Siniperca Chuatsi Research Articles

Identification of hub genes in digestive system of mandarin fish (Siniperca chuatsi) fed with artificial diet by weighted gene co-expression network analysis

Mandarin fish (Siniperca chuatsi) is a carnivorous freshwater fish and an economically important species. The digestive system (liver, stomach, intestine, pyloric caecum, esophagus, and gallbladder) is an important site for studying fish domestication. In our previous study, we found that mandarin fish undergoes adaptive changes in histological morphology and gene expression levels of the digestive system when subjected to artificial diet domestication. However, we are not clear which hub genes are highly associated with domestication. In this study, we performed WGCNA on the transcriptomes of 17 tissues and 9 developmental stages and combined differentially expressed genes analysis in the digestive system to identify the hub genes that may play important functions in the adaptation of mandarin fish to bait conversion. A total of 31,657 genes in 26 samples were classified into 23 color modules via WGCNA. The modules midnightblue, darkred, lightyellow, and darkgreen highly associated with the liver, stomach, esophagus, and gallbladder were extracted, respectively. Tan module was highly related to both intestine and pyloric caecum. The hub genes in liver were cp, vtgc, c1in, c9, lect2, and klkb1. The hub genes in stomach were ghrl, atp4a, gjb3, muc5ac, duox2, and chia2. The hub genes in esophagus were mybpc1, myl2, and tpm3. The hub genes in gallbladder were dyst, npy2r, slc13a1, and slc39a4. The hub genes in the intestine and pyloric caecum were slc15a1, cdhr5, btn3a1, anpep, slc34a2, cdhr2, and ace2. Through pathway analysis, modules highly related to the digestive system were mainly enriched in digestion and absorption, metabolism, and immune-related pathways. After domestication, the hub genes vtgc and lect2 were significantly upregulated in the liver. Chia2 was significantly downregulated in the stomach. Slc15a1, anpep, and slc34a2 were significantly upregulated in the intestine. This study identified the hub genes that may play an important role in the adaptation of the digestive system to artificial diet, which provided novel evidence and ideas for further research on the domestication of mandarin fish from molecular level.

Characterization of myosin heavy chain (MYH) genes and their differential expression in white and red muscles of Chinese perch, Siniperca chuatsi

Fish skeletal muscle is composed of two anatomically and functionally different fiber layers, white or fast and red or slow muscles. Myosin, the major structural protein of fish skeletal muscle, contains multiple myosin heavy chain (MYH) isoforms involved in the high plasticity of muscle in response to varying functional demands and/or environmental changes. In this study, we comparatively assayed the cellular and ultrastructural feature of white and red skeletal muscles. Then, a total of 28 class II myosin heavy chain genes were identified in by searching the Chinese perch genome database. Among them, 14 genes code for the fast-muscle-type myosin heavy chain, and 7 genes code for the slow-muscle-type myosin heavy chain. Further, the different isoform gene structures, function domains, phylogenetic relations, and muscle-fiber type-specific expression were characterized. This is the first systematic work on the molecular characterization of class II myosin heavy chain isoforms and the differential analysis of their expression in red and white muscle tissues in Chinese perch Siniperca chuatsi. Our work provided valuable information for a better understanding of myh genes and their molecular characteristics, and the correlations of multiple myosin isoforms with potential functions in response to varying functional demands and/or environmental changes.

Effects of flow velocity on the growth performance, antioxidant activity, immunity and intestinal health of Chinese Perch (Siniperca chuatsi) in recirculating aquaculture systems.

