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Sox9 is a multifunctional gene and plays crucial roles in vertebrate development including chondrogensis. In teleost, due to the genome duplication event, there are two co-orthologs sox9a and sox9b. In this study, CRISPR/Cas9 technology was performed to disrupt the function of either sox9a or sox9b. All sox9a mutants (sox9aΔ10 and sox9aΔ67) and sox9b mutants (sox9bΔ11 and sox9bΔ20) lost HMG domain and Q/S domain, however, only sox9a mutant larvae had mis-shaped pectoral fins and lacked the scapulocoracoid cartilage. sox9b mutant larvae showed normal cartilages similar to wild type larvae. The results suggested that sox9a, not sox9b was required for cartilage development in zebrafish, which was different from the sox9b-mutant phenotype induced by N-ethyl-N-nitrosourea (ENU) treatment, gamma radiation treatment or morpholino injection. This study confirmed that ancestral sox9 gene functions partitioned between the two paralogs in zebrafish.

Fish interferon (IFN)-mediated antiviral innate immunity is the first line of defense against virus invasion. In the present study, we identify two fish IFN genes (here tentatively named IFNa and IFNc) with different-sized 3' UTRs from clone F strain of gibel carp Carassius auratus gibelio. Carp IFNa has a relatively short 3'UTR without AU-rich elements (AREs) but IFNc has a long one with 9 AREs. Functionally, carp IFNa and IFNc display significantly antiviral potential to viral infection, likely through induction of downstream IFN-stimulated genes (ISGs). Both carp IFN genes are induced by viral infection, poly(I:C) treatment and IRF3/7, which are ascribed to the IFN-sensitive response elements (ISRE) within their promoters. Carp IFN genes are also induced by each other and by themselves, indicating existence of a positive feedback loop in fish IFN-mediated antiviral immune response. Comparative analyses of 3'UTR-mediated expression regulation at mRNA and protein levels show that the ARE-containing 3'UTR of carp IFNc rather than the short 3'UTR of carp IFNa promotes mRNA decay but instead results in high-level protein expression, indicating that 3'UTR of fish IFN mRNAs might be a potential factor for regulation of IFN-mediated antiviral immune response. Considering a fact that a given protein function is largely related to its protein level, these results suggest that both promoter and 3'UTR contribute to the transcription and translation of fish IFN genes, thus shaping their eventually antiviral potential.

In mammals and fish, emerging evidence highlights that TRIM family members play important roles in the interferon (IFN) antiviral immune response. Fish TRIM family has undergone an unprecedented expansion leading to generation of finTRIM subfamily, which is exclusively specific to fish. Our recent results have shown that FTRCA1 (finTRIM C. auratus 1) is likely a fish species-specific finTRIM member in crucian carp C. auratus and acts as a negative modulator to downregulate fish IFN response by autophage-lysosomal degradation of protein kinase TBK1. In the present study, we found that FTRCA1 also impedes the activation of crucian carp IFN promoter by IRF7 but not by IRF3. Mechanistically, FTRCA1 attenuates IRF7 transcription levels likely due to enhanced decay of IRF7 mRNA, leading to reduced IRF7 protein levels and subsequently reduced fish IFN expression. E3 ligase activity is required for FTRCA1 to negatively regulate IRF7-mediated IFN response, because ligase-inactive mutants and the RING-deleted mutant of FTRCA1 lose the ability to block the activation of crucian carp IFN promoter by IRF7. These results together indicate that FTRCA1 is a multifaceted modulator to target different signaling factors for shaping fish IFN response in crucian carp.

Sinorhodeus microlepis (S. microlepis) is recently described as a new species and represents a new genus Sinorhodeu of the subfamily Acheilognathinae. In this study, we first sequenced the complete mitogenome of S. microlepis and compared with the other 29 bitterling mitogenomes. The S. microlepis mitogenome is 16,591 bp in length and contains 37 genes. Gene distribution pattern is identical among 30 bitterling mitogenomes. A significant linear correlation between A+T% and AT-skew were found among 29 bitterling mitogenomes, except S. microlepis shows unusual AT-skew with slightly negative in tRNAs and PCGs. Bitterling mitogenomes exhibit highly conserved usage bias of start codon, relative synonymous codons and amino acids, overlaps and non-coding intergenic spacers. Phylogenetic trees constructed by 13 PCGs strongly support the polyphyly of the genus Acheilognathus and the paraphyly of Rhodeus and Tanakia. Together with the unusual characters of S. microlepis mitogenomes and phylogenetic trees, S. microlepis should be a sister species to the genus Rhodeu that might diverge about 13.69 Ma (95% HPD: 12.96–14.48 Ma).

