Development and evaluation of immunological effects of a DNA vaccine encoding phosphoketolase family protein against Nocardia seriolae in hybrid snakehead.

Fish nocardiosis is a chronic disease mainly caused by Nocardia seriolae, which occurs in a variety of economically cultured freshwater and marine fish. Studies have shown that DNA vaccine is an effective treatment to protect fish from bacterial infection. In our previous experiment, an in vivo-induced gene of N. seriolae, encoding phosphoketolase (PK) family protein, was identified by in vivo-induced antigen technology. In the present study, the antigenic gene encoding PK family protein was analyzed by bioinformatics and further inserted into the eukaryotic expression vector pcDNA3.1-myc-his-A for DNA vaccine development. The immunological effects of pcDNA-PK DNA vaccine were assessed in hybrid snakehead (Channa maculata ♀ × Channa argus ♂), showing induction in several serum enzyme activity parameters (including LZM, SOD, ACP and AKP), increasing in specific-antibody IgM levels, as well as up-regulation in six immune-related genes (CD4, CD8α, TNFα, IL-1ß, MHCIα and MHCIIα). Moreover, an immune-protection with a relative survival rate was provided at 53.82 % following artificial challenge with N. seriolae in vaccinated fish in comparison to the control group. In summary, these results indicate that pcDNA-PK DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, which may be applied in aquaculture to control fish nocardiosis.

Dietary kelp meal improves serum antioxidants, intestinal immunity, and lipid metabolism in hybrid snakehead (Channa maculata ♀ × Channa argus ♂).

BACKGROUND: Dietary kelp possesses a variety of useful biological qualities but does not have a toxic effect on the host. In this study, we examine how kelp dietary supplementation enhances the serum biochemistry, intestinal immunity, and metabolism of hybrid snakehead. A total of 810 juvenile hybrid snakeheads (Channa maculata ♀ × Channa argus ♂), with an initial average weight of 11.4 ± 0.15 g, were allocated randomly to three treatment groups (three replicates per group). The fish were fed for 60 days with isonitrogenous and isolipidic diets. The groups were the control group (C) (20% high-gluten flour), the medium replacement group (MR) (10% high-gluten flour and 10% kelp meal), and the full replacement group (FR) (0% high-gluten flour and 15% kelp meal). RESULTS: The results showed that dietary kelp increased the activity of serum antioxidant enzymes significantly and decreased the content of serum malondialdehyde (MDA) in hybrid snakeheads, with significant changes in the FR group (P < 0.05). The intestinal morphology results showed that dietary kelp helped to increase the specific surface area of intestinal villi, which was beneficial for intestinal digestion and absorption. According to transcriptome and quantitative real-time polymerase chain reaction (qRT-PCR) analysis, dietary kelp can improve the expression of intestinal immunity and metabolism-related pathways. Among them, immune-related genes MHC1 and HSPA1 were significantly up-regulated, and IGH, MHC2, and IL-8 were significantly down-regulated (P < 0.05). Lipid metabolism-related genes DGAT2, FABP2, RXRα, and PLPP1 were all significantly up-regulated (P < 0.05). CONCLUSION: Dietary kelp can effectively improve the antioxidant function of hybrid snakeheads, improve intestinal morphology, reduce intestinal inflammation, and promote intestinal lipid synthesis and transportation, thereby improving intestinal immunity and metabolic functions. © 2024 Society of Chemical Industry.

Establishment and characterization of a kidney cell line from hybrid snakehead (male Channa argus × female Channa maculata) and its susceptibility to hybrid snakehead rhabdovirus (HSHRV).

Hybrid snakehead (male Channa argus × female Channa maculata) is an emerging fish breed with increasing production levels. However, infection with hybrid snakehead rhabdovirus (HSHRV) critically affects hybrid snakehead farming. In this study, a fish cell line called CAMK, derived from the kidneys of hybrid snakehead, was established and characterized. CAMK cells exhibited the maximum growth rate at 28 °C in Leibovitz's-15 medium supplemented with 10% fetal bovine serum(FBS). Karyotyping revealed diploid chromosomes in 54% of the cells at the 50th passage (2n = 66), and 16S rRNA sequencing validated that CAMK cells originated fromhybrid snakehead, and the detection of kidney-specific antibodies suggested that it originated from kidney. .The culture was free from mycoplasma contamination, and the green fluorescent protein gene was effectively transfected into CAMK cells, indicating their potential use for in vitro gene expression investigations. Furthermore, qRT-PCR and immunofluorescence analysis revealed that HSHRV could replicate in CAMK cells, indicating that the cells were susceptible to the virus. Transmission electron microscopy revealed that the viral particles had bullet-like morphology. The replication efficiency of HSHRV was 107.33 TCID50/mL. Altogether, we successfully established and characterized a kidney cell line susceptible to the virus. These findings provide a valuable reference for further genetic and virological studies.