The cultivation of Chinese Perch (Siniperca chuatsi) in recirculating aquaculture systems (RASs) has become a common trend. To explore the effect of flow velocity on the growth performance, antioxidant activity, immunity and intestinal health of Chinese Perch in RAS, 240 Chinese Perch with an initial weight of 70.66±0.34g were selected and randomly divided into 4 groups: control group [CK, 0 body length per second (bl/s)], low flow velocity (LF, 0.4 bl/s), middle flow velocity (MF, 0.8 bl/s) and high flow velocity (HF, 1.2 bl/s) for a 56-days experiment. The results showed that the flow velocity significantly increased the weight gain rate and feed intake in Chinese Perch. At 1.2 bl/s, the flow velocity increased the intestinal trypsin content and intestinal villus length. Furthermore, the relative expression of appetite-related genes showed a tendency to increase, and the relative expression of appetite-inhibiting genes had a significant decrease in HF. Regarding immune-related indicators, the activities of alanine aminotransferase (ALT) and aspartate transaminase (AST) were significantly higher in MF and HF. However, the activities of lysozyme (LZM) significantly decreased. Moreover, the activities of total superoxide dismutase (T-SOD) and catalase (CAT) were significantly higher in the CK group than in the other groups. Excessive flow velocity also caused the mRNA level of most immune-relevant genes to markedly decrease. With regard to intestinal health, the intestinal content sequencing results showed that MF could increase the intestinal diversity index of Chinese Perch. In addition, with increasing flow velocity, the relative abundance of Proteobacteria gradually increased, while the proportion of Firmicutes decreased. In conclusion, although the high flow velocity could promote growth, feeding, and digestion, inhibit fat deposition and increase the intestinal microbial abundance, the flow velocity caused stress, which leads to a decline in immunity and increases the death rate and the risk of intestinal disease in Chinese Perch. These findings provide theoretical support for the development of RASs for Chinese Perch.

Interaction of Teleost Fish TRPV4 with DEAD Box RNA Helicase 1 Regulates Iridovirus Replication.

Viral diseases are a significant risk to the aquaculture industry. Transient receptor potential vanilloid 4 (TRPV4) has been reported to be involved in regulating viral activity in mammals, but its regulatory effect on viruses in teleost fish remains unknown. Here, the role of the TRPV4-DEAD box RNA helicase 1 (DDX1) axis in viral infection was investigated in mandarin fish (Siniperca chuatsi). Our results showed that TRPV4 activation mediates Ca2+ influx and facilitates infectious spleen and kidney necrosis virus (ISKNV) replication, whereas this promotion was nearly eliminated by an M709D mutation in TRPV4, a channel Ca2+ permeability mutant. The concentration of cellular Ca2+ increased during ISKNV infection, and Ca2+ was critical for viral replication. TRPV4 interacted with DDX1, and the interaction was mediated primarily by the N-terminal domain (NTD) of TRPV4 and the C-terminal domain (CTD) of DDX1. This interaction was attenuated by TRPV4 activation, thereby enhancing ISKNV replication. DDX1 could bind to viral mRNAs and facilitate ISKNV replication, which required the ATPase/helicase activity of DDX1. Furthermore, the TRPV4-DDX1 axis was verified to regulate herpes simplex virus 1 replication in mammalian cells. These results suggested that the TRPV4-DDX1 axis plays an important role in viral replication. Our work provides a novel molecular mechanism for host involvement in viral regulation, which would be of benefit for new insights into the prevention and control of aquaculture diseases. IMPORTANCE In 2020, global aquaculture production reached a record of 122.6 million tons, with a total value of $281.5 billion. Meanwhile, frequent outbreaks of viral diseases have occurred in aquaculture, and about 10% of farmed aquatic animal production has been lost to infectious diseases, resulting in more than $10 billion in economic losses every year. Therefore, an understanding of the potential molecular mechanism of how aquatic organisms respond to and regulate viral replication is of great significance. Our study suggested that TRPV4 enables Ca2+ influx and interactions with DDX1 to collectively promote ISKNV replication, providing novel insights into the roles of the TRPV4-DDX1 axis in regulating the proviral effect of DDX1. This advances our understanding of viral disease outbreaks and would be of benefit for studies on preventing aquatic viral diseases.

A Safe and Efficient Double-Gene-Deleted Live Attenuated Immersion Vaccine to Prevent the Disease Caused by the Infectious Spleen and Kidney Necrosis Virus.

