A new species of Myxobolus (Cnidaria: Myxosporea: Myxobolidae) from the gibel carp Carassius gibelio (Cypriniformes: Cyprinidae).

Myxobolus zhaltsanovae n. sp., is described from the gills of gibel carp Carassius gibelio found during a survey of myxozoans from the watershed of Lake Baikal, Russia, based on morphological and molecular characterizations. Plasmodia of M. zhaltsanovae n. sp. develop extravascularly and measure 500-1000 µm long, 25-100 µm wide. The myxospore is circular to oval, measuring 13.23 ± 0.09 (11.3-14.8) µm (mean ± SD, range) in length, 10.19 ± 0.07 (9.1-11.4) µm in width, and 6.49 ± 0.12 (5.4-7.2) µm in thickness. Polar capsules are unequal and subspherical; measurements of polar capsules are: length 5.62 ± 0.06 (4.7-6.7), width 3.44 ± 0.04 (2.4-4.4) µm and length 3.42 ± 0.05 (2.5-4.1), width 1.94 ± 0.04 (1.3-3.3) µm. Phylogenetic analysis with the 18S rDNA gene shows M. zhaltsanovae n. sp. as a sister species of the subclade formed by M. musseliusae, M. tsangwuensis, and M. basilamellaris, which infect common carp Cyprinus carpio.

The first detection of Myxobolus lentisuturalis Dyková, Fiala et Nie, 2002, a highly pathogenic muscle-infecting parasite of gibel carp (Carassius auratus gibelio Berg, 1932) in Hungary.

Myxobolus lentisuturalis is a myxosporean parasite infecting the musculature both of goldfish (Carassius auratus auratus) and gibel carp (Carassius auratus gibelio). The species was originally described in China from gibel carp that is a common fish for sport fishing in Hungary meanwhile is one of the most popular farmed fish in China due to its high demand. Eighteen gibel carp with distortions were collected from a barrage pond in southern Hungary. All fish had large humps in the dorsolateral region due to infection of the muscle between the head and the dorsal fin. The swollen degenerated tissue was filled with myxozoan spores, which were collected for morphological and molecular studies. By size and morphology, the spores were consistent with morphological description of M. lentisuturalis. Histopathological examination showed that the formation of plasmodia containing myxospores leads to severe destruction of muscle tissue. The 18S ribosomal DNA and 28S ribosomal DNA data of the samples presented matched with previous sequences of M. lentisuturalis in GenBank. Phylogenetic analyses confirmed that our sequences belong to a monophyletic group with them supported by a high bootstrap. This study highlights the occurrence of a highly pathogenic myxozoan, M. lentisuturalis in Hungary as a new geographical location.

Proteomic and Phosphoproteomic Analyses Reveal Gibel Carp Responses to Cyprinid Herpesvirus 2 Infection.

Cyprinid herpesvirus 2 (CyHV-2) is a typical linear double-stranded DNA virus, which can induce severe herpesviral hematopoietic necrosis disease (HVHND) in gibel carp. However, the CyHV-2 infection mechanisms still remain unresolved till now. Here, we combined the isobaric tag for relative absolute quantitation (iTRAQ)-labeled quantitative proteomic and phosphoproteomic analysis enriched by Ti4+-immobilized titanium ion affinity chromatography (IMAC) to uncover the host responses to CyHV-2 infection in the kidneys of symptomatic and diseased gibel carp. We totally identified 192 differential expression proteins and 951 high-confident phosphopeptides involved in 657 proteins. After being infected with CyHV-2, the proteins involved in energy generation and ion balance were significantly downregulated in the host, and the phosphorylated proteins induced by viral infection mainly participated in the regulation for RNA processing, translation, cytoskeleton organization, immunoreaction, etc. Furthermore, 11 phosphorylated CyHV-2 viral proteins were found in the diseased group by the host proteome. The virus-host protein-protein interactions were investigated, in which the potential host kinases casein kinase II (CK-II) and cyclin-dependent kinase (CDK) that interacted with viral ORF88 or ORF89 were identified and can serve as candidate targets for disease treatment in the future. Overall, our study provides a comprehensive understanding of CyHV-2-induced perturbations at the protein and phosphorylation levels in gibel carp, forming a base for the treatment of HVHND.

Functional Divergence of Multiple Duplicated Foxl2 Homeologs and Alleles in a Recurrent Polyploid Fish.