Recent studies have related the membrane-associated RING-CH-type finger (MARCH) family proteins to host innate immune response. Zebrafish (Danio rerio) MARCH8 is reported to target SVCV glycoprotein for degradation; however, little is known about whether fish MARCH8 is involved in innate interferon (IFN) response. In this study, zebrafish march8 was significantly induced by SVCV infection. Overexpression of MARCH8 diminished fish IFN-mediated antiviral response, thus promoting the replication of SVCV and GCRV in fish cells. Mechanistically, MARCH8 interacts with and degrades MITA and TBK1 proteins to inhibit IFN response. Moreover, MARCH8 has an E3 ligase activity and enhances MITA and TBK1 polyubiquitination. Our findings reveal a mechanism whereby zebrafish MARCH8 downregulates fish IFN response and facilitates viral replication by targeting MITA and TBK1 for protein degradation.

The sex-determining genes are found to be variable among different fish species. Yellow catfish (Pelteobagrus fulvidraco) is an important aquaculture fish species in China with XX/XY sex-determining type. Recently, YY super-male yellow catfish has been successfully produced by combining hormonal-induced sex reversal method with sex chromosome-linked markers. Here, we identified a novel PDZ domain-containing gene in yellow catfish designated as pfpdz1, in whose intron the sex-linked marker was located. The coding sequence of pfpdz1 in Y chromosome was identical to that in X chromosome except a missense SNP (A/T) that changes an amino acid (E8V) in the N-terminal region. Pfpdz1 displayed male-specific expression during sex differentiation. Overexpression of pfpdz1 using additive transgenesis induces XX ovary to differentiate into testis-like tissue, while the targeted inactivation of pfpdz1 in Y chromosome using CRISPR/Cas9-mediated mutagenesis triggers ovarian differentiation. Furthermore, we demonstrated that pfpdz1 initiates testicular differentiation through upregulating expression of amh, dmrt1 and sox9a1, as well as downregulating expression of cyp19a1, foxl2 and wnt4. Our data provide functional evidence that pfpdz1 is significant for male differentiation and maintenance in yellow catfish.

Diverse immunoglobulin (Ig) domain-containing protein (DICP) family is a novel bony fish-specific multi-gene family encoding diversified immune receptors. However, their function and the implication of binding partners remain unknown. In this study, we first identified 28 DICPs from three gibel carp gynogenetic clones and revealed their high variability and clone-specific feature. After crucian carp herpesvirus (CaHV) infection, these DICPs were significantly upregulated in head kidney, kidney and spleen. The up-regulation folds in clone A+, F and H were related to the susceptibility to CaHV, progressively increasing from resistant clone to susceptible clone. Overexpression of gibel carp DICPs inhibited interferon (IFN) and viperin promoter-driven luciferase activity. The additions of E. coli extracts and lipid A significantly enhanced the inhibition effect. In addition, gibel carp DICPs can interact with SHP-1 and SHP-2. These findings suggest that gible carp DICPs, as inhibitory receptors, might specifically recognize lipid A, and then interact with SHP-1 and SHP-2 to inhibit the induction of IFN and ISGs.

Fish biology has been developed for more than 100 years, but some important breakthroughs have been made in the last decade. Early studies commonly concentrated on morphology, phylogenetics, development, growth, reproduction manipulation, and disease control. Recent studies have mostly focused on genetics, molecular biology, genomics, and genome biotechnologies, which have provided a solid foundation for enhancing aquaculture to ensure food security and improving aquatic environments to sustain ecosystem health. Here, we review research advances in five major areas: (1) biological innovations and genomic evolution of four significant fish lineages including non-teleost ray-finned fishes, northern hemisphere sticklebacks, East African cichlid fishes, and East Asian cyprinid fishes; (2) evolutionary fates and consequences of natural polyploid fishes; (3) biological consequences of fish domestication and selection; (4) development and innovation of fish breeding biotechnologies; and (5) applicable approaches and potential of fish genetic breeding biotechnologies. Moreover, five precision breeding biotechniques are examined and discussed in detail including gene editing for the introgression or removal of beneficial or detrimental alleles, use of sex-specific markers for the production of mono-sex populations, controllable primordial germ cell on-off strategy for producing sterile offspring, surrogate broodstock-based strategies to accelerate breeding, and genome incorporation and sexual reproduction regain-based approach to create synthetic polyploids. Based on these scientific and technological advances, we propose a blueprint for genetic improvement and new breed creation for aquaculture species and analyze the potential of these new breeding strategies for improving aquaculture seed industry and strengthening food security.