Infectious diseases seriously threaten sustainable aquaculture development, resulting in more than $10 billion in economic losses annually. Immersion vaccines are emerging as the key technology for aquatic disease prevention and control. Here, a safe and efficacious candidate immersion vaccine strain (Δorf103r/tk) of infectious spleen and kidney necrosis virus (ISKNV), in which the orf103r and tk genes were knocked out by homologous recombination, is described. Δorf103r/tk was severely attenuated in mandarin fish (Siniperca chuatsi), inducing mild histological lesions, a mortality rate of only 3%, and eliminated within 21 days. A single Δorf103r/tk immersion-administered dose provided long-lasting protection rates over 95% against lethal ISKNV challenge. Δorf103r/tk also robustly stimulated the innate and adaptive immune responses. For example, interferon expression was significantly upregulated, and the production of specific neutralizing antibodies against ISKNV was markedly induced postimmunization. This work provides proof-of-principle evidence for orf103r- and tk-deficient ISKNV for immersion vaccine development to prevent ISKNV disease in aquaculture production. IMPORTANCE Global aquaculture production reached a record of 122.6 million tons in 2020, with a total value of 281.5 billion U.S. dollars (USD). However, approximately 10% of farmed aquatic animal production is lost due to various infectious diseases, resulting in more than 10 billion USD of economic waste every year. Therefore, the development of vaccines to prevent and control aquatic infectious diseases is of great significance. Infectious spleen and kidney necrosis virus (ISKNV) infection occurs in more than 50 species of freshwater and marine fish and has caused great economic losses to the mandarin fish farming industry in China during the past few decades. Thus, it is listed as a certifiable disease by the World Organization for Animal Health (OIE). Herein, a safe and efficient double-gene-deleted live attenuated immersion vaccine against ISKNV was developed, providing an example for the development of aquatic gene-deleted live attenuated immersion vaccine.

Nanopeptide C-I20 as a novel feed additive effectively alleviates detrimental impacts of soybean meal on mandarin fish by improving the intestinal mucosal barrier.

Antibacterial peptide has been widely developed in cultivation industry as feed additives. However, its functions in reducing the detrimental impacts of soybean meal (SM) remain unknown. In this study, we prepared nano antibacterial peptide CMCS-gcIFN-20H (C-I20) with excellent sustained-release and anti-enzymolysis, and fed mandarin fish (Siniperca chuatsi) with a SM diet supplemented with different levels of C-I20 (320, 160, 80, 40, 0 mg/Kg) for 10 weeks. 160 mg/Kg C-I20 treatment significantly improved the final body weight, weight gain rate and crude protein content of mandarin fish and reduced feed conversion ratio. 160 mg/Kg C-I20-fed fish maintained appropriate goblet cells number and mucin thickness, as well as improved villus length, intestinal cross-sectional area. Based on these advantageous physiological changes, 160 mg/Kg C-I20 treatment effectively reduced multi-type tissue (liver, trunk kidney, head kidney and spleen) injury. The addition of C-I20 did not change the muscle composition and muscle amino acids composition. Interestingly, dietary 160 mg/Kg C-I20 supplementation prevented the reduction in myofiber diameter and change in muscle texture, and effectively increased polyunsaturated fatty acids (especially DHA + EPA) in muscle. In conclusion, dietary C-I20 in a reasonable concentration supplementation effectively alleviates the negative effects of SM by improving the intestinal mucosal barrier. The application of nanopeptide C-I20 is a prospectively novel strategy for promoting aquaculture development.

Effects and regulatory pathway of proopinmelanocortin on feeding habit domestication in mandarin fish