Identifying the in vivo-induced antigenic genes is a strategy to develop DNA vaccine against Nocardia seriolae in hybrid snakehead (Channa maculata ♀ × Channa argus ♂).

Nocardia seriolae has been identified as the causative agent of fish nocardiosis, resulting in serious economic losses in aquaculture. With an aim to screen potential candidates for vaccine development against N. seriolae, the in vivo-induced genes of N. seriolae in hybrid snakehead (Channa maculate ♀ × Channa argus ♂) model were profiled via in vivo-induced antigen technology (IVIAT) in the present study, and 6 in vivo-induced genes were identified as follows: IS701 family transposase (is701), membrane protein insertase YidC (yidC), ergothioneine biosynthesis glutamate-cysteine ligase (egtA), molybdopterin respectively-dependent oxidoreductase (mol), phosphoketolase family protein (Ppl), hypothetical protein 6747 (hp6747). Additionally, the yidC was inserted into eukaryotic expression vector pcDNA3.1-myc-his-A to construct a DNA vaccine named as pcDNA-YidC to evaluate immunoprotection in hybrid snakehead after artificial challenge with N. serioale. Results showed that the transcription of yidC was detected in spleen, trunk kidney, muscle and liver in vaccinated fish, suggesting that this antigenic gene can be recombinantly expressed in fish. Meanwhile, indexes of humoral immunity were evaluated in the vaccinated fish through assessing specific-antibody IgM and serum enzyme activities, including lysozyme (LZM), superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Quantitative real-time PCR analysis indicated that pcDNA-YidC DNA vaccine could notably enhance the expression of immune-related genes (CD4、CD8α、MHCIIα、TNFα、IL-1ß and MHCIα) in 4 tissues (spleen, trunk kidney, muscle and liver) of the vaccinated fish. Finally, an immuno-protection with a relative survival rate of 65.71 % was displayed in vaccinated fish in comparison to the control groups. Taken together, these results indicate that pcDNA-YidC DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, indicating that IVIAT is a helpful strategy to screen the highly immunogenic antigens for vaccine development against fish nocardiosis.

A novel defensin-like peptide C-13326 possesses protective effect against multidrug-resistant Aeromonas schubertii in hybrid snakehead (Channa maculate ♀ × Channa argus ♂).

The purpose of this study was to investigate whether a defensin-like antimicrobial peptide (C-13326 peptide) identified in Hermetia illucens could possess protective effect against multidrug-resistant Aeromonas schubertii in hybrid snakehead (Channa maculate ♀ × Channa argus ♂). The cDNA of C-13326 peptide comprised 243 nucleotides encoding 80 amino acids, with six conserved cysteine residues and the classical CSαß structure. The recombinant expression plasmid pPIC9K-C-13326 was constructed and transformed into GS115 Pichia pastoris, and the C-13326 peptide was expressed by induction with 1% methanol. The crude extract of C-13326 peptide was precipitated by ammonium sulfate, assayed by Braford method, detected by tricine-SDS-PAGE, evaluated by BandScan software and identified by liquid chromatography-mass spectrometry. The C-13326 peptide was shown to have inhibitory activity against the growth of multidrug-resistant A. schubertii DM210910 by using the minimum growth inhibitory concentration and Oxford cup method. In addition, scanning electron microscopy analysis suggested that C-13326 peptide inhibited the growth of A. schubertii DM210910 by damaging the bacterial cell membrane. To explore the role of peptide C-13326 in vivo, hybrid snakehead was fed with peptide C-13326 as feed additives for 7 days. The results revealed that C-13326 peptide could significantly down-regulate the expression levels of IL-1ß, IL-8, IL-12 and TNF-α (p < .05), and significantly improved the survival rate of hybrid snakehead after challenging with A. schubertii DM210910. Therefore, the C-13326 peptide is a promising antimicrobial agent for A. schubertii treatment in aquaculture.

