Bioaccumulation of arsenic in fish (Labeo rohita) in presence of periphyton: ameliorative effect on oxidative stress, physiological condition, immune response and risk assessment.

The present study explores the use of periphyton to ameliorate toxic properties of arsenic (As) to Labeo rohita and also assesses the human food safety aspects. Fish were introduced to arsenite [As(III)] contaminated water (0.3 and 3 mg/L) along with periphyton. Biochemical, physiological and immunological parameters, including gene expression, were assessed after 30 days of exposure. Periphyton incorporation significantly improved (p < 0.05) the adverse effects of As on respiration, NH3 excretion and brain AChE activity by reducing oxidative stress and As bioaccumulation. The presence of periphyton in As(III) exposed fish (3 mg/L) increased the immune response (Immunoglobulin M and Complement C3) in the serum and the regulation of the respective immune genes in the anterior kidney was found to be similar to the control. A speciation study using LC-ICP-MS confirmed the high accumulation of As by periphyton (5.0-31.9 µg/g) as arsenate [As (V)], resulting in a lower amount of As in fish muscle. The calculated human health risk indices, Target Hazard Quotient (THQ) and Target Cancer risk (TCR) indicate that fish grown in periphyton-treated water may lower the human health risks associated with As. The study signifies the importance of periphyton-based aquaculture systems in As contaminated regions for safe fish production with enhanced yield.

Dietary arginine regulates the growth performance, antioxidant capacity, and immune response in Culter alburnus.

Culter alburnus is sensitive to stressors. Arginine is a precursor of nitric oxide, which can effectively relieve the level of oxidative stress and improve the antioxidant and immune capacity of fish. The effect of different arginine levels on topmouth culter (Culter alburnus) fry development performance, liver antioxidant capacity, and immune parameters were investigated in this study. Five diets (1.96%, ARG1, control group; 2.28%, ARG2; 2.52%, ARG3; 2.81%, ARG4; 3.09%, ARG5) were used to feed fry (initial weight 0.31 ± 0.01 g) for 8 weeks. The data showed that the final weight (FW), weight gain rate (WGR), and specific growth rate (SGR) of the ARG3 and ARG4 groups were significantly improved, while the feed conversion ratio (FCR) reduced significantly. Compared with the ARG1 group, all groups remarkably reduced the crude ash content of the whole body. The activity of hepatic superoxide dismutase (SOD) and the content of hepatic glutathione (GSH) were significantly increased in the ARG3 and ARG4 groups, while the malondialdehyde (MDA) content was significantly decreased. Compared with the ARG1 group, arginine levels in ARG2, ARG3, and ARG4 groups up-regulated the expression levels of Nrf2, down-regulated the gene expression level of Keap1 in the liver. And the expression of Nrf2/Keap1 pathway downstream genes Mn-SOD and CAT was up-regulated in ARG2 and ARG3 groups. Furthermore, the expression levels of MyD88 and IL-1ß were down-regulated, and the anti-inflammatory gene TGF-ß expression levels were up-regulated in the ARG2, ARG3, and ARG4 groups. Additionally, compared to the ARG1 group, there was a significant increase in the relative expression levels of the C3 and C4 genes in the ARG4 group. In conclusion, 2.28-2.81% dietary arginine levels improved the growth performance, promoted antioxidant capacity, and enhance immune response. The optimal level of arginine was determined by the quadratic regression analysis of SGR and FCR to be 2.55% of diet (5.43% of dietary protein) and 2.53% of diet (5.38% of dietary protein), accordingly.

Dietary papaya peel extract ameliorates the crowding stress, enhances growth and immunity in Labeo rohita fingerlings.