Proopiomelanocortin (POMC) is a hormone precursor, and has been reported to participate in domestication. However, its effects on feeding habit domestication in fish are poorly understood. Mandarin fish (Siniperca chuatsi) feeds solely on live prey fish since first-feeding. In the present study, the high expression of pomc in mandarin fish, both the pomc siRNA and MC4R inhibitor treatments increased the success rate of domestication from live prey fish to dead prey fish and food intake of dead prey fish, suggesting the role of pomc on the special feeding habit of live prey fish in mandarin fish. In addition, one c-fos binding site was identified in the region that from −1053 bp to −931 bp upstream of the transcription start site of pomc, and this region exhibited positive promoter activity. The mandarin fish brain cells treated with c-fos siRNA displayed suppressed pomc mRNA expression, indicating that c-fos positively regulated pomc expression. Furthermore, the mRNA expression of c-fos was higher in the mandarin fish which were more difficult to domesticate. The results of ChIP assay and inhibitor treatment confirmed that the activation of c-fos gene by histone H3K4me3 was catalyzed by Setd1b in mandarin fish. Three open peaks were found at the upstream regulatory region of setd1b by ATAC-seq, and the mRNA expression of setd1b was higher in the mandarin fish which were more difficult to domesticate. These results indicated that Setd1b could methylate histone H3K4 to activate the c-fos transcription, maintaining the high expression of pomc, which might contribute to the special feeding habit of mandarin fish.

Isolation, identification, and drug resistance of a partially isolated bacterium from the gill of Siniperca chuatsi.

This study was envisaged to identify a strain of bacteria isolated from the gill of mandarin fish. Identification and characterization of the bacterial strain were performed using morphological characteristics, growth temperature, physiological and biochemical tests, antibiotic sensitivity tests, artificial infection tests, and 16S rRNA gene sequencing homology analysis. The results showed that the bacterium was Gram-negative, with flagella at the end and the side. The bacterium exhibited a light brownish-gray colony on the Luria-Bertani culture and white colony on the blood agar plate without hemolytic ring. Normal growth was achieved at 42°C, and growth could be delayed in 7% NaCl broth medium. By homology comparison and analysis, the phylogenetic tree was constructed using MEGA7.0, and the bacterium was preliminarily identified as Achromobacter. The antibiotic sensitivity test showed that the strain was sensitive to piperacillin, carbenicillin, cefoperazone, cefazolin, ofloxacin, gentamicin, kanamycin, amikacin, neomycin, erythromycin, minocycline, doxycycline, polymyxin B, tetracycline, chloramphenicol, and other drugs. However, it was resistant to penicillin, ampicillin, oxacillin, ceftriaxone, cefradine, cefalexin, cefuroxime sodium, ciprofloxacin, norfloxacin, vancomycin, compound sulfamethoxazole, clindamycin, medimycin, and furazolidone.

Dietary host-associated Bacillus subtilis supplementation improves intestinal microbiota, health and disease resistance in Chinese perch (Sinipercachuatsi).

The purpose of this study was to evaluate the potential of a host-associated Bacillus subtilis 1-C-7 as a probiotic for Chinese perch (Siniperca chuatsi). Four test diets were formulated to contain graded levels of B.subtilis 1-C-7 at 0 (CY), 0.85×108 (Y1), 0.95×109 (Y2) and 0.91×1010 (Y3) CFU/kg diet. The test fish with initial weight 30.0±1.2g were fed the 4 test diets with 3 replicates in an indoor water-flow aquaculture system with 12 net cages (40 fish/cage) for 10wk. At the conclusion of the feeding trial, the probiotic effects of B.subtilis on Chinese perch were analyzed based on growth performance, serum biochemical indices, histologic morphology of liver and gut, gut microbiota and the resistance to Aeromonas hydrophila. The results showed that the percentage of weight gain had no significant change in the Y1 and Y2 groups (P>0.05) but decreased in the Y3 group compared to that in the CY group (P<0.05). The fish in the Y3 group displayed the highest activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) among these 4 groups (P<0.05). The fish in the CY group had the highest value of malondialdehyde in the liver (P<0.05) and showed severe nuclear migration and vacuolization of hepatocytes. The morphology indicated that all test fish had poor intestinal health. However, the fish in the Y1 group had a relatively normal intestinal histologic structure. The mid gut microbial diversity analysis showed that dietary B.subtilis supplementation increased the abundance of probiotics such as Tenericutes and Bacteroides, whereas it reduced the abundance of pernicious bacteria such as Proteobacteria, Actinobacteria, Thermophilia and Spirochaetes. The challenge test showed that dietary B.subtilis supplementation increased the resistance to A.hydrophila in Chinese perch. In conclusion, dietary supplementation of 0.85×108CFU/kg B.subtilis 1-C-7 could improve the intestinal microbiota, intestinal health and disease resistance in Chinese perch, but more or excessive supplementation could reduce growth performance and have negative effects on health.