Evolutionary fates of duplicated genes have been widely investigated in many polyploid plants and animals, but research is scarce in recurrent polyploids. In this study, we focused on foxl2, a central player in ovary, and elaborated the functional divergence in gibel carp (Carassius gibelio), a recurrent auto-allo-hexaploid fish. First, we identified three divergent foxl2 homeologs (Cgfoxl2a-B, Cgfoxl2b-A, and Cgfoxl2b-B), each of them possessing three highly conserved alleles and revealed their biased retention/loss. Then, their abundant sexual dimorphism and biased expression were uncovered in hypothalamic-pituitary-gonadal axis. Significantly, granulosa cells and three subpopulations of thecal cells were distinguished by cellular localization of CgFoxl2a and CgFoxl2b, and the functional roles and the involved process were traced in folliculogenesis. Finally, we successfully edited multiple foxl2 homeologs and/or alleles by using CRISPR/Cas9. Cgfoxl2a-B deficiency led to ovary development arrest or complete sex reversal, whereas complete disruption of Cgfoxl2b-A and Cgfoxl2b-B resulted in the depletion of germ cells. Taken together, the detailed cellular localization and functional differences indicate that Cgfoxl2a and Cgfoxl2b have subfunctionalized and cooperated to regulate folliculogenesis and gonad differentiation, and Cgfoxl2b has evolved a new function in oogenesis. Therefore, the current study provides a typical case of homeolog/allele diversification, retention/loss, biased expression, and sub-/neofunctionalization in the evolution of duplicated genes driven by polyploidy and subsequent diploidization from the recurrent polyploid fish.

Molecular cloning and tissue distribution of the leptin gene in gibel carp (Carassius auratus gibelio): Regulation by postprandial and long-term fasting treatment.

Leptin is a multifunctional hormone that serves as a feeding regulator in mammals. However, the effect of leptin on fish remains unclear. We sequenced the leptin gene from gibel carp (Carassius auratus gibelio) and designated it gLEP. The length of the gLEP cDNA sequence was 562 bp, including an open reading frame (ORF) of 516 bp. The ORF putatively encodes a peptide of 171 amino acids, including a signal peptide of 20 amino acids. gLEP shared low primary amino acid sequence homology with leptin genes in vertebrates, whereas three-dimensional (3D) structural modeling revealed strong identity with the structures in other vertebrates. gLEP mRNA was widely distributed in all of the tissue that we examined, with the highest levels of expression in the hepatopancreas. Hepatopancreas gLEP mRNA expression levels showed no changes following postprandial treatment. However, hepatopancreas gLEP mRNA expression levels greatly decreased (P < 0.05) after fasting but substantially increased (P < 0.05) after refeeding in the long-term fasting treatment. In summary, these results indicate that leptin expression could be influenced by the regulation of food intake. These results provide the initial step toward elucidating the appetite regulatory systems associated with leptin in gibel carp.

Histopathology and transcriptome analysis reveals the gills injury and immunotoxicity in gibel carp following acute deltamethrin exposure.

More and more evidences proved that deltamethrin (Del) exposure induced adverse effects and damaged immune function to the aquatic animals in the parasite killing process with increasing insecticide application. However, little is currently known of the negative effect on mucosal immunity, especially in gills tissue. Therefore, this study was aimed to reveal the tissue injury and immunotoxicity in the gill of gibel carp following acute deltamethrin exposure. The LC50 of deltamethrin on gibel carp at 96 h was determined to be 6.194 µg/L, and then juvenile gibel carp (Carassius auratus gibelio) (8.8 ± 1.0 g) were exposed to four Del exposure groups (0.61, 1.22, 2.44, and 4.88 µg/L) for 12 h and 24 h. We measured the lysozyme (LYZ) contents and myeloperoxidase (MPO) activities and found that with increased concentration of Del exposure, the LYZ contents were found to increase in the 1.22 µg/L Del group initially significantly and then gradually significantly decrease in the 4.88 µg/L Del group. And the activities of MPO were significantly lifted in a dose-dependent manner. The histological analysis showed that Del exposure caused serious desquamation and necrosis in the surface of epithelial cells, accompanied by interlamellar cellular mass degenerative. In addition, the mucous cells were significantly decreased in the high Del concentration group (2.44 µg/L and 4.88 µg/L Del group) by AB-PAS staining. Additionally, totally 2857 DEGs (including 1624 up-regulated and 1233 down-regulated genes) were identified between the control group and 4.88 µg/L Del exposure group using transcriptional analysis. Among these, some genes involved in innate immune molecules, complement activation, apoptosis-related molecules, cytokine, and adaptive immune molecules, were also down-regulated. Importantly, we found immune system process and tumor necrosis factor receptor (superfamily) binding pathways were downregulated based on the GO and KEGG enrichment analysis. Meanwhile, we detected the expression of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1ß, and IL-8), anti-inflammatory cytokines (TGF-ß), LYZ, IgM, and Hsp70 in the gills tissue at 12 h and 24 h after Del exposure, which were consistent with our sequencing results. Collectively, these results demonstrated that the gills injury and immunotoxicity were induced by Del exposure and provided novel insight for explaining to some extent why Del-exposure fish are more susceptible to concurrent or secondary viral or bacterial infections.