This study was designed to determine the effects of papaya peel extract (PPE) supplementation on the growth and immunophysiological responses of rohu fingerlings at different stocking densities. In this study, three isonitrogenous (307.2-309.8 g kg-1 protein) and isocaloric diets (16.10-16.16 MJ digestible energy kg-1) were prepared using three different inclusion levels (0, 5, and 10 g kg-1) of PPE. Four hundred and five rohu fingerlings (mean weight: 4.24 g ± 0.12) were randomly distributed into nine treatment groups in triplicates viz. low (10nos 75 L-1 or ≈ 0.565 kg/m3), medium (15nos 75 L-1 or ≈ 0.848 kg/m3), and high (20nos 75 L-1 or ≈ 1.13 kg/m3) following a completely randomized design. The study found that increasing stocking density negatively affected fish growth indices, such as weight gain percentage (WG%), feed efficiency ratio (FER), specific growth rate (SGR) and survival. In contrast, dietary PPE supplementation improved growth indices and survival (p < 0.05). We also observed that aminotransferase, lactate (LDH), and malate dehydrogenase (MDH) activity increased with stocking density, whereas 5 and 10 g kg-1 PPE supplementation reduced LDH and MDH activity (p < 0.05). PPE supplementation positively affected serum indices, decreased glucose levels, and increased respiratory burst activity (p < 0.05). Interferon-gamma (IFN-γ) expression was highest in the low- and medium-stocking density groups fed with 5 g kg-1 PPE, which also increased total immunoglobulin and myeloperoxidase activity while decreasing malondialdehyde concentration (p < 0.05). The results revealed that 5 g kg-1 dietary PPE supplementation could be used as a growth promoter and immunostimulant to improve immuno-physiological responses at low and medium stocking densities.

Impact of dietary leaf extracts of Black pepper Piper nigrum L on the growth, hematological and immunological parameters of Labeo rohita (Hamilton, 1822) cultured in glass aquaria / Impacto de extratos de folhas da dieta de pimenta-do-reino Piper nigrum L sobre o crescimento, parâmetros hematológicos e imunológicos de Labeo rohita (Hamilton, 1822) cultivado em aquários de vidro

A study was conducted to evaluate the effect of Piper nigrum (black pepper) leaf extract on on the growth performance, proximate composition, hematological parameters, and immune response of Labeo rohita fingerlings with an average weight of 22.14 ± 0.98g. After acclimation for two weeks, fish (n=25) were randomly selected and placed in four glass aquaria (T0, T1, T2 and T3) at constant water temperature (30.0 ± 1.0 °C), pH (7.50 ± 0.5) and total hardness (200 ± 2.0 mgL,-¹) for a period of 12 weeks, with three replicates each. Fish were fed with P. nigrum leaf extract supplemented feed @ 0.0%, 1.0%, 2.0% and 3.0% in T0, T1, T2 and T3, respectively. At the end of experiment, five fish were randomly selected from each aquaria for proximate composition, gut and skin microbial load, hematological parameters. Total proteins, albumins, and globulins were also recorded to evaluate immunological memory. The result revealed that fish in T2 showed better growth performance with an average weight gain of 56.11 ± 0.51 g. Thus, it had been concluded that Piper nigrum, a medicinal plant, can also be used to enhance the growth performance and immune response of Labeo rohita as attractive alternatives against antibiotics and vaccines and has shown no negative side effects on fish health as well as on its environment.

Um estudo foi conduzido para avaliar o efeito do extrato da folha de Piper nigrum (pimenta-do-reino) sobre o desempenho de crescimento, composição centesimal, parâmetros hematológicos e resposta imune de alevinos de Labeo rohita com peso médio de 22,14 ± 0,98g. Após aclimatação por duas semanas, os peixes (n = 25) foram selecionados aleatoriamente e colocados em quatro aquários de vidro (T0, T1, T2 e T3) em temperatura constante da água (30,0 ± 1,0 °C), pH (7,50 ± 0,5) e dureza total (200 ± 2,0 mgL-¹) por um período de 12 semanas, com três repetições cada. Os peixes foram alimentados com ração suplementada com extrato de folha de P. nigrum @ 0,0%, 1,0%, 2,0% e 3,0% em T0, T1, T2 e T3, respectivamente. Ao final do experimento, cinco peixes foram selecionados aleatoriamente de cada aquário para composição centesimal, carga microbiana intestinal e cutânea e parâmetros hematológicos. Proteínas totais, albuminas e globulinas também foram registradas para avaliar a memória imunológica. O resultado revelou que os peixes em T2 apresentaram melhor desempenho de crescimento com ganho de peso médio de 56,11 ± 0,51 g. Assim, concluiu-se que Piper nigrum, uma planta medicinal, também pode ser usado para melhorar o desempenho de crescimento e resposta imunológica de Labeo rohita como alternativas atraentes contra antibióticos e vacinas e não mostrou efeitos colaterais negativos na saúde dos peixes, bem como sobre seu ambiente.