Identification and characterization of immune-related microRNAs in hybrid snakehead(Channa maculata♀ × Channa argus♂)after treated by Echinacea purpurea (Linn.) Moench.

Echinacea purpurea (Linn.) Moench (EP) is a globally popular herbal medicine, which showed effects on growth promotion, antioxidant and immunomodulatory activities in fish culture world widely. However, there are few studies about the effects on miRNAs by EP in fish. The hybrid snakehead fish (Channa maculate♀ × Channa argus ♂) was new important economic specie of freshwater aquaculture in China with high market value and demand while there were only a few reports about its miRNAs. To overview immune-related miRNAs of the hybrid snakehead fish and to further understand the immune regulating mechanism of EP, we herein constructed and analyzed three small RNA libraries of immune tissues including liver, spleen and head kidney of the fish with or without EP treatment via Illumina high-throughput sequencing technology. Results showed that EP can affect the immune activities of fish by the miRNA-regulated ways. Totally, 67 (47 up and 20 down) miRNAs in liver, 138 (55 up and 83 down) miRNAs in spleen, and 251 (15 up and 236 down) miRNAs in spleen were detected, as well as 30, 60, 139 kinds of immune-related miRNAs belonging to 22, 35 and 66 families of the three tissues respectively. The expressions of 8 immune-related miRNA family members were found in all the three tissues, including miR-10, miR-133, miR-22 and etc. Some miRNAs have been identified involved in the innate and adaptive immune responses, such as the miR-125, miR-138, and miR-181 family. Ten miRNA families with antioxidant target genes were also discovered, including miR-125, miR-1306, and miR-138, etc. Results from Gene Ontology (GO) and KEGG pathway analysis further confirmed there are a majority immune response targets of the miRNAs involved in the EP treatment process. Our study deepened understanding roles of miRNAs in fish immune system and provides new ideas for the study of immune mechanism of EP.

Simultaneous Isolation and Identification of Largemouth Bass Virus and Rhabdovirus from Moribund Largemouth Bass (Micropterus salmoides).

Largemouth bass is an important commercially farmed fish in China, but the rapid expansion of its breeding has resulted in increased incidence of diseases caused by bacteria, viruses and parasites. In this study, moribund largemouth bass containing ulcer foci on body surfaces indicated the most likely pathogens were iridovirus and rhabdovirus members and this was confirmed using a combination of immunohistochemistry, cell culture, electron microscopy and conserved gene sequence analysis. We identified that these fish had been co-infected with these viruses. We observed bullet-shaped virions (100−140 nm long and 50−100 nm in diameter) along with hexagonal virions with 140 nm diameters in cell culture inoculated with tissue homogenates. The viruses were plaque purified and a comparison of the highly conserved regions of the genome of these viruses indicated that they are most similar to largemouth bass virus (LMBV) and hybrid snakehead rhabdovirus (HSHRV), respectively. Regression infection experiments indicated fish mortalities for LMBV-FS2021 and HSHRV-MS2021 were 86.7 and 11.1%, respectively. While co-infection resulted in 93.3% mortality that was significantly (p < 0.05) higher than the single infections even though the viral loads differed by >100-fold. Overall, we simultaneously isolated and identified LMBV and a HSHRV-like virus from diseased largemouth bass, and our results can provide novel ideas for the prevention and treatment of combined virus infection especially in largemouth bass.

Characterization of the pathogenesis and immune response to a highly virulent Edwardsiella tarda strain responsible for mass mortality in the hybrid snakehead (Channa maculate ♀ × Channa argus ♂).

Edwardsiella tarda is reported as the causative agent of the systemic disease Edwardsiellosis in fish, which lead to huge economic losses in aquaculture. The pathogenicity and immune response to a highly virulent E. tarda isolate responsible for mass mortality in hybrid snakehead were performed. After species identification, morphology and virulence gene detection of Edwardsiella isolated from hybrid snakehead, the pathogenicity of the strain and histopathological changes in infected fish were analyzed. The infected fish exhibited typical acute hemorrhagic symptoms and enlarged internal organs. Histopathology revealed that the liver, spleen, kidney and intestinal tissues of diseased fish exhibited marked inflammatory with vacuolar degeneration and cell necrosis. Subsequently, humoral immune factors such as superoxide dismutase, lysozyme and acid phosphatase activities were detected as serum indicators, and real-time quantitative PCR was used to investigate immune-related genes (STAT1, HSP70, IgM, IL-6, IL-8, TRAF2, CD40, HLA-DMA and LCK) expression patterns in liver, spleen and head kidney. The results showed that these enzyme activity indicators and immune-related gene expression were significantly activated compared with healthy fish. These data provide insight into the pathogenic mechanisms and host immune responses of E. tarda, which could be useful for the future prevention and treatment of Edwardsiellosis in fish.