Analysis of Lipid Molecule Profiling and Conversion Pathway in Mandarin Fish (Siniperca chuatsi) during Fermentation via Untargeted Lipidomics

Lipids are the key aroma contributors and nutrients in fermented fish products. A total of 376 lipid molecules were identified in mandarin fish during fermentation by untargeted lipidomics, including glycerolipids, glycerophospholipids, lysoglycerophospholipid, sphingolipids, fatty acids (FAs), and sterol lipids. Both lipid composition and content changed dynamically during fermentation. Triglyceride (TAG, 30.05%) and phosphatidylcholine (PC, 14.87%) were the two major lipids, with especially 39.36% saturated FAs in PCs and 35.34% polyunsaturated FAs in TAGs. The content of TAGs and PCs reached a peak point at 0 and 6 days, respectively. Fermented mandarin fish expressed a high nutritional value, and the ratio of total linoleic acid/total linolenic acid was about 5:1. Glycerophospholipid metabolism was a potential metabolic pathway, and the oxidation of derived FAs contributed to flavor. These data progress in understanding lipid dynamic variation during fermentation and provide thoughts on controlling the flavor quality and safety of fermented fish products.

The Inactivated ISKNV-I Vaccine Confers Highly Effective Cross-Protection against Epidemic RSIV-I and RSIV-II from Cultured Spotted Sea Bass Lateolabrax maculatus.

The genus Megalocytivirus of the family Iridoviridae is composed of two distinct species, namely, infectious spleen and kidney necrosis virus (ISKNV) and scale drop disease virus (SDDV), and both are important causative agents in a variety of bony fish worldwide. Of them, the ISKNV species is subdivided into three genotypes, namely, red seabream iridovirus (RSIV), ISKNV, and turbot reddish body iridovirus (TRBIV), and a further six subgenotypes, RSIV-I, RSIV-II, ISKNV-I, ISKNV-II, TRBIV-I, and TRBIV-II. Commercial vaccines derived from RSIV-I , RSIV-II and ISKNV-I have been available to several fish species. However, studies regarding the cross-protection effect among different genotype or subgenotype isolates have not been fully elucidated. In this study, RSIV-I and RSIV-II were demonstrated as the causative agents in cultured spotted seabass, Lateolabrax maculatus, through serial robust evidence, including cell culture-based viral isolation, whole-genome determination and phylogeny analysis, artificial challenge, histopathology, immunohistochemistry, and immunofluorescence as well as transmission electron microscope observation. Thereafter, a formalin-killed cell (FKC) vaccine generated from an ISKNV-I isolate was prepared to evaluate the protective effects against two spotted seabass original RSIV-I and RSIV-II. The result showed that the ISKNV-I-based FKC vaccine conferred almost complete cross-protection against RSIV-I and RSIV-II as well as ISKNV-I itself. No serotype difference was observed among RSIV-I, RSIV-II, and ISKNV-I. Additionally, the mandarin fish Siniperca chuatsi is proposed as an ideal infection and vaccination fish species for the study of various megalocytiviral isolates. IMPORTANCE Red seabream iridovirus (RSIV) infects a wide mariculture bony fish and has resulted in significant annual economic loss worldwide. Previous studies showed that the phenotypic diversity of infectious RSIV isolates would lead to different virulence characteristics, viral antigenicity, and vaccine efficacy as well as host range. Importantly, it is still doubted whether a universal vaccine could confer the same highly protective effect against various genotypic isolates. Our study here presented enough experimental evidence that a water in oil (w/o) formation of inactivated ISKNV-I vaccine could confer almost complete protection against RSIV-I and RSIV-II as well as ISKNV-I itself. Our study provides valuable data for better understanding the differential infection and immunity among different genotypes of ISKNV and RSIV isolates in the genus Megalocytivirus.