Changes in immune genes expression, immune response, digestive enzymes -antioxidant status, and growth of catla (Catla catla) fed with Astragalus polysaccharides against edwardsiellosis disease.

The effect of four level of Astragalus polysaccharides (APs) supplementation diets, (CD: control diet and three experiment diet (E), EA: 100 mg kg-1 APs; EB: 200 mg kg-1 APs; EC: 300 mg kg-1 APs) on growth, changes in haemato-biochemical parameters and metabolic-digestive enzymes, enhancement of antioxidant activity, innate-adaptive immune response, and cytokine gene expression were studied in catla (Catla catla) against Edwardsiella tarda. The healthy and challenged groups fed the CD displayed no mortality, while fish fed EA or EC revealed 10% mortality, but the mortality was only 5% in diet EB. Fish fed diet EB and EC revealed significantly better growth rates and high RBC count during the experimental period. Albumin and globulin levels were significant improved when fish were fed the diet EB and EC from weeks 6-8. The superoxide dismutase (SOD) was significant ameliorated by EB feeding from weeks 4-8. In contrast, serum myeloperoxidase (MPO), catalase (CAT), malondialdehyde (MDA)/lipid peroxidation (LPO), glutathione peroxidase (GPx), respiratory burst activity (RBA), bactericidal action (BCA), serum lysozyme activity (SLA), nitric oxide synthase (NOS), head kidney leukocytes response proliferation (HKLP), hemolytic action (HLA), hydrogen peroxides (H2O2), and immunoglobulin (Ig) were significantly improved from week 6-8. Groups fed the APs enriched diets had significant ameliorated interleukin (IL)-1ß and interferon (IFN)-γ mRNA expression after 6 and 8 weeks of feeding. However, IL-10 and major histocompatibility complex (MHC)-1 mRNA expressions were significant enhanced in catla fed all APs diets on week 8. APs enriched diets revealed significant improved tumor necrosis factor (TNF)-α and TNF receptor-associated factor-6 (TRAF6) mRNA expression on week 4, but toll-like receptor-2 (TLR2) and TLR4 mRNA expression were significant enhanced by diet EB and EC after weeks 6 and 8. Similarly, the lysozyme (Lyz)-C and Lyz-G mRNA levels in the head kidney (HK) increased by APs feeding on weeks 6 and 8, whereas the EB diet, the expression of nucleotide binding oligomerization domain-1 (NOD1) was significantly improved on weeks 6 and 8, but NOD2 mRNA expression was only significant enhanced after 8 weeks of diet EB. By feeding healthy catla and E. tarda challenged fish fed diet EB, resulted in significantly increased growth, haemato-biochemical indices, metabolic-digestive enzymes, antioxidant activities, innate-adaptive immune responses, and cytokine gene expression mainly between 6 and 8 weeks.

Impact of Hygrophila auriculata supplementary diets on the growth, survival, biochemical and haematological parameters in fingerlings of freshwater fish Cirrhinus mrigala (Hamilton, 1822).

The present study investigates the effects of Supplementary diet Hygrophila auriculata on the growth, survival, biochemical and haematological parameters of Cirrhinus mrigala. The seaweed was administered to the fish possessing an initial average weight of 14.063 ± 1.828 g. Fish were fed with supplementary diet H. auriculata exhibited significant difference (P < 0.05) in the growth performance, haematological indices such as RBC count, haematocrit volume, haemoglobin, WBC, MCV, MCH and MCHC concentration in contrast to the control after a period of 8 weeks. Also, there were significant differences in biochemical parameters (P < 0.05), between the fish supplemented with dietary H. auriculata extract and the control group. These findings suggest that the administration of H. auriculata extract has a positive effect on the immunological indices and the immune system activity in Mrigal fish.

Transgenerational effects of ß-glucan on thermal tolerance, growth performance, and immune gene expression of endangered cyprinid Tor putitora progeny.