Effects of partial replacement of dietary flour meal with seaweed polysaccharides on the resistance to ammonia stress in the intestine of hybrid snakehead (Channa maculatus ♀ × Channa argus ♂).

The purpose of this study was to evaluate the effects of partial replacement of dietary flour meal with seaweed polysaccharides on survival rate, histology, intestinal oxidative stress levels, and expression of immune-related genes in hybrid snakeheads under acute ammonia stress. Four experimental diets were set: (C) basal diet with 0% of seaweed polysaccharides as the control group, (MR) basal diet with 10% of seaweed polysaccharides, (HR) basal diet with 15% of seaweed polysaccharides, (HF) basal diet with 10% of fish oil. After 60 days of feeding, fish fed with the diet of C group were sampled as the control group, and other fish were exposed to ammonia nitrogen for 48 h. Two concentrations of total ammonia nitrogen (TAN) were used in this study: 120 mg/L TAN (low concentration exposure group), and 1200 mg/L TAN (high concentration exposure group). After exposure to ammonia nitrogen for 48 h, fish were sampled. The results indicated that adding seaweed polysaccharides to the diet could improve the survival rate of hybrid snakeheads under high concentration of ammonia stress. Histopathological analysis demonstrated multiple abnormalities in gills and intestines after exposure to two concentrations of TAN. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and lactate dehydrogenase (LDH) were all increased in the MR group under two concentrations of TAN stress. The mRNA abundance of immune-related genes in fish intestinal tissues was significantly induced or inhibited. These results suggested that partial replacement of dietary flour meal with seaweed polysaccharides improved the ability of hybrid snakeheads to resist ammonia stress.

Interactions effects of nano-microplastics and heavy metals in hybrid snakehead (Channa maculata ♀ × Channa argus ♂).

The interaction between microplastics and contaminants has potentially generated new undefined risks on animals and ecosystems, and nano-microplastics are considered to be more harmful than microplastics. This experiment investigated the interactions and effects of nano-microplastics with heavy metals cadmium in hybrid snakehead. Different concentrations of nano-microplastics 80 nm (50 µg/L and 500 µg/L) and Cd (50 µg/L) were used for exposure, and four sampling points were set for 24 h, 48 h, 96 h and clear-48 h. Results indicated that the morphology of gill was altered under the influence of nano-microplastics and cadmium, and the damage was aggravated with time. Nano-microplastics and Cd can cause oxidative damage to fish liver partly by effect the activities of antioxidant enzyme, and significantly suppressed the expressions of genes related to the inflammation (IL-1ß and TNF-α) and as well as significantly up-regulated the expression of genes HSP70 and SOD. Additionally, the mRNA levels of MT gene can be speculated that the heavy metal cadmium may accumulated in the body over time. And the concentration of heavy metals will also affect their accumulation in the body. Our study elucidated the nano-microplastics and Cd will increase the impact on environmental and organisms that the nano-microplastics contribute to the bioaccumulation of metals, which served as a new support for study the interaction between environmental contaminants.

Molecular Identification of Nocardia seriolae and Comparative Analysis of Spleen Transcriptomes of Hybrid Snakehead (Channa maculata Female × Channa argus Male) With Nocardiosis Disease.

