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Concept: Mandarin fish

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Abstract The Chinese mandarin fish (Siniperca chuatsi) is currently one of the most important economic freshwater fish in China, whereas the wild resource has declined dramatically in recent years. In this study, we examined the genetic structure and diversity of five populations from the middle reach of the Yangtze River using mitochondrial cytochrome b sequences and microsatellite markers. This research revealed high genetic diversity and low genetic differentiation of S. chuatsi from these regions. The pairwise Fst values of the two markers showed low and no-significant differentiation among populations. AMOVA analysis of two markers and the haplotype genealogy of the Cytb gene confirmed these results. The STRUCTURE analysis of the microsatellite marker implied that the dam upon the tributary of the Yangtze River blocked the gene flow among those regions. This research will be useful in breeding programs and conservation management of this species.

Concepts: DNA, Genetics, Evolution, Biology, China, Population genetics, Shanghai, Mandarin fish

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Mandarin fish refuse dead prey fish or artificial diets and can be trained to transform their inborn feeding habit. To investigate the effect of memory on feeding habit transformation, we compared the reaction time to dead prey fish and the success rate of feeding habit transformation to dead prey fish with training of mandarin fish in the 1st experimental group (trained once) and the 2nd experimental group (trained twice). The mandarin fish in the 2nd group had higher success rate of feeding habit transformation (100%) than those in the 1st group (67%), and shorter reaction time to dead prey fish (<1 s) than those in the 1st group (>1 s). Gene expression of cAMP responsive element binding protein I (Creb I), brain-derived neurotrophic factor (Bdnf), CCAAT enhancer binding protein delta (C/EBPD), fos-related antigen 2 (Fra2), and proto-oncogenes c-fos (c-fos) involved in long-term memory formation were significantly increased in the 2nd group after repeated training, and taste 1 receptor member 1 (T1R1), involved in feeding habit formation, was significantly increased in brains of the 2nd group after repeated training. DNA methylation levels at five candidate CpG (cytosine⁻guanine) sites contained in the predicted CpG island in the 5′-flanking region of T1R1 were significantly decreased in brains of the 2nd group compared with that of the 1st group. These results indicated that the repeated training can improve the feeding habit transformation through the memory formation of accepting dead prey fish. DNA methylation of the T1R1 might be a regulatory factor for feeding habit transformation from live prey fish to dead prey fish in mandarin fish.

Concepts: DNA, Gene, Gene expression, Molecular biology, Memory, DNA methylation, Brain-derived neurotrophic factor, Mandarin fish

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Teleost fish are unique in having type I and type II interferons (IFNs) only, and the type I IFNs are classified into Group one and Group two based on the presence of two or four cysteines respectively, and are further classified into seven subgroups. In the present study, three distinct type I IFNs, IFNc, IFNd and IFNh, have been identified in the genome sequences of a perciform fish, the mandarin fish Siniperca chuatsi. These IFNs are induced following the stimulation of Polyinosinic polycytidylic acid (poly(I:C)) and Resiquimod (R848) either in vivo or in vitro. But, the infectious spleen and kidney necrosis virus (ISKNV) infection caused a delayed response of IFNs, which may be resulted from the viral inhibition of type I IFN production and related signalling. The three receptor subunits, cytokine receptor family B 1 (CRFB1), CRFB2 and CRFB5 are also expressed in a similar manner as observed for the IFNs, and IFNc, IFNd and IFNh use preferentially the receptor complex, CRFB2 and CRFB5, CRFB1 and CRFB5, CRFB1 and CRFB5 respectively for their effective signalling in the induction of IFN-stimulated genes (ISGs). Moreover, the IFNs are able to induce their own expression, and also the IRF3 and IRF7 expression, leading to the amplification of IFN cascade. It is further revealed that these three IFNs are transcribed differently by IRF7 and IRF3. The composition, function, signalling and transcription of type I IFNs have been investigated in detail in a teleost fish.