Nutritional programming signifies a process in which broodstock feeding approaches have long-term effects on the subsequent progeny. The present study aimed to elucidate whether supplementing golden mahseer, Tor putitora broodstock diets with ß-glucan affects progeny growth performance, survival, thermal tolerance, and non-specific immunity. Initially, the growth performance of progeny produced from brooders fed with different levels of ß-glucan was non-significant. However, on the 15th and 35th DPH, the maximum weight was observed in fry obtained from the brooders fed with 0.5% followed by 1.0% ß-glucan. Furthermore, on 50th DPH, significantly higher weight was registered in the fry from the 0.5% ß-glucan fed group while 1.0% ß-glucan group had no transgenerational effect on growth. The condition factor of fry obtained from golden mahseer brooders fed with a 0.5% ß-glucan diet was greater than the control and 1.0% ß-glucan fed group. On the other hand, we did not find any significant transgenerational influence of ß-glucan on the survival of the progeny. The thermal tolerance of fry produced from brooders fed with ß-glucan was significantly modulated at both end-points (CTmax and CTmin). Expression of interleukin-1ß was significantly up-regulated in fry obtained from ß-glucan fed brooders. In contrast, the expression level of tumor necrosis factor-α was significantly higher only in fry produced from 1.0% ß-glucan fed brooders. The expression of immunoglobulin light chain and serum amyloid A gene was significantly higher in fry produced from 0.5% ß-glucan fed brooders. Overall results suggest that the dietary provisioning of ß-glucan in golden mahseer brooders can be a strategy to produce healthy and robust fry in captivity for stock enhancement and conservation programs.

Role of Zingiber officinale and autochthonous probiotic Bacillus coagulans in feeds of Catla catla (Hamilton, 1822) for growth promotion, immunostimulation, histoprotection, and control of DNA damage.

Experiment was conducted to assess the impact of ginger (Zingiber officinale) as a dietary supplement with probiotic bacterium Bacillus coagulans on growth performance, hematological parameters, and non-specific immune parameters in Catla catla. An attempt was also made to investigate their effects on histology and extent of DNA damage against pathogenic bacterium Aeromonas hydrophila in C. catla. Ten dietary treatments were designed in triplicate in which five groups of fingerlings fed on diets containing different incorporation level of dried ginger at 0 g kg-1 (C1; basal diet 1 with duckweed as major protein source), 1 g kg-1 (G1), 5 g kg-1 (G2), 10 g kg-1 (G3), and 15 g kg-1 (G4) and other five groups of fingerlings fed on diets (G5-G9) containing different incorporation level of dried ginger at 0, 1, 5, 10, and 15 g kg-1 along with probiotic bacterium B. coagulans @ 3000 CFU ml-1 for 90 days. Results clearly revealed significantly (P < 0.05) high values of weight gain, growth % gain in body weight, specific growth rate (SGR), RBC and WBC count, phagocytic activity, respiratory burst activity, serum protein, and serum bactericidal activity in group of fingerlings fed on ginger supplemented diets which further improved with the inclusion of probiotic with best results in treatment G8. Histopathological study and COMET assay reflected that Z. officinale and probiotics protect extent of DNA damage and the digestive organs from the detrimental effects of pathogenic bacteria affirming their positive role and harmonious effects of probiotic bacterium with Z. officinale-supplemented diets.

Efficacy of ulvan on immune response and immuno-antioxidant gene modulation in Labeo rohita against columnaris disease.

This study reports the effect of ulvan enriched diet on the influence of growth, changes in hemato-biochemical indices, improvement of antioxidant system, enhancement of innate-adaptive immunity and modification of immuno-antioxidant genes expression in Labeo rohita against Flavobacterium columnaris. The weight gain (WG) was significantly high (P > 0.05) in unchallenged normal and challenged fish fed with diets enriched with 25 and 50 mg kg-1 ulvan; the FCR was better (P > 0.05) when fed with 50 mg kg-1 enriched diet. In normal fish fed with or without ulvan supplementation was noted 100% survival rate (SR). In both groups, the red blood cell (RBC) and while blood cell (WBC) counts increased significantly (P > 0.05) when fed with 50 mg kg-1 ulvan diet whereas the hemoglobin (Hb) level increased significantly on being fed with 25 and 50 mg kg-1 ulvan diets. The SOD activity was enhanced significantly in both groups fed with any dose of ulvan diets whereas the MDA and GPx activity increased only with 25 and 50 mg kg-1 ulvan diets. The phagocytic (PC) activity significantly increased with any enriched diet and control diet groups while the respiratory burst (RB) activity increased only with 50 mg kg-1 ulvan diet. The alternate complement pathway (ACP), activity of lysozyme (Lyz), and immunoglobuline M (IgM) were better in both groups fed with 50 mg kg-1 ulvan diet. The SOD and GPx antioxidant gene expression were significantly high in both groups fed with any ulvan diet while the Nrf2 gene expression was high with 50 mg kg-1 ulvan diet. The IL-1ß, TNFα, hepcidin, Lyz, and IgM cytokines or proteins mRNA expression were significant in both groups fed with all ulvan supplement diet whereas the ß-2M expression was significant only with 50 mg kg-1 ulvan diet. The present research indicates that both L. rohita groups fed with 50 mg kg-1 ulvan diet significantly improved growth, antioxidant system, immune defense system, and immuno-antioxidant related gene expression against F. columnaris.