Longsnout catfish (Leiocassis longirostris), an important commercial fish in China, shows significant sexual size dimorphism. However, the sex determination system and mechanism of longsnout catfish remain unknown. In this study, an effective protocol of artificial gynogenesis using heterologous sperm was successfully developed and male heterogametic XX/XY sex determination system was revealed in longsnout catfish. Subsequently, sex-specific markers were developed based on isolated homologous X-specific and Y-specific sequences by 2b-RAD sequencing and PCR amplification. Finally, the Y-specific and XY-shared primers were developed and validated in a full-sib family, a gynogenetic population and two local populations, which further verify the sex determination system. Therefore, this study not only reveals XX/XY sex determination system in longsnout catfish, but also provides applicable sex-specific markers for production of all-male population for aquaculture in the future.

Viperin is increasingly thought as a key protein in antiviral response. However, the in vivo antiviral effects of fish viperin remains unresolved. Hexaploid gibel carp (Carassius gibelio) is an important aquaculture species in China and its culture industry has suffered enormous economic losses due to an epizootic disease caused by C. auratus herpesvirus (CaHV) since 2012. In our previous studies, two duplicated viperin homeologs (Cgviperin-A and Cgviperin-B) were identified and their divergent antiviral mechanisms were revealed through in vitro overexpression in CAB (C. auratus blastulae embryonic) cell. Here, we tried to explore their function difference in vivo by using CRISPR/Cas9 editing. Firstly, we uncovered their biased expression patterns and differential promoter activities. Then we mutated them singly or simultaneously in resistant clone H of gibel carp by using CRISPR/Cas9 and finally confirmed that they cooperatively participated in the antiviral battle between gibel carp and CaHV through virus challenge and comparative transcriptome analysis. CgViperin-A depletion mainly affected immune-related pathways and Cgviperin-B deficiency influenced autophagy-associated pathways. Simultaneous disruption of them resulted in complete loss of resistance ability of gibel carp against CaHV. Significantly, the deficiency of Cgviperin-A influenced the expression of Cgviperin-B in Cgviperin-A−/−/− mutant, but Cgviperin-A transcripts kept a high level of expression in the Cgviperin-B−/−/− mutant, same as in the WT. Therefore, the current findings are not only the first time to clarify the antiviral activities of Viperin in the battle between teleost and virus in vivo, but also explain the detailed evolutionary divergence of a duplicated gene in a recurrent polyploid fish.

Hybridization is an efficient method to breed new strains of aquatic animals. In the present study, we produced a hybrid puffer by crossing female obscure puffer with male tiger puffer. The hybrid puffer could live in fresh water like obscure puffer and exhibited growth superiority. The averaged body weight of 4- and 6-month-old hybrid puffer were respectively 38.06% and 38.93% higher than that of obscure puffer. Then, we analyzed the underlying genetic basis for the growth advantage of hybrid puffer by comparative transcriptome analysis. A total number of 4264 and 1285 differentially expressed genes (DEGs) were respectively identified from pituitary and liver transcriptome profiles between hybrid puffer and obscure puffer. Comprehensive analysis showed that the DEGs related with cell proliferation and differentiation, and protein synthesis and export, specifically showed higher expression levels in hybrid puffer, such as "ECM-receptor interaction", "focal adhesion", "protein export" and "protein processing in endoplasmic reticulum". While the DEGs involved in gametogenesis and carbohydrate and energy metabolism highly expressed in obscure puffer, such as "oxidative phosphorylation", "citrate cycle", "progesterone-mediated oocyte maturation" and "oocyte meiosis". Furthermore, a series of candidate genes related to the growth superiority of hybrid puffer were identified, such as fn1a, ptprc, plcg2, igf1, tgfβ1, bmp4, abl1, col1a2, col1a1a, and myl9a. These results will be beneficial to understand the molecular basis of growth superiority and helpful to the hybrid breeding of pufferfish.

