IRF9 inhibits CyHV-3 replication by regulating the PI3K-AKT signalling pathway in common carp (Cyprinus carpio) epithelial cells.

Interferon regulatory factor 9 (IRF9) is an important transcriptional regulator involved in innate and adaptive immunity. Cyprinid herpesvirus-3 (CyHV-3) is a virus causing widespread death and great economic loss in farmed common carp (Cyprinus carpio). However, the effect of IRF9 on CyHV-3 infection in common carp has not been reported. In this study, during CyHV-3 infection, IRF9 overexpression in common carp fin epithelial (CCF) cells significantly reduced the expression of viral factor thymidine kinase (TK) and open reading frame 72 (ORF72), and knockdown of IRF9 produced the opposite results (p < 0.05). In CCF cells. The IRF9 protein was expression in the nucleus and was rapidly induced in CCF cells by CyHV-3 infection. In addition, several genes associated with virus infection, including type I interferon (IFNI), IFN-stimulated gene 15 (ISG15), myxovirus resistance 1 (Mx1) and Viperin were induced in CCF cells overexpressing IRF9 upon CyHV-3 infection. IRF9 overexpression induced by CyHV-3 infection significantly increased the gene expression of Mx1 and phosphoinositide 3-kinase (PI3K) and the protein expression of protein kinase B (AKT) (p < 0.01). Interestingly, IRF9 did not significantly affect Mx1 gene expression when AKT protein levels remained unchanged during CyHV-3 infection of CCF cells. Furthermore, a significant resistance-related locus was found in the IRF9 sequence in "Longke-11" mirror carp (M11) and Yellow River carp (p < 0.05). These results indicated that IRF9 inhibited viral replication by upregulating the expression of Mx1 via the PI3K-AKT signalling pathway during CyHV-3 infection in CCF cells and provide some basis for the study of the antiviral molecular mechanisms of common carp.

Effects of Different Dietary Protein Levels on the Growth Performance, Physicochemical Indexes, Quality, and Molecular Expression of Yellow River Carp (Cyprinus carpio haematopterus).

A 12-week rearing trial was carried out to estimate effects on the growth performance, physicochemical indexes, quality, and the molecular expression of Yellow River Carp (Cyprinus carpio haematopterus) using five practical diets, including dietary protein levels of 220, 250, 280, 310, and 340 g/kg. The results illustrated that the fish's weight gain (WG) and specific growth rate (SGR) were significantly influenced, with an ascending dietary protein level of up to 250 g/kg (p < 0.05). The carp muscle contents of total saturated fatty acids (∑SFA), monounsaturated fatty acids (∑MUFA), polyunsaturated fatty acids (∑PUFA), and fatty acids (∑FA) decreased significantly with the ascending dietary protein levels, except for the 250 g/kg protein diet (p < 0.05). Only the glutamic acid and total essential amino acid (∑EAA) contents were significantly influenced by the ascending dietary protein levels (p < 0.05). The relative GH expression of the carp muscle significantly decreased with the increase in the dietary protein level up to 310 g/kg, and then it significantly increased (p < 0.05). In the intestines, the peak relative TOR expression was observed on the 220 g/kg protein diet, while the relative 4EBP1 expression was significantly influenced by the dietary protein level up to 250 g/kg (p < 0.05). In the muscle, the peak relative TOR and 4EBP1 expression levels were observed on the 250 g/kg protein diet. In gills, the lowest relative Rhag, Rhbg, and Rhcg1 expression levels were observed on the 250 g/kg protein diet. Based on all of the aforementioned results, the optimal dietary protein level for Cyprinus carpio haematopterus (160.24 ± 15.56 g) is 250-280 g/kg.

Integrative Transcriptomic Analysis Reveals the Immune Mechanism for a CyHV-3-Resistant Common Carp Strain.