Molecular cloning and expression mechanism of Mnp65 in Megalobrama amblycephala response to Aeromonas hydrophilia challenge.

p65 is one of the important subunits of the inflammation-related transcription factor NF-κB. In the present study, we cloned and identified the p65 from Megalobrama amblycephala (Mnp65) by homologous cloning and RACE technique. The full-length Mnp65 cDNA consisted of 2331 bp, and included one open reading frame encoding a 604-amino acid putative protein. The protein sequence included a DNA binding motif, a well conserved N-terminal Rel-homology domain (RHD), and a C-terminal IG-like plexins transcription (IPT). Mnp65 was closely related with the other p65 proteins of Cypriniformes and clearly distinct from that of Perciformes and Salmoniformes in terms of sequence homology. Mnp65 homodimer may interact with IκBα in the IPT domain based on the predicted 3D structure of IκBα/Mnp65 complex. Mnp65 was ubiquitously expressed in M. amblycephala tissues, and the highest levels were detected in muscle and liver. Intragastric infection with Aeromonas hydrophila caused respiratory burst and cytokine storm from 8 h to 48 h, showing significantly higher level of respiratory burst activities and significantly high cytokines levels, such as TNF-α, IL-1ß, IL-6, IL-8 etc., compared to 0 h. In addition, the bacterial challenge downregulated the IkBα, and upregulated Mnp65 and TNF-α in the liver. IkBα-Mnp65 was regulated by the negative feedback of cytokine storm, to increase IkBα and decrease Mnp65. Then cytokine storm was relieved at 96 h. Finally, severe intestinal inflammation was observed from 24 h to 48 h after infection, characterized by extensive villous necrosis, epithelial hyperplasia and lymphocyte infiltration, all of which were relieved at 96 h. Taken together, Mnp65 plays a crucial role in the physiological response of teleost fish to bacterial infection.

Mannose Receptor Mediates the Activation of Chitooligosaccharides on Blunt Snout Bream (Megalobrama amblycephala) Macrophages.

Chitooligosaccharide (COS) is an important immune enhancer and has been proven to have a variety of biological activities. Our previous research has established an M1 polarization mode by COS in blunt snout bream (Megalobrama amblycephala) macrophages, but the mechanism of COS activation of blunt snout bream macrophages remains unclear. In this study, we further explored the internalization mechanism and signal transduction pathway of chitooligosaccharide hexamer (COS6) in blunt snout bream macrophages. The results showed that mannose receptor C-type lectin-like domain 4-8 of M. amblycephala (MaMR CTLD4-8) could recognize and bind to COS6 and mediate COS6 into macrophages by both clathrin-dependent and caveolin-dependent pathways. In the inflammatory response of macrophages activated by COS6, the gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and nitric oxide synthase 2 (NOS2) was significantly inhibited after MaMR CTLD4-8-specific antibody blockade. However, even if it was blocked, the expression of these inflammation-related genes was still relatively upregulated, which suggested that there are other receptors involved in immune regulation. Further studies indicated that MaMR CTLD4-8 and Toll-like receptor 4 (TLR4) cooperated to regulate the pro-inflammatory response of macrophages caused by COS6. Taken together, these results revealed that mannose receptor (MR) CTLD4-8 is indispensable in the process of recognition, binding, internalization, and immunoregulation of COS in macrophages of blunt snout bream.

Novel insights into the immune regulatory effects of Megalobrama amblycephala intelectin on the phagocytosis and killing activity of macrophages.