Genome Characteristics of Two Ranavirus Isolates from Mandarin Fish and Largemouth Bass.

Ranaviruses are promiscuous pathogens that threaten lower vertebrates globally. In the present study, two ranaviruses (SCRaV and MSRaV) were isolated from two fishes of the order Perciformes: mandarin fish (Siniperca chuatsi) and largemouth bass (Micropterus salmoides). The two ranaviruses both induced cytopathic effects in cultured cells from fish and amphibians and have the typical morphologic characteristics of ranaviruses. Complete genomes of the two ranaviruses were then sequenced and analyzed. Genomes of SCRaV and MSRaV have a length of 99, 405, and 99, 171 bp, respectively, and both contain 105 predicted open reading frames (ORFs). Eleven of the predicted proteins have differences between SCRaV and MSRaV, in which only one (79L) possessed a relatively large difference. A comparison of the sequenced six ranaviruses from the two fish species worldwide revealed that sequence identities of the six proteins (11R, 19R, 34L, 68L, 77L, and 103R) were related to the place where the virus was isolated. However, there were obvious differences in protein sequence identities between the two viruses and iridoviruses from other hosts, with more than half lower than 55%. Especially, 12 proteins of the two isolates had no homologs in viruses from other hosts. Phylogenetic analysis revealed that ranaviruses from the two fishes clustered in one clade. Further genome alignment showed five groups of genome arrangements of ranaviruses based on the locally collinear blocks, in which the ranaviruses, including SCRaV and MSRaV, constitute the fifth group. These results provide new information on the ranaviruses infecting fishes of Perciformes and also are useful for further research of functional genomics of the type of ranaviruses.

Infection tracing and organ tropism of Siniperca chuatsi rhabdovirus expressing enhanced green fluorescent protein

Siniperca chuatsi Rhabdovirus (SCRV) is one of the important pathogens that infect mandarin fish. Recovery of recombinant viruses expressing fluorescent protein is an important method for virology research. In this study, the enhanced green fluorescent protein (EGFP) gene was inserted between the leader and the N gene of the SCRV genome. The recombinant SCRV of rS3-LeNEGFP expressing the EGFP was rescued, and the virus titer was as high as 2 × 106 PFU/mL in EPC cells. The insertion of the EGFP gene in the genome of SCRV was stable. EGFP was expressed efficiently after 10 passages in EPC cells. In vitro growth characterization, pathogenicity and organ tropism in vivo were studied. Compared to the parental rS3, rS3-LeNEGFP was defective in vitro, but not attenuated in vivo. The mortality of red zebrafish caused by rS3-LeNEGFP was the same as the parental SCRV rS3. The earliest expression of EGFP was detectable at 2 h, and clear fluorescence spots appeared at 8 h post-infection in EPC cells. The liver, heart, and brain were the major organ targets of rS3-LeNEGFP in vivo, and the kidney, gill, and spermatocyte were also invaded. The organ infection in vitro was different from that in vivo. This study is of great significance for revealing the pathogenesis of SCRV and developing vaccines and antiviral drugs for SCRV in the future.

The feasibility of Chinese mitten crab (Eriocheir sinensis), mandarin fish (Siniperca chuatsi), and mud carp (Cirrhinus molitorella) polyculture and their effects on growth performance, water quality, antioxidant, and nonspecific immunological capacity

The feasibility of Chinese mitten crab (Eriocheir sinensis), mandarin fish (Siniperca chuatsi), and mud carp (Cirrhinus molitorella) polyculture and their effects on growth performance, water quality, antioxidant, and nonspecific immunological capacity

Transcriptomic responses of the liver of mandarin fish (Siniperca chuatsi) under hypoxic stress.