Enhancement of growth, antioxidative status, nonspecific immunity, and disease resistance in gibel carp (Carassius auratus) in response to dietary Flos populi extract.

This study investigated the effects of dietary Flos populi extract (FPE) on the growth, antioxidation capability, innate immune response, and disease resistance in gibel carp. A total of 480 fish were fed with five different diets containing 0, 0.5, 1.0, 1.5, or 2.0 g kg-1 FPE (designated as control, D0.5, D1.0, D1.5, or D2.0 groups) for 45 days. The fish were challenged with A. hydrophila after the feeding trial. Compared with the control, the feed efficiency (FE), weight gain (WG), final body weight (FBW), and specific growth rate (SGR) were significantly improved in groups D1.0 and D1.5. Dietary FPE significantly increased serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, as well as glutathione (GSH) content. The contents of protein carbonyl (PCC) and malondialdehyde (MDA) in serum decreased significantly. Additionally, FPE supplementation in diets resulted in significant improvement in serum lysozyme (LZM) and myeloperoxidase (MPO) activities, as well as immunoglobulin M (IgM) and complement 3 (C3) concentrations. The hepatic antioxidant enzymes (CAT and SOD) activities increased, whereas content of MDA decreased in fish treated with dietary FPE than those of control both pre- and post-challenged. After 12 h-challenge, an obvious downregulation of hepatic Kelch-like-ECH-associated protein 1 (Keap1), splenic tumor necrosis factor-α (TNF-α), interleukin (IL)-8, IL-1ß, and toll-like receptor 2 (TLR2) mRNA levels was observed in fish treated with dietary FPE, whereas hepatic Nrf2 transcription level was upregulated compared to the control. Furthermore, compared to group D0.5, higher relative percent survival (RPS) was observed in gibel carp fed dietary 1.0-2.0 g/kg FPE. Our results reveal that FPE supplemented diet has a stimulatory effect on antioxidant capacity and nonspecific immune response, along with improved growth performance and enhanced resistance against A. hydrophila infection in juvenile gibel carp.

Comparative transcriptomic analysis reveals an association of gibel carp fatty liver with ferroptosis pathway.

BACKGROUND: Fatty liver has become a main problem that causes huge economic losses in many aquaculture modes. It is a common physiological or pathological phenomenon in aquaculture, but the causes and occurring mechanism are remaining enigmatic. METHODS: Each three liver samples from the control group of allogynogenetic gibel carp with normal liver and the overfeeding group with fatty liver were collected randomly for the detailed comparison of histological structure, lipid accumulation, transcriptomic profile, latent pathway identification analysis (LPIA), marker gene expression, and hepatocyte mitochondria analyses. RESULTS: Compared to normal liver, larger hepatocytes and more lipid accumulation were observed in fatty liver. Transcriptomic analysis between fatty liver and normal liver showed a totally different transcriptional trajectory. GO terms and KEGG pathways analyses revealed several enriched pathways in fatty liver, such as lipid biosynthesis, degradation accumulation, peroxidation, or metabolism and redox balance activities. LPIA identified an activated ferroptosis pathway in the fatty liver. qPCR analysis confirmed that gpx4, a negative regulator of ferroptosis, was significantly downregulated while the other three positively regulated marker genes, such as acsl4, tfr1 and gcl, were upregulated in fatty liver. Moreover, the hepatocytes of fatty liver had more condensed mitochondria and some of their outer membranes were almost ruptured. CONCLUSIONS: We reveal an association between ferroptosis and fish fatty liver for the first time, suggesting that ferroptosis might be activated in liver fatty. Therefore, the current study provides a clue for future studies on fish fatty liver problems.