Determining the sex and controlling the sex ratio are essential aspects of fish genetics that can assist in developing successful fish breeding programs. High quality genome assembly and annotations are prerequisites to determine sex-specific genes and their expression. In addition, analysis of resequencing data can identify genomic difference between male and female fishes. In this study, we performed chromosome-level genome assembly in female Ancherythroculter nigrocauda fish having low heterozygosity using PacBio reads. High-throughput chromatin conformation capture (HiC) yielded a genome of size 1054.05 Mb, with a contig N50 length of 3.40 Mb and a scaffold N50 length of 42.68 Mb. In addition, we sequenced 5 female and 5 male A. nigrocauda samples and identified sex-specific regions on LG20 Furthermore, diet-specific amino acid mutation were found on 582 genes between herbivorous and carnivorous fishes, with 26 of them exhibiting significantly different expression patterns in the liver tissue of these two types of fishes. The chromosome-level genome assembly of A. nigrocauda provides valuable resources for conducting in-depth comparative genomic studies with immense applications in fish genetic breeding and farming. Similarly, the diet-specific amino acid mutations are useful in the breeding of new strains of carnivorous fishes with an herbivorous diet.

Signal transducer and activator of transcription 5b (STAT5b) has been identified as a key downstream mediator of growth hormone (GH) signaling in somatic growth of mammalian. However, the corresponding homologue gene of Stat5b is unknown in fish species. In this study, we generated loss-of-function mutants in stat5.1 and stat5.2, two stat5 homologues existing in zebrafish. In stat5.1-deficient zebrafish, a significant reduction of body length and body weight was detected in the embryos/larvae and adults compared with the wild-type control fish, and sexual size dimorphism in adult zebrafish was also eliminated. However, the stat5.2-deficient zebrafish displayed a normal developmental phenotype during all lifespan. Chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) method was adopted to further investigate the potential transcriptional targets of Stat5 protein and cast much light upon the biological function of Stat5. We identified more than 800 genes as transcriptional targets of Stat5 during zebrafish embryogenesis. KEGG analysis indicated that the Stat5 target gene network is predominantly linked to the metabolic pathways, neuroactive ligand-receptor interaction and JAK-STAT signaling pathways. Further validation studies suggested that Stat5.1 protein could directly regulate the expression of gh1, and stat5.1-mutated zebrafish showed a reduction of gh1 mRNA level. In the present study, stat5.1 was revealed as the corresponding homologue gene of Stat5b in fish species. Additionally, we found a novel molecular interaction between Stat5.1/Stat5b and GH, and unraveled a positive feedback loop Stat5.1-GH-Stat5.1 which is necessary for somatic growth and body development in zebrafish.

Gonadal-specific aromatase encoded by cyp19a1a is the important enzyme controlling estrogen biosynthesis in teleosts. In the present study, the cDNA sequence of cyp19a1a was cloned and characterized from miiuy croaker Miichthys miiuy. The cDNA encoded a protein of 519 amino acids with five structural regions. Higher identities of amino acid sequences and conserved structural regions were found between Mmcyp19a1a and other cyp19a1a genes. In addition, Mmcyp19a1a was clustered together with other seawater fishes. Immunohistochemical analysis revealed that Mmcyp19a1a was localized exclusively in the cytoplasmic of thecal and granulosa cells surrounding the oocytes. Both the protein and mRNA levels of Mmcyp19a1a were increased significantly at the stage III follicles (mid-vitellogenic) and then decreased along with vitellogenesis. Interestingly, strong immunoreactive signals were also detected in the supporting cells of connective tissues during ovarian development. A 1777bp promoter fragment of Mmcyp19a1a was also isolated, and functional analysis using an EGFP reporter fusion in zebrafish larvae presented positive signals in the above of yolk sac, where is the region of pronephros and germ plasm occur. The Mmcyp19a1a:EGFP expression pattern was generally consistent with the endogenous cyp19a1a genesis. These results indicate that the Mmcyp19a1a gene plays an important role during vitellogenesis and oocyte maturation. The constructor of Mmcyp19a1a:EGFP may provide a useful tool for genetic analysis of gonad development in teleost.

