolism, and skeletal development of juvenile mirror carp (Cyprinus carpio) fed low phosphorous diets. In the current study, we tested the hypothesis that 1% AKG dietary supplementation reduces the dietary phosphorus requirements of juvenile mirror carp. A total of 12 experimental isoproteic and isolipidic diets containing available phosphorus levels of 0.21%, 0.38%, 0.55%, 0.72%, 0.89%, and 1.07% dry matter with either 0 or 1% AKG supplementation were used in the study. A total of 1080 juvenile fish of similar initial weight (0.90 ± 0.03 g) were selected and randomly assigned to 36 tanks. There were three replicates for each experimental group, with a density of 30 fish per tank. Fish were fed to satiation for 8 weeks. The results indicated that fish fed the diet supplemented with 1% AKG showed a significant increase in final body weight (FBW), weight gain rate (WGR), feed intake (FI) and phosphorus intake (PI) compared to the diet without AKG (P < 0.05). FBW and WGR increased significantly with increasing available phosphorus levels from 0.21% to 0.89% (P < 0.05). The mRNA expression of ZO-1, claudin 11, and occludin was significantly increased by dietary AKG and phosphorus (P < 0.05). The mRNA expression of Nrf2, GPx1a, and CAT in the Nrf2 signaling pathway was significantly increased by dietary AKG and phosphorus (P < 0.05). The expression levels of IL-10 and TGF-ß2 were significantly increased by dietary AKG and phosphorus, but the expression levels of IL-1ß, IL-8, IL-10, TNF-a and NF-κB were significantly decreased with dietary AKG and phosphorus supplementation (P < 0.05). Based on second-order polynomial regression analysis of WGR against dietary phosphorus levels, the optimal dietary phosphorus level was found to be 0.79% of dry feed for juvenile mirror carp fed a diet with 1% AKG supplementation and 0.93% of dry feed without AKG supplementation. This study confirmed that AKG supplementation can reduce the phosphorus requirements of juvenile mirror carp by promoting growth performance, intestinal tight junctions, Nrf2 signaling pathways and immune response.

PRKX down-regulates TAK1/IRF7 signaling in the antiviral innate immunity of black carp Mylopharyngodon piceus.

TGF-ß-activated kinase-1 (TAK1), tightly related to innate immunity, is phosphorylated and activated by X-linked protein kinase (PRKX) in humans and mammals, which belongs to the c-AMP-dependent protein kinase family. However, the relationship between PRKX and TAK1 remains unknown in teleost. It has been reported in vertebrates for the first time that TAK1 of black carp (bcTAK1) interacts with bcIRF7 and is capable to up-regulate bcIRF7-mediated IFN signaling in our previous study. In this study, the role of PRKX homologue of black carp (Mylopharyngodon piceus) (bcPRKX) in bcTAK1/IFN signaling has been explored. Overexpression of bcPRKX suppressed the transcription of interferon promoters but enhanced the transcription of NF-κB promoter. Mylopharyngodon piceus kidney (MPK) cells transfected with shRNA targeting bcPRKX gene presented enhanced antiviral activity against spring viremia of carp virus (SVCV), in which the mRNA levels of the antiviral proteins were increased, including MX1, Viperin and PKR. Overexpressed bcPRKX dampened bcTAK1/bcIRF7/IFN signaling in the luciferase reporter assay and plaque assay. The interaction between bcTAK1 and bcPRKX has been identified by the immunofluorescence (IF) staining and co-immunoprecipitation (co-IP) assay. In addition, we found that bcPRKX can trigger the degradation of bcTAK1. However, the lysosome inhibitor chloroquine, but not the proteasome inhibitor MG-132, prevented the bcTAK1 degradation mediated by bcPRKX. Thus, we conclude that bcPRKX inhibits bcTAK1/bcIRF7/IFN signaling during the innate immune activation by targeting bcTAK1 and triggers lysosome-dependent degradation of bcTAK1.

Two Duplicated Ptpn6 Homeologs Cooperatively and Negatively Regulate RLR-Mediated IFN Response in Hexaploid Gibel Carp.

Src homology region 2 domain-containing phosphatase 1 (SHP1), encoded by the protein tyrosine phosphatase nonreceptor type 6 (ptpn6) gene, belongs to the family of protein tyrosine phosphatases (PTPs) and participates in multiple signaling pathways of immune cells. However, the mechanism of SHP1 in regulating fish immunity is largely unknown. In this study, we first identified two gibel carp (Carassius gibelio) ptpn6 homeologs (Cgptpn6-A and Cgptpn6-B), each of which had three alleles with high identities. Then, relative to Cgptpn6-B, dominant expression in adult tissues and higher upregulated expression of Cgptpn6-A induced by polyinosinic-polycytidylic acid (poly I:C), poly deoxyadenylic-deoxythymidylic (dA:dT) acid and spring viremia of carp virus (SVCV) were uncovered. Finally, we demonstrated that CgSHP1-A (encoded by the Cgptpn6-A gene) and CgSHP1-B (encoded by the Cgptpn6-B gene) act as negative regulators of the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via two mechanisms: the inhibition of CaTBK1-induced phosphorylation of CaMITA shared by CgSHP1-A and CgSHP1-B, and the autophagic degradation of CaMITA exclusively by CgSHP1-A. Meanwhile, the data support that CgSHP1-A and CgSHP1-B have sub-functionalized and that CgSHP1-A overwhelmingly dominates CgSHP1-B in the process of RLR-mediated IFN response. The current study not only sheds light on the regulative mechanism of SHP1 in fish immunity, but also provides a typical case of duplicated gene evolutionary fates.