Development and evaluation of a convenient immunochromatographic strip test for rapid detection of cyprinid herpesvirus 2 (CyHV-2).

Cyprinid herpesvirus 2 (CyHV-2) has become a serious threat to the gibel carp Carassius auratus gibelio industry and has led to enormous losses worldwide. We have therefore developed an immunochromatographic strip (ICS) to enable rapid on-site detection of CyHV-2 by aquaculture facility staff. The ICS employs 2 monoclonal antibodies (MAbs 2C3-1E6 and 3H2-1G5) against the ORF25 protein, a CyHV-2 membrane protein, as the capture and detection antibodies, respectively. Indirect immunofluorescence assay (IIFA) and Western blotting of CyHV-2-infected fathead minnow cells indicated that the 2 MAbs could specifically bind CyHV-2 by recognizing ORF25 antigen. Sandwich ELISA showed that the detection limit of ORF25 protein halved when MAb 2C3-1E6 served as the capture antibody compared to MAb 3H2-1G5. The test for detecting purified CyHV-2 using the ICS could be completed in 10 min and the sensitivity was 1 µg ml-1. Sensitivity of the ICS remained stable following storage at 4, 25 and 37°C for 6 mo. Tissue homogenate from gibel carp with and without obvious gill hemorrhages was subjected to CyHV-2 detection using the ICS: the results were in good accordance with conventional PCR. Our ICS does not require highly trained technicians or specialized equipment, making it suitable for rapid diagnosis of CyHV-2 infection both in the laboratory and in the field.

Does the Anionic Surfactant Isopropylamine Dodecylbenzene Sulfonate Induce Hepatic Oxidative Stress and Impairment in Carassius auratus gibelio?

Isopropylamine dodecylbenzene sulfonate (IDS) is a new kind of anionic surfactant (ANS). To preliminarily evaluate the aquatic toxicity of IDS, this study took gibel carp (Carassius auratus gibelio) as the research object. The well-acclimatized fish were divided into six groups and exposed to 0 mg/L, 0.5 mg/L, 1.0 mg/L, 2.0 mg/L, 4.0 mg/L, or 8.0 mg/L of IDS for 7, 14, 21 or 28 days. Our results showed that the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content of the liver were unaffected by IDS exposure, while glutathione peroxidase (GSH-Px) activity was significantly inhibited. Hepatic tissue exhibited pathological damage, characterized by nuclear migration and dissolution and cell boundary blurring. The results suggest that IDS does not cause oxidative stress in the liver, but cause hepatic histopathological damage. GSH-Px can be considered as a biomarker of IDS exposure in gibel carp.

Dietary supplementation of Geotrichum candidum improves growth, gut microbiota, immune-related gene expression and disease resistance in gibel carp CAS â ¢ (Carassius auratus gibelio).

Geotrichum candidum is a filamentous fungus mostly used in the dairy industry for cheese ripening and flavoring. The current study was designed to evaluate the potential probiotics effect of dietary supplementation of G. candidum on growth, digestive enzymes activities, gut microbiota and immune-related gene expression and disease resistance in Gibel carp CAS Ⅲ (Carassius auratus gibelio) against A. hydrophila infections. For this purpose, 420 healthy fish (initial body weight: 7.09 ± 0.02g) were randomly divided into 7 feeding groups in triplicates of 20 fish in each tank. Seven isocaloric and isonitrogenous diets were prepared, supplemented with 6 different doses of G. candidum viz; C: 0, T1:106, T2:107, T3:108, T4:109, T5:1010, T6:1011 CFU/kg diet and fed to fish twice a day for 60 days. Results showed significantly higher weight gain (WG) and feed efficiency (FE) in groups fed 106 and 108 CFU/kg diet compared to the control (P < 0.05). All G. candidum fed groups showed higher intestinal α-amylase activity (P < 0.05) while no difference in lipase and trypsin activity compared to the control group. Before challenge test, gut microbial diversity analysis revealed relatively more abundance of bacteria belonging to phylum Proteobacteria, Actinobacteria, Firmicutes and Bacteriodetes in group fed G. candidum supplemented diet and higher percentage of pathogenic bacteria belonging to order Aeromonadales and Vibrionales in control post-challenge. After challenge test immune response expressed numerically higher survival rate (P > 0.05) and significantly higher (P < 0.05) Respiratory Burst Activity(RBA), Immunoglobulin M level (IgM), Aspartate Transaminase activity (AST) and improved (P > 0.05) lysozyme activity, and Alanine Transaminase activity (ALT) in groups fed 106 and 108 CFU/kg G. candidum supplemented diet. The expression of immune related gene (IL-I ß, TNF- α, HSP70 and TLR-2) in liver of the fish were significantly affected (P < 0.05) by supplementation of G. candidum post bacterial challenge. The results showed that G. candidum is a potential probiotic as it could improve feed utilization, immunity and cause no harmful effects on growth of gibel carp at higher supplementation levels. The productive results can be achieved by using the lower dose as 106-108 CFU/kg diet.