Dead end (dnd), vertebrate-specific germ cell marker, had been demonstrated to be essential for primordial germ cell (PGC) migration and survival, and the link between PGC number and sex change had been revealed in some teleost species, but little is known about dnd in hermaphroditic vertebrates. In the present study, a protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides) dnd homologue (Ecdnd) was identified and characterized. Quantitative real-time PCR and in situ hybridization analysis revealed a dynamic and sexually dimorphic expression pattern in PGCs and germ cells of gonads. During sex changing, the Ecdnd transcript sharply increased in early transitional gonad, reached the highest level at late transitional gonad stage, and decreased after testis maturation. Visualization of zebrafish PGCs by injecting with RFP-Ecdnd-3'UTR RNA and GFP-zfnanos3-3'UTR RNA confirmed importance of Ecdnd 3'UTR for the PGC distribution. In addition, knockdown of EcDnd by using antisense morpholinos (MO) caused the ablation of PGCs in orange-spotted grouper. Therefore, the current data indicate that Ecdnd is essential for PGCs survival and may serve as a useful germ cell marker during gametogenesis in hermaphroditic grouper.

Genome polyploidy has been revealed to result in evolutionary advantages and novelties, and therefore, polyploid aquatic animals may possess excellent traits of economic interest including rapid growth, extensive adaptability and disease resistance. For this reason, numerous species of natural polyploid fishes, such as common carp, gibel carp, crucian carp, salmon, and sturgeon, were chosen as important target species for aquaculture. Many artificial polyploids have been commercially utilized for aquaculture and most of them were created from natural polyploid fishes of the Cyprinidae and Salmonidae. Thanks to the easy mass production and better economic traits in growth and flesh quality, the synthetized autopolyploids or allopolyploids from natural polyploid species in cyprinid fishes have been extensively applied to aquaculture throughout China. This review outlines polyploidy advantages and innovative opportunities, lists natural polyploid species used in aquaculture, and summarizes artificial polyploids that have been induced or synthetized, and used in aquaculture. Moreover, some main research trends on polyploid utilization and ploidy manipulation of aquaculture animals are also introduced and discussed in the review.

Various genes have been screened on extra chromosomes, but their molecular characterization, expression pattern and biological function are still unclear. Here, we utilized a male-specific sequence of polyploid gibel carp (Carassius gibelio) to identify a novel male-specific SET (Su(var)3–9, Enhancer-of-zeste, Trithorax) domain-containing gene setdm on extra microchromosomes of gibel carp males. And setdm was characterized in molecule and expression aspects, in which its expression was specific to testis, and had relative high transcription during middle/late stages of testis development. Moreover, prominent expression of Setdm in spermatogenic cells was observed in testis through immunofluorescence co-localization analysis. These results suggest that biological function of setdm might be related to testis development and spermatogenesis of gibel carp. Additionally, the homeologous gene setdmf of setdm, was also characterized, and its expression was gonad-specific, in which its expressed product was detected to mainly distribute in gametogenic cells of testis and ovary, and to have dynamic expression pattern similar to that of setdm. Based on the current results, we propose that the novel male-specific setdm on extra microchromosomes might be functional divergence gene of the gonad-specific setdmf. Therefore, these findings will help us to further understand evolutionary fate and functional role of genes on extra microchromosomes.

Interferon regulatory factor 7 (IRF7) is a key mediator in regulating the type Ι IFN response. Although irf7 has been identified in more than twenty fish species, alternative splicing has not been found in teleost irf7. Alternative splicing is an important mechanism expanding the transcriptomic and proteomic diversity, and has been found in several IRF family members. Here, three alternative splicing variants of irf7 were identified and characterized in obscure puffer. The first splicing transcript (Toirf7v1) was predicted to encode 428 amino acids with a DNA-binding domain (DBD), an interaction-associated domain (IAD) and a serine-rich domain (SRD). Toirf7v2 encoded 430 amino acids caused by the intron retention, and contained the whole conserved domains. Toirf7v3 encoded a truncated protein with 337 amino acids resulting from the alternative 5' splice-site selection, and lacked part of IAD domain and the entire SRD domain. Functional studies demonstrated that all of the three isoforms could activate the expression of type I IFN and IFN-stimulated genes (ISGs). Nevertheless, the two variants (Toirf7v2 and Toirf7v3) exhibited much less ability to induce transcription of IFN and ISGs compared to the Toirf7v1. Our findings suggest that these splicing variants may have distinct roles in the regulation of immune response. These results will be beneficial to understand the functional characteristics of irf7 variants in fish.