Previous studies have found that the expression level of Megalobrama amblycephala intelectin (MaINTL) increased significantly post Aeromonas hydrophila infection, and recombinant MaINTL (rMaINTL) protein could activate macrophages and enhance the phagocytosis and killing activity of macrophages. In order to reveal the immune regulatory mechanisms of MaINTL, primary M. amblycephala macrophages were treated with endotoxin-removed rMaINTL and GST-tag proteins, then total RNA were extracted and used for comparative Digital Gene Expression Profiling (DGE). 1247 differentially expressed genes were identified by comparing rMaINTL and GST-tag treated macrophage groups, including 482 up-regulated unigenes and 765 down-regulated unigenes. In addition, eleven randomly selected differentially expressed genes were verified by qRT-PCR, and most of them shared the similar expression patterns as that of DGE results. GO enrichment revealed that the differentially expressed genes were mainly concentrated in the membrane part and cytoskeleton of cellular component, the binding and signal transducer activity of molecular function, the cellular process, regulation of biological process, signaling and localization of biological process, most of which might related with the phagocytosis and killing activity of macrophages. KEGG analysis revealed the activation and involvement of differentially expressed genes in immune related pathways, such as Tumor necrosis factor (TNF) signaling pathway, Interleukin 17 (IL-17) signaling pathway, Toll-like receptor signaling pathway, and NOD like receptor signaling pathway, etc. In these pathways, TNF-ɑ, Activator protein-1 (AP-1), Myeloid differentiation primary response protein MyD88 (MyD88), NF-kappa-B inhibitor alpha (ikBɑ) and other key signaling factors were significantly up-regulated. These results will be helpful to clarify the immune regulatory mechanisms of fish intelectin on macrophages, thus providing a theoretical basis for the prevention and control of fish bacterial diseases.

Developmental thyroid disruption causes long-term impacts on immune cell function and transcriptional responses to pathogen in a small fish model.

Current evidence suggests thyroid hormones (THs) impact development of the immune system, but few studies have explored the connection between the thyroid and immune systems, especially in fish. This is important as some environmental contaminants disrupt TH homeostasis and may thus have negative impacts on the immune system. To determine the long-term consequences of early life stage (ELS) hypothyroidism on immune function, fathead minnows were exposed to the model thyroid hormone suppressant propylthiouracil (PTU) from < 1 to 30 days post hatch. Fish were transferred to clean water and raised to adulthood (5-7 months post hatch) at which time, several aspects of immune function were evaluated. Ex vivo assessment of immune cell function revealed significant decreases (1.2-fold) in the phagocytic cell activity of PTU-treated fish relative to the controls. Fish were also injected with Yersinia ruckeri to evaluate their in vivo immune responses across a suite of endpoints (i.e., transcriptomic analysis, leukocyte counts, spleen index, hematocrit, bacterial load and pathogen resistance). The transcriptomic response to infection was significantly different between control and PTU-treated fish, though no differences in bacterial load or pathogen resistance were noted. Overall, these results suggest that early life stage TH suppression causes long-term impacts on immune function at the molecular and cellular levels suggesting a key role for TH signaling in normal immune system development. This study lays the foundation for further exploration into thyroid-immune crosstalk in fish. This is noteworthy as disruption of the thyroid system during development, which can occur in response to chemicals present in the environment, may have lasting effects on immune function in adulthood.

Establishment of a rare minnow (Gobiocypris rarus) model for evaluation of experimental vaccines against a disease induced by grass carp reovirus genotype II.

Vaccination is the most effective way to control the grass carp haemorrhagic disease (GCHD) with the primary pathogen grass carp reovirus genotype II (GCRV-II). However, due to the large difference in breeding conditions and unclear genetic background of grass carp, the results of the experiment were not reliable, which further hinders the effective prevention and control of GCHD. The rare minnow (Gobiocypris rarus) is highly sensitive to GCRV. Its small size, easy feeding, transparent egg membrane, and annual spawning are in line with the necessary conditions for an experimental aquatic animals culture object. In this study, immunogenicity and protective effects of attenuated and inactivated viruses for grass carp and rare minnow were evaluated in parallel. The expression of immune-related genes increased statistically significant after immunization. With the rise of specific serum antibody titers, the results of rare minnow and grass carp were consistent. In addition, there was no significant residue of adjuvant observed in both fish species injected with an adjuvanted and inactivated virus. Challenge of immunized grass carp and rare minnow with the isolate HuNan1307 resulted in protection rates of 95.8% and 92.6% for attenuated virus, 81.4% and 77.7% for inactivated virus, respectively, as well as the viral load changed consistently. The results indicated that rare minnow can be used as a model for evaluation of experimental vaccines against GCHD.