Anti-disease breeding is becoming the most promising solution to cyprinid herpesvirus-3 (CyHV-3) infection, the major threat to common carp aquaculture. Virus challenging studies suggested that a breeding strain of common carp developed resistance to CyHV-3 infection. This study illustrates the immune mechanisms involved in both sensitivity and anti-virus ability for CyHV3 infection in fish. An integrative analysis of the protein-coding genes and long non-coding RNAs (lncRNAs) using transcriptomic data was performed. Tissues from the head kidney of common carp were extracted at days 0 (the healthy control) and 7 after CyHV-3 infection (the survivors) and used to analyze the transcriptome through both Illumina and PacBio sequencing. Following analysis of the GO terms and KEGG pathways involved, the immune-related terms and pathways were merged. To dig out details on the immune aspect, the DEGs were filtered using the current common carp immune gene library. Immune gene categories and their corresponding genes in different comparison groups were revealed. Also, the immunological Gene Ontology terms for lncRNA modulation were retained. The weighted gene co-expression network analysis was used to reveal the regulation of immune genes by lncRNA. The results demonstrated that the breeding carp strain develops a marked resistance to CyHV-3 infection through a specific innate immune mechanism. The featured biological processes were autophagy, phagocytosis, cytotoxicity, and virus blockage by lectins and MUC3. Moreover, the immune-suppressive signals, such as suppression of IL21R on STAT3, PI3K mediated inhibition of inflammation by dopamine upon infection, as well as the inhibition of NLRC3 on STING during a steady state. Possible susceptible factors for CyHV-3, such as ITGB1, TLR18, and CCL4, were also revealed from the non-breeding strain. The results of this study also suggested that Nramp and PAI regulated by LncRNA could facilitate virus infection and proliferation for infected cells respectively, while T cell leukemia homeobox 3 (TLX3), as well as galectin 3 function by lncRNA, may play a role in the resistance mechanism. Therefore, immune factors that are immunogenetically insensitive or susceptible to CyHV-3 infection have been revealed.

Transcriptomic profiling revealed key signaling pathways for cold tolerance and acclimation of two carp species.

BACKGROUND: Closely related species of the carp family (Cyprinidae) have evolved distinctive abilities to survive under cold stress, but molecular mechanisms underlying the generation of cold resistance remain largely unknown. In this study, we compared transcriptomic profiles of two carp species to identify key factors and pathways for cold tolerance and acclimation. RESULTS: Larvae of Songpu mirror carp and Barbless carp that were pretreated at 18 °C for 24 h significantly improved their survival rates under lethal cold temperature at 8 °C or 10 °C, indicating that two carp species possess the ability of cold acclimation. However, Songpu mirror carp exhibited stronger abilities of cold tolerance and acclimation than Barbless carp. Transcriptomic profiles of Songpu mirror carp and Barbless carp larvae at 28 °C and 18 °C were compared during cold acclimation through RNA-seq. Differentially expressed genes that are closely associated with the differences in cold acclimation between two carp species were identified through bioinformatics and Venn's diagram analysis. GO enrichment analysis of these genes indicated that cellular component assembly involved in morphogenesis, secondary alcohol metabolism and drug transport were the most up-regulated biological processes during cold acclimation of Songpu mirror carp. Conversely, positive regulation of macroautophagy, intracellular protein transport, and organonitrogen compound catabolism were the most down-regulated biological processes during cold acclimation of Barbless carp. KEGG enrichment analysis revealed that factors in the FoxO-related signaling pathways are mainly responsible for the development of differences in cold tolerance and acclimation between two carp species since altering the phosphorylation of key proteins in the FoxO-related signaling pathways with inhibitors or an activator significantly decreased the cold tolerance and acclimation of Songpu mirror carp. These data provided key clues for dissection of molecular mechanisms underlying the development of cold tolerance and acclimation in carps. CONCLUSIONS: These findings indicate that larvae of two carp species possess different abilities of cold tolerance and can build cold acclimation under mild low temperature. Multiple biological processes and FoxO-related signaling pathways are closely associated with the development of differences in cold tolerance and acclimation between two carp species.

Characterization of the mitochondrial genome of Megalobrama terminalis in the Heilong River and a clearer phylogeny of the genus Megalobrama.