Effects of dietary fish meal replacement by fermented moringa (Moringa oleifera Lam.) leaves on growth performance, nonspecific immunity and disease resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS III).

This study was aimed to evaluate the effects of partial replacement of fish meal by fermented moringa leaves (FMLs) on growth performance, serum biochemistry, antioxidant status, nonspecific immunity, and resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS III). Four isonitrogenous and isoenergetic balanced diets, including three FML diets (substituting 20%, 40%, 60% of the fish meal in basal diet, F20, F40 and F60, respectively) and a basal diet (a diet containing 10% fish meal) were used. Each diet was randomly allocated to four fish groups (F20, F40, F60 and control) reared in a recirculating system. After 50 days of the feeding trial, fish were challenged by A. hydrophila. The result revealed that final mean body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), feed efficiency (FE) and survival rate (SR) were significantly increased (P < 0.05) in F20 and F40 groups compared with the control group. Decreased hepatosomatic index (HSI), body crude lipid, serum aspartate transaminase (AST) and serum alanine aminotransferase (ALT) activities, and increased serum alkaline phosphatase (AKP) and serum glutathione peroxidase (GPx) activities were observed in F40 and F60 groups compared with the control and F20 groups. All FMLs-supplemented groups increased (P < 0.05) serum superoxide dismutase (SOD), catalase (CAT) and lysozyme activities, complement component 3 (C3) and serum immunoglobulin M (IgM) concentration, or decreased serum malondialdehyde (MDA) and protein carbonyl (PCC) contents (P < 0.05). After the challenge test, the significant downregulation of toll-like receptors2 (TLR2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-8 mRNA transcription levels was observed in spleens of FMLs supplemented groups. Dietary F40 and F60 showed higher (P < 0.05) relative percent survival (RPS) (48.72% and 43.59%, respectively) against A. hydrophila infection than control. These results indicate that, as a dietary fish meal substitute, FMLs enhance the growth, and antioxidant and immune response, and regulate the expression of immune-related genes and increase disease resistance against A. hydrophila via TLR2 pathway in gibel carp, with greatest effects of 40% fish meal substitution.

Development of monoclonal antibodies against serum immunoglobulins from gibel carp (Carassius auratus gibelio) and their applications in serodiagnosis of inapparent infection and evaluation of vaccination strategies.

The disease outbreak caused by viral infection and bacterial pathogens has hampered the sustainable development of the gibel carp (Carassius auratus gibelio) industry, lack of monoclonal antibodies against serum immunoglobulin (Ig) of gibel carp has impeded the development of nonfatal immunoassays in detection of pathogen infection and understanding of fish immune response post vaccination. In the present study, serum Ig of gibel carp was purified by a combination of salting-out and DEAE Sepharose Column chromatography. The purified Ig had an apparent molecular weight of 74 kDa and 24 kDa in SDS-PAGE. Three monoclonal antibodies (MAbs) against Ig, designed as 2F4-1G10, 3H3-1E8 and 7H11-1C8, were developed with purified Ig preparations, which were selected on the basis of the indirect enzyme-linked immunosorbent assay (ELISA). Western blotting showed that MAbs 2F4-1G10 and 7H11-1C8 reacted with the heavy chain of IgM, while MAb 3H3-1E8 showed a reaction with the light chain. MAb 7H11-1C8 could react with surface Ig-positive (sIg+) lymphocytes under indirect immunofluorescence assay. Fluorescence-activated cell sorter analysis revealed that the percentage of sIg + lymphocytes were 32.68% in peripheral blood and 12.13% in pronephros. MAb 7H11-1C8 was proven to be effective in detecting the Cyprinid Herpesvirus 2-specific serum Ig, and determining the levels of Aeromonas hydrophila specific Ig in serum and immune organs under different vaccination strategies.