Molecular characterization of non-specific immune genes of endangered golden mahseer (Tor putitora) and their expression during embryonic and larval development.

The present study was undertaken to characterize and analyze the expression of non-specific immune genes to get an insight into the early immune status of endangered golden mahseer. In this study, the full-length mRNA sequence of IFNγ, TNFα, C3, and IL10 was 927, 1409, 5125 and 1177 bp with an ORF of 558, 765, 4938, and 540 bp, encoding a putative protein of 185, 254, 1645, and 179 amino acid residues, respectively. The deduced amino acid sequences of these genes shared highly conserved structures with those from other cyprinids. Ontogenic real-time qPCR results indicated that expression of IFNγ and TNFα was lower until the morula stage and increased from blastula stage and found maximum at the organogenesis stage. Expression of the C3 gene was lower until the gastrula stage, followed by a linear increase from organogenesis to the pre-metamorphosis stage. The expression of IL10 was significantly lower during early developmental stages (till gastrula stage) and reached maximum at organogenesis. The level of IL1ß was found maximum in unfertilized eggs and remained elevated till the morula stage. TLR4 expression remained lower during the initial developmental stages and reached the maximum at the organogenesis stage. The expression level of defensin1 was substantially low until the organogenesis stage. In comparison, hepcidin1 was found considerably high until the blastula stage and remained significantly lower during later stages of development. Overall, the data generated improves knowledge on the immune status of endangered golden mahseer during embryonic and larval development, which may help develop effective immunomodulatory interventions during nursery rearing of golden mahseer to produce fry with better fitness.

Non-Specific Immunity Associated Gut Microbiome in Aristichthys nobilis under Different Rearing Strategies.

To understand the intestinal microbial diversity and community structure of bighead carp (Aristichthys nobilis) under different feeding strategies, 39 fish from three groups (A: 9 fish, natural live food only; B: 15 fish, natural live food + fish formulated feeds; C: 15 fish, natural live food + fish formulated feed + lactic acid bacteria) were obtained for the high throughput 16S rRNA gene sequencing. We first examined five non-specific immunity indications of the carp-lysozyme (LZM), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD). Interestingly, the composition of gut microbiota and related non-specific immune indices were affected by the feeding treatment of the bighead carp. Notably, all enzyme activity indexes were significantly different (p < 0.01) in the spleen and three enzyme activity indexes (LZM, GSH-PX, and SOD) had significant differences in the hepatopancreas (p < 0.001) of the carp from the three groups. The 16S rRNA gene sequencing showed higher diversity in groups B and C. Compared to group A, the relative abundance of Actinobacteria increased significantly and the relative abundance of Proteobacteria and Firmicutes decreased significantly in groups B and C at the phylum level. Functional analysis revealed the association between non-specific immune indicators and import genera in the hepatopancreas and spleen of bighead carp. This study provides new insights into the gut microbiomes and non-specific immune of bighead carp.

Viperin_sv1 promotes RIG-I expression and suppresses SVCV replication through its radical SAM domain.

SVCV infection is known to activate the host's innate immune responses, including the production of interferon (IFN) and interferon-stimulated genes (ISGs). Viperin_sv1 is a novel splice variant of viperin, which is induced during SVCV infection and proves to positively regulate the IFN activation and production. However, the underlying mechanism remains unsolved. In this study, the P protein of SVCV was identified to be the key to induce the mRNA modification and production of viperin_sv1 during the virus infection. Besides, Viperin_sv1 was able to trigger the RLR signaling cascades to activate type-1 interferon response. Additional analysis revealed that viperin_sv1 promoted the stability and function of RIG-I, which result in the production of IFN and ISGs. Moreover, the central SAM domain of viperin_sv1 was demonstrated to be essential for regulating RIG-I protein expression and inducing IFN production. Furthermore, this study also showed that SVCV replication could be inhibited by the viperin_sv1 SAM domain. In conclusion, our study demonstrates that viperin_sv1 reduces the replication of SVCV by promoting the RIG-I protein expression. Our findings identified the antiviral function played by the SAM domain of viperin_sv1 and suggested an antiviral mechanism that is conserved among different species.