Lanzhou catfish (Silurus lanzhouensis), an important commercial fish in China, takes three years to be sexually mature and males display faster growth performance than females. Thus, shortening of breeding cycle and production of all-male population in Lanzhou catfish have great significance in aquaculture. Here, we first developed an optimal method for artificial gynogenesis, through which the male heterogametic XX/XY sex determination system was revealed in Lanzhou catfish. Subsequently, sex-specific markers were identified via 2b-RAD-seq and verified in artificial breeding and wild populations. Intriguingly, a hermaphrodite Lanzhou catfish with XY genotype was occasionally obtained, whose gonad contained both mature ovarian and testicular tissues. The eggs and sperm from the intersexual gonad were used for artificial fertilization to establish a self-fertilization family, which contained 21 XX females, 25 XY males, and 12 YY males in the offspring. And the YY males are able to mate with XX females to produce all-male progenies with XY genotype. Compared with the conventional way of expending two-generation time, self-fertilization of the XY hermaphrodite saved one-generation time to produce YY males. Therefore, this study not only reveals XX/XY sex determination system in Lanzhou catfish, but also directly obtains YY males for future production of all-male population in aquaculture.

STAT (Signal Transducers and Activators of Transcription) gene family members have been revealed to be involved in cell growth and differentiation in vertebrates. Despite their physiological importance, their functions are poorly studied at organ and systemic levels. In this study, we performed a genome-wide analysis using data from invertebrates to vertebrates to identify STAT genes and analyze their evolutionary history. Interestingly, the STAT gene family undergoes genome duplications during the evolutionary history with STAT3 homologues firstly appearing in the basal extant vertebrate, sea lamprey, suggesting its possible roles in spine formation. To investigate the functions of stat3 in fish species, TALEN technology was performed to generate mutant zebrafish lines. Stat3 mutant zebrafish showed no obvious defects at early developmental stage but displayed severe lateral and vertical curvature of the spine (scoliosis), spine fracture and the incomplete bone joints with narrower junction between vertebrae at early juvenile stage, as indicated by Alizarin red and Alcian blue staining, radiography and micro-computed tomography (MicroCT) analysis. Transcriptome analysis reveals dramatic alterations in a number of genes involved in immune and infection response, skeletal development and somatic growth, especially downregulated expression of collagen gene family, in the juvenile stat3 mutant zebrafish. Moreover, most of the collagen genes were detected to have abnormal expression pattern during the formation of spine deformities in stat3 mutants. Our data reveal that stat3 is specially expressed in vertebrates and required for normal spine development and immune function in zebrafish.

Sperm motility is a critical determinant of male fertility in vertebrates including fish species. Chloroquine, an autophagy inhibitor, has been reported to own debated influence on sperm quality of mammalians. The objectives of this study were to determine the effect of chloroquine on sperm motility and fertilizing efficiency in yellow catfish, an important freshwater fish species in China. Gradient doses of chloroquine were intraperitoneally injected into yellow catfish that resulted in an increasing expression level of LC3-II and inhibition of autophagy. Subsequently, various sperm parameters were assessed with the computer-assisted sperm analysis (CASA) system. Chloroquine injection significantly increased straight line velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP) of sperm compared with the control. Consequently, 1 μM chloroquine/g body weight was revealed to be an optimal dose for in vivo treatment, which led to elevation of fertility rate, whereas no effect on the hatching rate. The comparative transcriptome analysis between chloroquine-treated and control testes were conducted in order to figure out the possible molecular mechanism of chloroquine to regulate sperm motility. Interestingly, Gene Ontology (GO) analysis indicated that chloroquine treatment not only inhibited autophagy pathway but also significantly reduced toll-like receptor signaling pathway, suggesting a possible trade-off between male reproduction and immunity. KEGG analysis showed up-regulation of many pathways including PI3K-AKT signaling pathway which has been reported to be correlated with spermatogenesis and sperm maturation in yellow catfish. Our data reveals a potential trade-off between reproductive traits and immune function, and suggests that the application of chloroquine could improve sperm quality and fertilization efficiency in broodstock fish. Statement of relevance Our research demonstrated that an optimal dose of chloroquine could efficiently inhibit autophagy and improve sperm motility and fertilization efficiency. The comparative transcriptome analysis between chloroquine-treated and control testes were conducted in order to figure out the possible molecular mechanism of chloroquine to regulate sperm mobility.