Concepts: Gene, Gene expression, Transcription, Virus, RNA, Interferon, Mandarin fish, Siniperca

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Infectious spleen and kidney necrosis virus (ISKNV) has been recognized as the causative agent of the most serious disease in cultured mandarin fish, Siniperca chuatsi, in China. Disease outbreaks have resulted in substantial losses to the aquaculture industry. Currently, reliable laboratory detection and identification methods are available for this virus. However, rapid detection methods applicable for on-site diagnosis of this infectious agent are unavailable. To address this need, a nearly instrument-free, cost-effective and simple detection method was developed and optimized and incorporates cross priming amplification coupled with vertical flow visualization for rapid identification of ISKNV (ISKNV-CPA-VF). Results show that cross circulation amplification targeting the conserved region of the major capsid protein (MCP) regiment of the ISKNV genome had a sensitivity 10 times greater than traditional PCR at 64 °C within 60 min. The optimized concentration of dNTPs and the concentration for Mg2+ were 1.0 mmol/L and 10 mmol/L, respectively. No cross-reactions with other viruses or bacteria were observed. When combined with the nucleic acid strip detection technology, visual detection of ISKNV amplified products was realized within 3 ∼ 5 min following amplification. The simplicity and nearly instrument-free method for this ISKNV-CPA-VF assay shows great potential for on-site diagnostics of ISKNV infection in Siniperca chuatsi.

Concepts: DNA, Infectious disease, Bacteria, Microbiology, Virus, Infection, Mandarin fish, Siniperca

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Interferon regulatory factors (IRFs) are a family of mediators in various biological processes including immune modulation of interferon (IFN) and proinflammatory cytokine expression. However, the data on the complete composition of IRFs is rather limited in teleost fish. In the present study, all IRF members, i.e. IRF1‒11 with two IRF4, IRF4a and IRF4b have been characterised in an aquaculture species of fish, the mandarin fish, Siniperca chuatsi, in addition to the previous report of IRF1, IRF2, IRF3 and IRF7 from the fish. These IRFs are constitutively expressed in various organs/tissues of the fish, and their expression can be induced following the stimulation of polyinosinic:polycytidylic acid (poly(I:C)) and the infection of infectious spleen and kidney necrosis virus (ISKNV), a viral pathogen of mandarin fish in aquaculture. The ISKNV infection induced the significant increase in the expression of some IRF genes, i.e. IRF2, IRF4a, IRF7, IRF9, IRF10 at 24 or 36 h post-infection (hpi) in spleen and head-kidney, and the significant increase of some other IRF genes, e.g. IRF1, IRF3, IRF4b, IRF5, IRF6, IRF8 at later stage of infection from 72, or 96, or even 120 hpi, which may imply the inhibitory effect of ISKNV on fish immune response. It is considered that the present study provides the first detailed analysis on all IRF members in an aquaculture species of fish, and can be served as the base for further investigation on the role of IRFs in teleost fish.

Concepts: Immune system, DNA, Gene expression, Bacteria, Virus, Transcription factors, Interferon regulatory factors, Mandarin fish

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The mandarin fish (Siniperca chuatsi) is an important and widely cultured fish in China. However, the lack of selective breeding of mandarin fish in previous decades has resulted in a decline in the growth rate of pond-cultured fish, a shortened period of sexual maturity, and reduced disease resistance; these issues seriously affect the quality and safety of the fish products. Therefore, it is necessary to establish a selective breeding program for the mandarin fish to improve the economical traits of the fish and to sustain the development of the mandarin fish industry.

Concepts: Reproduction, Genetic linkage, Map, Breeding, Mandarin fish, Siniperca, Percichthyidae, Breeding program

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In recent years, mandarin fish had a high mortality rate associated with abnormal swimming, exophthalmia, corneal opacity and eye hemorrhage on a fish farm located at Foshan city, Guangdong province, China. Three isolates of Gram-positive, chain-forming cocci were recovered from moribund fish, and designated as SS131025-1, SS131025-2, and SS131025-3. These isolates were identified as Streptococcus uberis according to their morphologic and physio-biochemical characteristics as well as phylogenetic analysis based on their 16S rRNA and GapC gene sequences. The pathogenicity of S. uberis to mandarin fish was determined by challenge experiments. Results of artificial challenge showed S. uberis infected healthy mandarin fish and lead to death by eyeball injection or immersion route, and the LD50 of SS131025-1 with eyeball injection was 2.0 × 10(6.42) CFU per fish. Moreover extracellular product (ECP) of the isolated S.uberis induced CPB cell apoptosis and cause death of mandarin fish. In addition, these S. uberis strains could also infect tilapia, but not grass carp and crucian carp, and grew in brain-heart infusion broth with an optimal temperature of 37 °C, pH of 7.0, and salinity of 0%. Antibiotic sensitivity testing indicated that these isolates were susceptible to rifampicin and furazolidone but resistant to 20 kinds of antibiotics. Histopathologically, infection with S. uberis could cause serious pathological changes in brain tissues such as vacuoles in matrix, swollen mitochondria with lysis of cristae and disintegration, and lots of coccus was observed both under electron and light microscope. These results shed some light on the pathogenicity of the isolates and how to prevent and control S. uberis infection in mandarin fish.