RIPK3 collaborates with RIPK1 to inhibit MAVS-mediated signaling during black carp antiviral innate immunity.

Both the activation and attenuation of MAVS/IFN signaling are critical for host defensing against viral infection and thus lead to an elaborate regulation of MAVS-mediated signaling. However, the regulatory mechanisms concerning MAVS/IFN signaling in teleost fish are not well understood. RIPK3 has been identified as a key regulator of necroptosis, apoptosis, and inflammatory signaling in human and mammals. Here we report the identification of the RIPK3 homologue from black carp Mylopharyngodon piceus (bcRIPK3) and describe its role in regulating MAVS/IFN signaling. qPCR results demonstrated that bcRIPK3 was transcriptionally activated in response to poly (I:C) or LPS stimulation. Immunoblot assay and immunofluorescent staining assay showed that bcRIPK3 was a cytosolic protein with molecular weights of 47 kDa. Like its mammalian counterparts, bcRIPK3 exhibited a conserved function in inducing cell death. The reporter assay and plaque assay showed that overexpression of bcRIPK3 restricted bcMAVS-activated transcription of the interferon promoters of black carp and zebrafish, and suppressed bcMAVS-mediated antiviral activity. Notably, EPC cells co-expressing bcRIPK3, bcRIPK1 and bcMAVS presented much attenuated antiviral activity than the cells co-expressing bcRIPK3 and bcMAVS; and the subsequent co-IP assay identified the interaction between bcRIPK3 and bcRIPK1. Our findings collectively elucidate for the first time in teleost that black carp RIPK3 interacts with RIPK1 to inhibit MAVS-mediated antiviral signaling.

A C-type lectin with a single CRD from Onychostoma macrolepis mediates immune recognition against bacterial challenge.

C-type lectins (CTL) are a large group of pattern-recognition proteins and to play important roles in glycoprotein metabolism, multicellular integration, and immunity. Based on their overall domain structure, they can be classified as different groups that possess different physiological functions. A typical C-type lectin (named as OmLec1) was identified from the fish, Onychostoma macrolepis, an important cultured fish in China. Open reading frame of OmLec1 contains a 570 bp, encoding a protein of 189 amino acids that includes a signal peptide and a single carbohydrate-recognition domain. The phylogenetic analysis showed that OmLec1 could be grouped with C-type lectin from other fish. OmLec1 was expressed in all the tissues in our study, and the expression level was highest in liver. And its relative expression levels were significantly upregulated following infection with Aeromonas hydrophila. The recombinant OmLec1 protein (rOmLec1) could agglutinate some Gram-negative bacteria and Gram-positive bacteria in vitro in the presence of Ca2+, showing a typical Ca2+-dependent carbohydrate-binding protein. Furthermore, rOmLec1 purified from E. coli BL21 (DE3), strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-dependent manner. These results indicate that OmLec1 plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis.

Epidermal Club Cells in Fishes: A Case for Ecoimmunological Analysis.

Epidermal club cells (ECCs), along with mucus cells, are present in the skin of many fishes, particularly in the well-studied Ostariophysan family Cyprinidae. Most ECC-associated literature has focused on the potential role of ECCs as a component of chemical alarm cues released passively when a predator damages the skin of its prey, alerting nearby prey to the presence of an active predator. Because this warning system is maintained by receiver-side selection (senders are eaten), there is want of a mechanism to confer fitness benefits to the individual that invests in ECCs to explain their evolutionary origin and maintenance in this speciose group of fishes. In an attempt to understand the fitness benefits that accrue from investment in ECCs, we reviewed the phylogenetic distribution of ECCs and their histochemical properties. ECCs are found in various forms in all teleost superorders and in the chondrostei inferring either early or multiple independent origins over evolutionary time. We noted that ECCs respond to several environmental stressors/immunomodulators including parasites and pathogens, are suppressed by immunomodulators such as testosterone and cortisol, and their density covaries with food ration, demonstrating a dynamic metabolic cost to maintaining these cells. ECC density varies widely among and within fish populations, suggesting that ECCs may be a convenient tool with which to assay ecoimmunological tradeoffs between immune stress and foraging activity, reproductive state, and predator-prey interactions. Here, we review the case for ECC immune function, immune functions in fishes generally, and encourage future work describing the precise role of ECCs in the immune system and life history evolution in fishes.