Dietary Scenedesmus ovalternus improves disease resistance of overwintering gibel carp (Carassius gibelio) by alleviating toll-like receptor signaling activation.

This study was conducted to investigate the effect of dietary Scenedesmus ovalternus on the growth and disease resistance of gibel carp (Carassius gibelio) during overwintering. Gibel carp (initial body weight: 90.39 ± 0.33 g) were fed with diets containing 0% or 4% Scenedesmus ovalternus (DS0 and DS4) for 4 weeks during the early overwintering period, and then all fish were left unfed during the late overwintering period. A bacterial challenge test using Aeromonas hydrophila was subsequently conducted. The 4% Scenedesmus ovalternus diet had no effect on the growth of gibel carp (P > 0.05), but did improve the survival rate after the challenge (P ≤ 0.05). In the DS0 group, the bacterial challenge decreased the contents of complement 3 (C3), immunoglobulin M (IgM), interleukin 2 (IL2) and tumor necrosis factor α (TNFα) in fish (P < 0.05); in the DS4 group, the challenge increased total antioxidant capacity (T-AOC) and myeloperoxidase (MPO) activity but decreased IL2 and TNFα contents (P < 0.05). The activities of MPO and contents of C3, IgM and TNFα were higher in the DS4 group than that fed the DS0 diet after bacterial challenge (P < 0.05). Compared to pre challenge, the expression levels of toll like receptor 2 (TLR2), toll like receptor 3 (TLR3), toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), TIR-domain-containing adapter-inducing interferon ß (TRIF), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα), transforming growth factor ß (TGFß), interleukin 1ß (IL1ß), tumor necrosis factor α1 (TNFα1) and interleukin 10 (IL10) in the head kidney of gibel carp were induced after challenge (P < 0.05). Gibel carp fed the DS4 diet showed lower expression of TGFß in head kidney before the challenge and lower expression of TLR2, TLR3, TLR4, TIRAP, TRIF, IκBα, TNFα1, IL10 and TGFß after the challenge than that fed the DS0 diet (P < 0.05). Overall, Scenedesmus ovalternus supplement enhanced the resistances of gibel carp against A. hydrophila after overwintering via the TLR signaling pathway.

Transcriptome analysis provides insights into the antiviral response in the spleen of gibel carp (Carassius auratus gibelio) after poly I: C treatment.

Gibel carp (Carassius auratus gibelio) is an important commercial fish that has become one of the most cultured fishes in the region of Yangtze River in China. However, the fish faces increasing hazard due to cyprinid herpesvirus 2 (CyHV-2) infection, which has caused great economic losses. In this study, healthy gibel carp were intraperitoneally injected with different doses of poly I:C at 24 h before CyHV-2 challenge. Results showed that the mortality decreased and peak death time appeared later in the fish injected with poly I:C at a dose of 10 µg/g body weight. To explore what gene plays an important role after poly I:C treatment, the transcriptome analysis of the gibel carp spleen was further performed. Compared with the PBS group, 1286 differentially expressed genes (DEGs) were obtained in the poly I:C-treated fish, including 1006 up-regulated and 280 down-regulated DEGs. GO analysis revealed that the most enriched DEGs responded to "biological regulation", "regulation of cellular process" and "regulation of biological process". Meanwhile, KEGG enrichment analysis showed that the DEGs were mainly mapped on the immune pathways like "TNF signal pathway", "p53 signal pathway" and "JAK-STAT signal pathway", suggesting that these signal pathways may be responsible for the delayed peak of CyHV-2 infection in gibel carp after poly I:C treatment. Taken together, this study provides insights into the immune protection effect of poly I:C against CyHV-2 infection, as well as providing useful information for antiviral defense in gibel carp.

Molecular and light microscopy evidence for the transfer of Myxobolus honghuensis from Carassius auratus gibelio broodfish to progeny.