Hybrid snakehead (Channa maculata female × Channa argus male) is a new freshwater aquaculture fish species in southern China. During intensive aquaculture, hybrid snakeheads are often infected by Nocardia seriolae. In this study, hybrid snakehead infected suspiciously by N. seriolae in an artificial breeding pond were examined. Diseased hybrid snakeheads swam slowly without food intake, and the clinical symptoms included skin wound, anal swelling and ascites, and white granulomatous in liver, spleen, and kidney of fish. Through bacterial isolation, 16S rDNA sequencing, fluorescence in situ hybridization (FISH) and artificial infection experiment, the pathogen was identified as N. seriolae. Furthermore, the spleen samples from diseased and healthy male hybrid snakeheads in the same pond were used for RNA-Seq analysis. A total of 3,512 unique transcripts (unigenes) were identified as differentially expressed genes (DEGs), and 1,886 of them were up-regulated in diseased fish. The expression patterns of 20 DEGs were verified by quantitative polymerase chain reaction (qPCR). Several immune-related pathways and many immune-related genes were identified. qPCR results showed that the expression patterns of immune-related genes in the liver and kidney of diseased fish were comparable to that in the spleen. This study provides deep-sequencing data of hybrid snakehead spleen and will help understand the immune response of hybrid snakehead to N. seriolae. It is also helpful for the biomarker screening of fish-borne Nocardia spp. and the breeding of nocardiosis-resistant fish species.

Immune Response and Apoptosis-Related Pathways Induced by Aeromonas schubertii Infection of Hybrid Snakehead (Channa maculata♀ × Channa argus♂).

Aeromonas schubertii is the etiological pathogen of internal organ nodules in snakehead fish. Infections with A. schubertii produce a significant economic loss in aquaculture. Therefore, it is important to examine the immune mechanisms by which snakeheads defend against A. schubertii infection. In this study, we established a hybrid snakehead infection model by intraperitoneal injection of A. schubertii that produced internal organ nodules. The splenic immune response of infected fish was examined at the transcriptome level by Illumina-seq analysis. Results showed 14,796 differentially expressed genes (DEGs) following A. schubertii infection, including 4441 up-regulated unigenes and 10,355 down-regulated unigenes. KEGG analysis showed 2084 DEGs to be involved in 192 pathways, 14 of which were immune-related. Twelve DEGs were used to validate quantitative real-time PCR results with RNA-seq data. Time-course expression analysis of six genes demonstrated modulation of the snakehead immune response by A. schubertii. Furthermore, transcriptome analysis identified a substantial number of DEGs that were involved in the apoptosis signaling pathway. TUNEL analysis of infected spleens confirmed the presence of apoptotic cells. This study provided new information for a further understanding of the pathogenesis of A. schubertii in snakeheads, which can be used to prevent and possibly treat A. schubertii infections.

MEKK3 in hybrid snakehead (Channa maculate ♀ ×Channa argus ♂): Molecular characterization and immune response to infection with Nocardia seriolae and Aeromonas schubertii.

Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) is a serine/threonine protein kinase that acts as a key regulator and is widely involved in various innate and acquired immune signaling pathways. In this study, we first cloned the complete open reading frame (ORF) of the MEKK3 gene (named CcMEKK3) in a hybrid snakehead (Channa maculate ♀ × Channa argus ♂). The full-length ORF of CcMEKK3 is 1851 bp, and encodes a putative protein of 616 amino acids containing a serine/threonine kinase catalytic (S-TKc) domain and a Phox and Bem1p (PB1) domain. A sequence alignment and phylogenetic tree analysis showed that CcMEKK3 is highly conserved relative to the MEKK3 proteins of other teleost species. CcMEKK3 was constitutively expressed in all the healthy hybrid snakehead tissues tested, with greatest expression in the immune tissues, such as the head kidney and spleen. The expression of CcMEKK3 was usually upregulated in the head kidney, spleen, and liver at different time points after infection with Nocardia seriolae or Aeromonas schubertii. Similarly, the dynamic expression levels of CcMEKK3 in head kidney leukocytes after stimulation revealed that CcMEKK3 was induced by LTA, LPS, and poly(I:C). In the subcellular localization analysis, CcMEKK3 was evenly distributed in the cytoplasm of HEK293T cells, and its overexpression significantly promoted the activities of NF-κB and AP-1. These results suggest that CcMEKK3 is involved in the immune defense against these two pathogens, and plays a crucial role in activating the NF-κB and MAPK signaling pathways.

Immunogenicity and efficacy of two DNA vaccines encoding antigenic PspA and TerD against Nocardia seriolae in hybrid snakehead.