Megalobrama terminalis distributed in Sino-Russian Heilong-Amur River basin has decreased dramatically in the last few decades. It has been listed in the Red Book of the Russian Federation as an endangered fish species. Here, the complete mitochondrial (mt) genome sequence of M. terminalis in the Heilong River (MTH) was first determined and characterized. Additionally, we identified a population-specific single nucleotide polymorphism (SNP) locus in MTH which could effectively separate MTH from the six other populations of the genus Megalobrama in the absence of hybridization. Moreover, phylogenetic analyses determined that the Xi River M. hoffmanni is located at the basal branch of the clade, and the rest of the group is divided into two assemblages, namely, one containing M. terminalis from Qiantang River and Jinsha River Reservoir/Longxi River M. Pellegrini/MTH and the other containing M. amblycephala from Liangzi Lake and Yi River. We clarify the intraspecies identity of MTH and construct a clearer phylogeny of the genus Megalobrama, which will contribute to the germplasm identification, protection and development of MTH in the future.

Performances of the hybrid between CyCa nucleocytplasmic hybrid fish and scattered mirror carp in different culture environments.

To improve the performance of growth traits and survival in common carp, CyCa nucleocytoplasmic hybrid fish (C) was used as parental fish for hybridization with Russian scattered mirror carp (R). Performances in morphological characters, growth traits and survival rate were compared among the purebreds (CC &RR) and crossbreds (RC &CR) at different time period in solitary and communal rearing system, respectively. The results demonstrated that both RC and CR crossbreds inherited the grey skin color type from the mirror carp, and got the full-scale pattern from the CyCa nucleocytoplasmic hybrid fish, which suggested that the grey color dominated to red color and full-scale dominated to scattered scale. With respect to yield, the RC crossbreds perform quite great compared to the RR and CC purebreds because they have quite high growth and survival rate. In contrast to RC crossbreds, the CR crossbreds performed poorly in growth traits, together with that crosses where scattered mirror carp was used as mother (RC and RR) achieved the greatest performance for all growth traits, suggested that the maternal influence also displayed an important role in growth traits. These results indicated that the RC crossbreds will be a potential carp variety for commercial production.

Mitochondrial genome of Ying hybrid carp (Russian scattered scale mirror carp ♀ × carp-goldfish nucleocytoplasmic hybrid ♂).

The complete mitogenomes of Ying hybrid carp (Russian scattered scale mirror carp ♀ × Carp-goldfish nucleocytoplasmic hybrid ♂) was sequenced by PCR-based method. The total of the mtDNA was 16,581 bp, and consisted of 13 protein-coding genes, 22 tRNAs, 2rRNA genes, and 1 control region (D-loop region). The overall base composition was as follows: A: 31.9%, T: 24.8%, C: 27.5%, and G: 15.8%, respectively. The overall AT and GC skew were found to be 0.125 and -0.271, respectively. A majority of the genes used ATG as the start codon, except that the codon GTG was found to initiated the CO I gene. Most of the genes ended with the complete stop codon TAA or TAG, while the COII, ND3, ND4, and Cytb genes terminated with the incomplete stop codons 'T--'.

Development of MHC class I and II B primers in common carp and its molecular characterization.

The major histocompatibility complex (MHC) has an important role in immune response and is known as the most polymorphic locus in vertebrates. We developed three pairs of polymerase chain reaction primers of the alpha-2 domain (exon 3) of MHC class I and the beta-2 (exon 3) and beta-3 domains (exon 4) of MHC class II B gene in the German mirror common carp (Cyprinus carpio L.). We analyzed the three loci in a population of 65 individuals that had suffered the serious disease of gill rot. Five to six variable nucleotide sites and two to six variable amino acid sites (71.43%) were detected in the exon sequence of the sampled populations, indicating that many of them corresponded to amino acids involved in antigen recognition. Deviation from Hardy-Weinberg equilibrium and linkage disequilibrium were differentially found in some loci, which will be important for further study of disease resistance/susceptibility and population evolution.