Environmental hypoxia is becoming more prevalent in aquatic environments of mandarin fish (Siniperca chuatsi) aquaculture because of eutrophication and climate change. Little information is available on the molecular mechanisms of the detrimental effects of hypoxia in this species. In this study, the authors assembled a transcriptome for mandarin fish exposed to lower oxygen conditions at different times (24 and 96 h). The antioxidant enzymatic activities of catalase, glutathione, superoxide dismutase, glutamic pyruvic transaminase and malondialdehyde significantly increased at 6 or 12 h but decreased after reaching a climax during 96 h hypoxia stress. The gene ontology study revealed 27,616 transcripts, whereas the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed 25 linked pathways. Significant changes in the expression of certain genes involved in protein processing in the endoplasmic reticulum, the calcium signalling system and inositol phosphate metabolism were discovered using the KEGG pathway analysis. In the liver, 97 genes were differentially expressed between the control and experimental groups. The expression level of 28 differentially expressed genes (DEGs) under different hypoxic stress conditions was detected using real-time PCR and compared to transcriptome sequencing results. The result showed that some genes in the experimental group associated with hypoxic stress, such as hif, ho-1a, ho-1b, igfbp1, hsp90α and hsp90 β, were significantly upregulated compared with those in the control group. The large amount of transcriptome data from this research has enlarged the mandarin fish gene and genome bioinformation. The identified DEGs and pathways are useful in further studies of biological responses to hypoxia.

METTL3-Mediated m6A Modification of TRIF and MyD88 mRNAs Suppresses Innate Immunity in Teleost Fish, Miichthys miiuy.

Methyltransferase (METTL3), the most important N6-methyladenosine (m6A) writer, plays a vital role in regulating immune-related signaling pathways. However, the underlying mechanism of METTL3 action remains largely unknown, especially in lower vertebrates. The results of this study show that METTL3 inhibits innate immune response and promotes the infection of miiuy croaker, Miichthys miiuy, by Siniperca chuatsi rhabdovirus and Vibrio anguillarum. Significantly, the function of METTL3 in inhibiting immunity depends on its methylase activity. Mechanistically, METTL3 increases the methylation level of trif and myd88 mRNA, rendering them sensitive to degradation by the YTHDF2/3 reader proteins. By contrast, we found that the YTHDF1 reader protein promotes the translation of myd88 mRNA. In summary, these results indicate that METTL3-mediated m6A modification of trif and myd88 mRNAs suppresses innate immunity by inhibiting the TLR pathway, unveiling a molecular mechanism by which RNA methylation controls innate immunity to pathogens in the teleost fish.

Molecular Characterization and Functional Analysis of Hypoxia-Responsive Factor Prolyl Hydroxylase Domain 2 in Mandarin Fish (Siniperca chuatsi).

With increased breeding density, the phenomenon of hypoxia gradually increases in aquaculture. Hypoxia is primarily mediated by the hypoxia-inducible factor 1 (HIF-1) signaling pathway. Prolyl hydroxylase domain proteins (PHD) are cellular oxygen-sensing molecules that regulate the stability of HIF-1α through hydroxylation. In this study, the characterization of the PHD2 from mandarin fish Siniperca chuatsi (scPHD2) and its roles in the HIF-1 signaling pathway were investigated. Bioinformation analysis showed that scPHD2 had the conserved prolyl 4-hydroxylase alpha subunit homolog domains at its C-terminal and was more closely related to other Perciformes PHD2 than other PHD2. Tissue-distribution results revealed that scphd2 gene was expressed in all tissues tested and more highly expressed in blood and liver than in other tested tissues. Dual-luciferase reporter gene and RT-qPCR assays showed that scPHD2 overexpression could significantly inhibit the HIF-1 signaling pathway. Co-immunoprecipitation analysis showed that scPHD2 could interact with scHIF-1α. Protein degradation experiment results suggested that scPHD2 could promote scHIF-1α degradation through the proteasome degradation pathway. This study advances our understanding of how the HIF-1 signaling pathway is regulated by scPHD2 and will help in understanding the molecular mechanisms underlying hypoxia adaptation in teleost fish.