Myxozoans usually have a complex life cycle involving indirect transmission between vertebrate and invertebrate hosts. The vertical transmission of these parasites in vertebrate hosts has not been documented so far. Here, we assessed whether the myxozoan parasite Myxobolus honghuensis is vertically transmitted in naturally infected allogynogenetic gibel carp Carassius auratus gibelio (Bloch). M. honghuensis infection of broodfish, fertilized eggs and laboratory-cultured progeny was monitored in 2018 and 2019. The presporogonic stage was microscopically observed in the pharynx of broodfish and their progeny. In situ hybridization confirmed the presence of M. honghuensis presporogonic stage in the pharynx of broodfish and progeny. Nested PCR results showed that M. honghuensis was present in tissues and eggs of broodfish, fertilized eggs and their corresponding progeny. The sequences obtained from broodfish and progeny showed 98.0-99.8% similarity with ITS-5.8S rDNA of M. honghuensis. This study provides molecular and light microscopy evidence for the transfer of M. honghuensis from broodfish to progeny via the eggs, but it is insufficient to assert that M. honghuensis can transmit vertically in naturally infected allogynogenetic gibel carp. This is the first record about vertical transfer of myxozoan in the vertebrate host.

Differential expression of innate and adaptive immune genes in the survivors of three gibel carp gynogenetic clones after herpesvirus challenge.

BACKGROUND: Accompanied with rapid growth and high density aquaculture, gibel carp has been seriously threatened by Carassius auratus herpesvirus (CaHV) since 2012. In previous study, distinct CaHV resistances and immune responses were revealed in the diseased individuals of three gibel carp gynogenetic clones (A+, F and H). However, little is known about the gene expression changes in the survivors after CaHV challenge, particularly their differences of innate and adaptive immune system between susceptible clone and resistant clone. RESULTS: We firstly confirmed the CaHV carrier state in the survivors of three gibel carp clones after CaHV challenge by evaluating the abundances of five CaHV genes. The assay of viral loads indicated the resistant clone H possessed not only stronger resistance but also higher tolerance to CaHV. Then, 2818, 4047 and 3323 differentially expressed unigenes (DEUs) were screened from the head-kidney transcriptome profiles of survivors compared with controls from clone A+, F and H. GO and KEGG analysis suggested that a persistent immune response might sustain in resistant clone H and F, while susceptible clone A+ had a long-term impact on the circulatory system which was consistent with the major symptoms of bleeding caused by CaHV. Among the top 30 enriched pathways of specifically up-regulated DEUs in respective clones, 26, 7 and 15 pathways in clone H, F and A+ were associated with infections, diseases, or immune-related pathways respectively. In addition, 20 pathways in clone F belonged to "metabolism" or "biogenesis", and 7 pathways involved in "circulatory system" were enriched in clone A+. Significantly, we revealed the differential expression changes of IFN system genes and immunoglobulin (Ig) genes among the survivors of three clones. Finally, myosins and Igs were identified as co-expression modules which were positively or negatively correlated to CaHV viral loads respectively. CONCLUSIONS: Our results revealed the common and distinct gene expression changes in immune and circulatory system in the survivors of three gibel carp gynogenetic clones with different CaHV resistances. The current study represents a paradigm of differential innate and adaptive immune reactions in teleost, and will be beneficial to the disease-resistance breeding of gibel carp.

RNA-seq analysis of local tissue of Carassius auratus gibelio with pharyngeal myxobolosis: Insights into the pharyngeal mucosal immune response in a fish-parasite dialogue.