Fish nocardiosis is a widespread chronic granulomatous disease in aquatic environment, which was particularly caused by Nocardia seriolae. The phage shock protein A (PspA) and tellurium resistance protein D (TerD) were identified to be the immunodominant antigens of the wild-type N. seriolae strain ZJ0503 in our previous study. In an attempt to develop effective DNA vaccines against this pathogen, PspA and TerD were used as candidates to ligate with pcDNA3.1-Flag plasmids, respectively. In addition, the abilities of these two DNA vaccines to elicit various immune responses in hybrid snakehead and supply protective efficacy against artificial challenge with N. seriolae were determined in the present study. The results showed that intramuscular injection with pcDNA-PspA and pcDNA-TerD did not exhibit cytotoxic activities in hybrid snakehead via histopathological examination. Besides, hybrid snakehead immunization with pcDNA-PspA and pcDNA-TerD could increase several non-specific immune paraments in serum, including LYZ, POD, ACP, AKP and SOD activities. Meanwhile, the pcDNA-TerD DNA vaccine could induce strongly specific antibody (IgM) titer in hybrid snakehead with a relative percent of survival (RPS) value of 83.14% against N. seriolae, while that of pcDNA-PspA DNA vaccine was displayed comparably low IgM titer with RPS value of 57.83%. Furthermore, quantitative real-time PCR assays presented that the expression of immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1ß and TNFα) were up-regulated to various degrees after vaccination with pcDNA-PspA or pcDNA-TerD, indicating that these two DNA vaccines were able to boost humoral and cell-mediated immune responses in hybrid snakehead. Taken together, both the pcDNA-PspA and pcDNA-TerD DNA vaccines were proved to be safe, immunogenic and effective in protecting hybrid snakehead against N. seriolae infection, which can promote the development and application of DNA vaccines to control fish nocardiosis in aquaculture.

Establishment of a brain cell line obtained from hybrids of Channa argus ×Channa maculata for the detection of tilapia lake virus.

A brain cell line (CAMB) derived from hybrid snakehead (Channa argus (♂) × Channa maculata (♀)) was established by trypsin and collagenase combined digestion. The culturing conditions and cell biological characteristics were systematically studied. For growth of the cells, M199 medium containing 10% fetal bovine serum was used and at 27 °C incubated. Based on morphological analysis, CAMB cells were confirmed to be epithelial. The cell line has been subcultured more than 80 times since its initial primary culture. Chromosome analysis revealed that CAMB cells had an abnormal chromosome number 2n = 64, whereas the chromosome number in the hybrid snakehead was 45. The suitability of CAMB for tilapia lake virus (TiLV) was demonstrated. A CPE was observed after infection with TiLV-2017A. The highest TiLV titer was observed after 12 days post infection (dpi) and reached 107.2 TCID50/mL. The virus replication was confirmed by electron microscopic observations. Additionally, immunofluorescence assay confirmed the presence of TiLV-2017A after infection of CAMB. Therefore, CAMB cells can be a useful tool for the investigation of the pathogenesis of the TiLV induced disease in tilapia.

Effects of Dietary Inclusion of Shrimp Paste on Growth Performance, Digestive Enzymes Activities, Antioxidant and Immunological Status and Intestinal Morphology of Hybrid Snakehead (Channa maculata ♀ × Channa argus ♂).

A nutritional feeding experiment was conducted to evaluate the effects of shrimp paste on feeding attractiveness, growth performance, digestive enzyme activities, immune-related genes and intestinal morphology in hybrid snakehead (Channa maculata ♀ × Channa argus ♂). Two diets were formulated with or without shrimp paste supplementation (D1:0% and D2: 3%) to feed fish for 8 weeks. Results showed that growth performance (FBW, WG and SGR) and feed intake (FI) significantly increased with shrimp paste supplemented (P < 0.05), while FCR and SR of hybrid snakehead fed diets supplemented with shrimp paste or not showed no significant difference (P > 0.05). Gut lipase and amylase activities were significantly higher in diet supplemented with shrimp paste than that in control one (P < 0.05). Hepatic antioxidant statuses of hybrid snakehead fed dietary shrimp paste or not showed no significant differences in total antioxidant capacity, malondialdehyde and superoxide dismutase of fish (P > 0.05). Results showed that fish fed diet with shrimp paste supplemented did not show significant difference in expression of GR, IκB, P65 and IL8 than that in control group (P > 0.05). There are significantly more goblet cells in shrimp paste supplemented diet than that in control diet (P < 0.05). However, villi length and muscle thickness showed no significant difference compared to control diet (P > 0.05). The results indicated that dietary 3% shrimp paste supplementation improved the growth performance of hybrid snakehead by enhancing feed intake (FI) while made no difference to antioxidant capacity and immunity.