Introduction of Artemia nauplii during Chinese perch (Siniperca chuatsi) first feeding: Effects on larvae growth, survival, gene expression and activity of critical digestive enzymes

This study investigates the effects of first feeding regime on growing, survival, and gene expression (appetite, digestion, absorption, and growth) and digestive enzymes activity in Chinese perch (Siniperca chuatsi) larvae. The fish larvae at 3 days post-hatch (DPH), were fed with prey fry (newly hatched zebrafish Danio rerio)/Artemia naupli from 3 to 15 DPH for different time periods as follows: (A)only prey fry;(B) 3–5 DPH Artemia/6–15 DPH prey fry;(C) 3–7 DPH Artemia/8–15 DPH prey fry; (D) a combination of Artemia and prey fry. Final performance parameters (total length, body weight, survival rate), gene expression and digestive enzymes were measured and compared among treatments at 15 DPH. The groups fed only prey fry (group A) (53.67 ± 1.76%) and combination of Artemia and prey fry (group D) (61.22 ± 1.18%), respectively, showed significantly (P < 0.05) higher survival specific growth rate (SGRTL) than other groups. Brain expression of npy and agrp were not significantly affected under different feeding regimes. The groups fed with Artemia till 7 DPH followed by feeding with prey fry till 15 DPH (group C) showed significantly (P < 0.05) higher pomc and cck expression level than the other groups. The expression of amy, ctrb, and pl was significantly higher in larvae fed combination of Artemia and prey fry (group D). pep had a high expression level in both group A and D. The highest expression of two genes related to intestinal development and maturation (alp, lap3, anpep) were observed in the fish fed a combination of Artemia and prey fry, but there was no significant difference compared to group A. The expression of absorption and growth (mtor, pept1 and igf-1) increased with combination of Artemia and prey fry in larval diet (group D), indicating a more efficient functionality of digestive structures. In conclusion, feeding with combination of Artemia and prey fry could be suggested as an economical feeding regime for first feeding of Chinese perch larvae as comparable growth to prey fry were achieved, and better survival.

Molecular cloning and functional analysis of polymeric immunoglobulin receptor, pIgR, gene in mandarin fish Siniperca chuatsi

Polymeric immunoglobulin receptor (pIgR) can bind and transport immunoglobulins (Igs), thus playing a role in mucosal immunity. In this study, pIgR gene was cloned in mandarin fish, Siniperca chuatsi, with the open reading frame (ORF) of 1011 bp, encoding 336 amino acids. The pIgR protein consists of a signal peptide, an extracellular domain, a transmembrane domain and an intracellular region, with the presence of two Ig-like domains (ILDs) in the extracellular domain, as reported in other species of fish. The pIgR gene was expressed in all organs/tissues of healthy mandarin fish, with higher level observed in liver and spleen. Following the immersion infection of Flavobacterium columnare, pIgR transcripts were detected in immune related, especially mucosal tissues, with significantly increased transcription during the first two days of infection. Through transfection of plasmids expressing pIgR, IgT and IgM, pIgR was found to be interacted with IgT and IgM as revealed by co-immunoprecipitation and immunofluorescence.

Interaction of Nmi and IFP35 Promotes Mutual Protein Stabilization and IRF3 and IRF7 Degradation to Suppress Type I IFN Production in Teleost Fish.

IFN-stimulated genes (ISGs) can act as effector molecules against viral infection and can also regulate pathogenic infection and host immune response. N-Myc and STAT interactor (Nmi) is reported as an ISG in mammals and in fish. In this study, the expression of Nmi was found to be induced significantly by the infection of Siniperca chuatsi rhabdovirus (SCRV), and the induced expression of type I IFNs after SCRV infection was reduced following Nmi overexpression. It is observed that Nmi can interact with IRF3 and IRF7 and promote the autophagy-mediated degradation of these two transcription factors. Furthermore, Nmi was found to be interactive with IFP35 through the CC region to inhibit IFP35 protein degradation, thereby enhancing the negative role in type I IFN expression after viral infection. In turn, IFP35 is also capable of protecting Nmi protein from degradation through its N-terminal domain. It is considered that Nmi and IFP35 in fish can also interact with each other in regulating negatively the expression of type I IFNs, but thus in enhancing the replication of SCRV.