Concepts: Bacteria, Infection, Carp, Commercial fish, Guangdong, Guangzhou, Mandarin fish, Siniperca

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Siniperca chuatsi (Basilewsky), a demersal piscivore, is an endemic freshwater fish species in China. For the purpose of genomics research, we have constructed the first bacterial artificial chromosome (BAC) library for S. chuatsi. The BAC library comprised a total of 84,480 clones with an average insert size of 124.6 kb and less than 2.5% empty clones, corresponding to a 10.5-fold coverage of the S. chuatsi genome. The probability of isolating genes of interest was more than 99%. To validate the library, we screened 220 superpools and found that 1-19 were positive for six SSR markers, while none was positive for two mitochondrial gene markers. Therefore, the S. chuatsi BAC library will provide useful genomics resources and tools for cloning, functional genomics research and identification of economically important genes in this species.

Concepts: DNA, Gene, Genetics, Bacteria, Molecular biology, Genome, Chromosome, Mandarin fish

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Nervous necrosis virus (NNV) is the causative agent of viral encephalopathy and retinopathy (VER), a neurological disease responsible for high mortality of fish species worldwide. Taking advantage of our established Chinese perch brain (CPB) cell line derived from brain tissues of Mandarin fish (Siniperca chuatsi), the susceptibility of CPB cell to Red-Spotted Grouper nervous necrosis virus (RGNNV) was evaluated. The results showed that RGNNV replicated well in CPB cells, resulting in cellular apoptosis. Moreover, the susceptibility of Mandarin fish to RGNNV was also evaluated. Abnormal swimming was observed in RGNNV-infected Mandarin fish. In addition, the cellular vacuolation and viral particles were also observed in brain tissues of RGNNV-infected Mandarin fish by Hematoxylin-eosin staining or electronic microscopy. The established RGNNV susceptible brain cell line from freshwater fish will pave a new way for the study of the pathogenicity and replication of NNV in the future.

Concepts: Brain, Fish, Apoptosis, Human brain, Neurology, Mandarin fish, Siniperca, Percichthyidae

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HSP90 plays important roles in multiple cellular stress responses. Here, two cytoplasmic HSP90 isoforms, ScHSP90α and ScHSP90β, were identified from Siniperca chuatsi. Their cDNA and gDNA structures, amino acid sequence features, and sequence identities and phylogenetic analysis with other species were described. Their expression profiles during embryonic development in different tissues and under stressful conditions were analyzed using real-time quantitative PCR. During embryogenesis, transcripts of both genes were detected at low levels during the early developmental stages and were up-regulated from appearance of myomere for ScHSP90a and closure of blastopore for ScHSP90β. ScHSP90α showed a tissue-specific variation with high expression in ovary and brain under non-stressed conditions, while ScHSP90β was ubiquitously highly expressed in different tissues. Acute heat shock resulted in a strong up-regulation of ScHSP90α in heart, liver, and head kidney, while it only weakly induced ScHSP90β in these tissues. ScHSP90α was also markedly induced in liver in a time-dependent manner under hypoxia, while the expression of ScHSP90β was not affected by hypoxia. Additionally, Aeromonas hydrophila infection markedly augmented ScHSP90α in head kidney and spleen and mildly up-regulated ScHSP90β in spleen, while suppressing ScHSP90β in head kidney. These results suggest that ScHSP90α and ScHSP90β are differently involved in embryogenesis and under different environmental conditions including high temperature, hypoxia, and bacterial infection. This study will benefit to further clarify the roles of fish HSP90 isoforms in embryogenesis and under stressful conditions and contribute to further study on enhancing stress tolerance and disease resistance of mandarin fish.

Concepts: DNA, Protein, Gene expression, Bacteria, Amino acid, Molecular biology, Mandarin fish, Siniperca