The lack of practical control measures for pharyngeal myxobolosis is becoming an important limiting factor for the sustainable development of the gibel carp (Carassius auratus gibelio) culture industry in China. Myxobolus honghuensis has been identified as the causative agent of this pandemic disease, which exclusively infects the pharynx of gibel carp, a potential important mucosal lymphoid-associated tissue (MLAT). Myxozoa generally initiate invasion through the mucosal tissues of fish, where some of them also complete their sporogonial stages. However, the pharynx-associated immune responses of teleost against myxosporeans infection remain unknown. Here, a de novo transcriptome assembly of the pharynx of gibel carp naturally infected with M. honghuensis was performed for the first time, using RNA-seq. Comparative analysis of severely infected and mildly infected pharyngeal tissues (SI group and MI group) from the same fish individuals and control pharyngeal tissues (C group) from the uninfected fish was carried out to investigate the potential mucosal immune function of the fish pharynx, and characterize the panoramic picture of pharynx local mucosal immune responses of gibel carp against the M. honghuensis infection. A total of 242,341 unigenes were obtained and pairwise comparison resulted in 13,009 differentially-expressed genes (DEGs) in the SI/C group comparison, 6014 DEGs in the MI/C group comparison, and 9031 DEGs in the SI/MI group comparison. Comprehensive analysis showed that M. honghuensis infection elicited a significant parasite load-dependent alteration of the expression of numerous innate and adaptive immune-related genes in the local lesion tissue. Innate immune molecules, including mucins, toll-like receptors, C-type lectin, serum amyloid A, cathepsins and complement components were significantly up-regulated in the SI group compared with the C group. Up-regulation of genes involved in apoptosis signaling pathway and the IFN-mediated immune system were found in the SI group, suggesting these two pathways played a crucial role in innate immune response to M. honghuensis infection. Up-regulation of chemokines and chemokine receptors and the induction of the leukocyte trans-endothelial migration pathways in the severely and mildly infected pharynx suggested that many leucocytes were recruited to the local infected sites to mount a strong mucosal immune responses against the myxosporean infection. Up-regulation of CD3D, CD22, CD276, IL4/13A, GATA3, arginase 2, IgM, IgT and pIgR transcripts provided strong evidences for the presence of T/B cells and specific mucosal immune responses at local sites with M. honghuensis infection. Our results firstly demonstrated the mucosal function of the teleost pharynx and provided evidences of intensive local immune defense responses against this mucosa-infecting myxosporean in the gibel carp pharynx. Pharyngeal myxobolosis was shaped by a prevailing anti-inflammatory response pattern during the advanced infection stages. Further understanding of the functional roles of fish immune molecules involved in the initial invasion and/or final sporogony site may facilitate future development of control strategies for this myxobolosis.

Identification, expression profiles and antiviral activities of a type I IFN from gibel carp Carassius auratus gibelio.

Type I interferons, as a class of multipotent cytokines, play a key role in host antiviral immune responses. In this study, a type I IFN coding gene of gibel carp, Carassius auratus gibelio, CagIFNa was cloned and sequenced. The full-length cDNA sequence of CagIFNa consists of 724 nucleotides that encode a predicted protein of 183 amino acids. CagIFNa has two highly conserved cysteine residues in the deduced protein, which is mostly conserved in the fish group I type I IFNs. CagIFNa was identified as a member of the IFNa subgroup of group I type I IFNs by phylogenetic analysis. CagIFNa transcripts were detected in all investigated tissues with higher levels in the liver, intestine, spleen and head kidney of gibel carp. Following injection with Cyprinid herpesvirus 2 (CyHV-2), CagIFNa gene expression was significantly inhibited in the spleen but delayed and then increased in head kidneys. Similarly, while CagIFNa expression was rapidly induced in gibel carp brain (GiCB) cells by poly I:C stimulation and its high induction level was delayed following CyHV-2 infection. CagIFNa overexpression in GiCB cells drastically reduced virus CPE and titer. Furthermore, several genes associated with type I IFN signaling pathway including IRF3, IRF7, IRF9, STAT1, Mx1 and PKR were induced in GiCB cells overexpressing CagIFNa upon CyHV-2 infection. These results show that CagIFNa plays a role in antiviral immune system in gibel carp.

Three forms of cocaine- and amphetamine-regulated transcript may be involved in food intake regulation in gibel carp (Carassius auratus gibelio).

In fish, as in mammals, several studies have demonstrated that the cocaine- and amphetamine-regulated transcript (CART) plays an important role in feeding. However, thus far, the function of CART in gibel carp (Carassius auratus gibelio) feeding regulation has not been reported. In our study, we first identified three forms of CART peptide precursors from gibel carp brain and named these CART-1, CART-2, and CART-3. The full-length cDNA sequences of CART-1, CART-2, and CART-3 were 616 bp, 705 bp, and 760 bp, respectively, encoding peptides of 118, 120, and 104 amino acid residues. We detected mRNA expression of CART-1, CART-2, and CART-3 in a wide range of peripheral and central tissues, with the highest expression detected in the brain. After a meal, mRNA expression of CART-1, CART-2, and CART-3 was significantly elevated, suggesting that CART-1, CART-2, and CART-3 may act as postprandial satiety signals. Moreover, mRNA expression of all three CART-1, CART-2, and CART-3 was significantly reduced during fasting and significantly elevated with refeeding. Our findings indicate that CART-1, CART-2, and CART-3 might function as a satiety factor in the gibel carp.