Epizootic ulcerative syndrome causes cutaneous dysbacteriosis in hybrid snakehead (Channa maculata♀ × Channa argus♂).

Cutaneous microbiota play an important role in protecting fish against pathogens. Aphanomyces infection causes epizootic ulcerative syndrome (EUS) in fish, and by perturbing the integrity of the cutaneous microbiota, increases the potential for infection by pathogenic bacteria. However, whether the composition of the cutaneous microbiota is altered in fish with EUS, and if so, which species are changed and how this might influence infected fish, is still largely unclear. Considering the importance of cutaneous microbiota in maintaining host health, we hypothesized that Aphanomyces infection significantly enhances the presence of certain bacterial pathogens in the cutaneous microbiota and causes cutaneous dysbacteriosis. To test this hypothesis, we compared the cutaneous microbiota compositions of hybrid snakehead (Channa maculata♀ × Channa argus♂) with and without Aphanomyces infection using Illumina Miseq sequencing of the 16S rRNA gene. Our results showed that the cutaneous microbiota of hybrid snakehead were significantly altered subsequent to EUS infection and that the numbers of potentially pathogenic bacteria classified into the genera Anaerosinus, Anaerovorax, Dorea, and Clostridium were significantly enhanced in the cutaneous microbiota of hybrid snakehead with EUS, whereas bacteria classified into the genera Arthrobacter, Dysgonomonas, Anoxybacillus, Bacillus, Solibacillus, Carnobacterium, Lactococcus, Streptococcus, Achromobacter, Polynucleobacter, Vogesella, and Pseudomonas were significantly reduced. These results imply that treatment for EUS should not only take into consideration the control of Aphanomyces reproduction but should also focus on regulating the cutaneous microbiota of infected fish.

Identification and expression analysis of miRNA in hybrid snakehead by deep sequencing approach and their targets prediction.

MicroRNAs (miRNAs) play important regulatory roles in numerous biological processes, but there is no report on miRNAs of hybrid snakehead. In this study, four independent small RNA libraries were constructed from the spleen, liver kidney and muscle of hybrid snakehead. These libraries were sequenced using deep sequencing technology, as result, a total of 1,067,172, 1,152,002, 1,653,941 and 970,866 clean reads from these four libraries were obtained. 252 known miRNAs and 63 putative novel miRNAs in these small RNA dataset were identified. The stem-loop RT-qPCR analysis indicated that eight known miRNAs and two novel miRNAs show different expression in eight different kinds of tissues. For better understanding the functions of miRNAs, 95,947 predicated target genes were analyzed by GO and their pathways, the results indicated that these targets of the identified miRNAs are involved in a broad range of physiological functions.

Transcriptome analysis of immune-related gene expression in hybrid snakehead (Channa maculata ♀ × Channa argus ♂) after challenge with Nocardia seriolae.

Hybrid snakehead fish (Channa maculata ♀ × Channa argus ♂), a new species used in freshwater aquaculture in China, is the common host of an epizootic bacterial infection by Nocardia seriolae. However, the information on the functions and mechanisms of hybrid snakehead immune pathways with the N. seriolae infection is limited. Thus, the peripheral blood lymphocytes from hybrid snakehead were used for transcriptome analysis to understand the host immune response after challenge with N. seriolae. A total of 49,839,332 and 50,059,283 raw reads were obtained from the N. seriolae-challenged group (Ns group) and phosphate-buffered saline control group (Ctr group), respectively. The 75.50% and 74.25% reads from the Ns and Ctr groups were matched to reference genomic sequence after cleaning the raw reads, respectively. Additionally, there were 2892 significant differentially expressed genes (DEGs) among the 17,196 expressed genes between the Ns and Ctr groups, including 1387 upregulated and 1505 downregulated genes. All the DEGs were classified into three gene ontology categories, and 2502 DEGs had significant matches, which were allocated to 246 Kyoto Encyclopedia of Genes and Genomes pathways. Immune-related genes were detected from immune system pathways among the top 20 enriched pathways. Moreover, the regulation of several observed effective genes was confirmed by real-time quantitative polymerase chain reaction. Altogether, this study offers deep-sequence data of hybrid snakehead peripheral blood lymphocyte via transcriptome analysis and lays the foundation for further study on the immunogenetics of hybrid snakehead. Moreover, it provides insights into the pathogenic mechanism of N. seriolae, facilitating the prevention and treatment of fish nocardiosis.