As a germ cell marker gene, Dead end (dnd) has been identified and characterized in many vertebrates. Recently, we created a complete germ cell-depleted gonad model by the dnd-specific morpholino-mediated knockdown approach, and revealed sex-biased gene expression alteration through utilizing unisexual gynogenetic superiority in polyploid gibel carp. However, dnd and its expression pattern are still unclear in the gibel carp. In this study, we further analyzed molecular characterization of gibel carp dnd and its dynamic expression pattern during gametogenesis and embryogenesis. Similar to other homologs in vertebrates, gibel carp dnd contains a conserved RRM motif and five other motifs, and is highly evolutionary conserved in genomic organization and neighborhood gene synteny. RT-PCR and Western blot analyses showed its gonad-specific expression intensively in testis and ovary. Section in situ hybridization (SISH) and immunofluorescence localization revealed its dynamic expression pattern specific to oogenic cells and spermatogenetic cells during oogenesis and spermatogenesis. Moreover, its temporal and spatial distribution specific to PGCs were also demonstrated by RT-PCR and whole mount in situ hybridization (WISH) during embryogenesis. Therefore, gibel carp Dnd is a conserved germ cell marker during gametogenesis, and its maternal transcript is also a useful marker for tracing PGC specification and migration.

In this study, a stable allotetraploid gibel carp strain was established from allogynogenetic populations of triploid gibel carp clone D. Its modal chromosome number was identified as 208, and the added chromosomes were demonstrated to originate from common carp by internal transcribed spacer-1 (ITS1) and transferrin (Tf) allele sequence analyses. Significantly, its morphological body type and morphometric traits were observed to be similar to that of the maternal gibel carp. And, the new allotetraploid showed remarkable growth superiority by growth rate comparison tests, in which an approximate 22.9-26.6% higher growth rate was found in the allotetraploid strain than that in the maternal allogynogenetic gibel carp. Overall, we have bred an allotetraploid strain that resembles a typical gibel carp body type, and possesses significant growth superiority. Therefore, this study provides an excellent material for fish polyploidy breeding and aquaculture practice. (C) 2016 Elsevier B.V. All rights reserved.

High-density genetic linkage map based on single nucleotide polymorphisms (SNPs) are essential for fine mapping of QTLs and identification of trait-related genes in aquaculture fish species. In this study, the first high-density genetic linkage map of red-tail catfish (Hemibagrus wyckioides) based on 2b-RAD sequencing technology was constructed using one F1 family composed of 165 offspring and their parents. A total of 2369 SNP markers were assigned to 29 linkage groups (LGs) and the map spanned 2067.35 cM with an average SNP marker interval of 0.87 cM and genome coverage of 98.16%. QTL mapping analysis revealed that 2 significant and 15 suggestive QTLs for all four morphometric traits were identified on 10 LGs, explaining 7.4–13.3% of phenotypic variations. Some candidate growth-related genes such as ttc39b, lrp1, gng3, aspp2, mgp and rusc2 were identified within QTL intervals, and a SNP in ttc39b was found to be significantly associated with body weight in different genetically unrelated populations of red-tail catfish. In addition, one genome-wide significance sex-related QTL was detected on the LG20 with a contribution to phenotypic variation of 44.2–85.7%. A total of 10 potential sex dimorphic genes, such as vps54, tsg10, pld6, psmd8 and gpa33, were also identified within or near the sex QTL markers in LG20. It suggests that the LG20 may be a sex-related LG in red-tail catfish. This high-density genetic map not only enables us to fine-map these genes related with the economic traits, but also provides effective information for future marker-assisted selection and sex-control breeding in this species.

In mammals, tripartite motif (TRIM)-containing proteins are involved in interferon (IFN)-mediated antiviral response as pivotal players endowed with antiviral effects and modulatory capacity. Teleost fish have a unique subfamily of TRIM, called finTRIM (fish novel TRIM, FTR) generated by genus- or species-specific duplication of TRIM genes. Herein, four TRIM genes are identified from Epithelioma papulosum cyprini (EPC) cells, and phylogenetically close to the members of finTRIM, thus named FTREPC1, FTREPC2, FTREPC3 and FTREPC4. Despite high similarity in nucleotide sequence, FTREPC1/2 genes encode two proteins with a typically consecutive tripartite motif followed by a C-terminal B30.2 domain, while FTREPC3/4-encoding proteins retain only a RING domain due to early termination of translation. They are induced by poly(I:C), GCRV and SVCV as IFN-stimulated genes (ISGs), and this induction is severely impaired by blockade of STAT1 pathway and is dependent on a typical ISRE motif within the 5' untranslated regions (5'UTRs) of FTREPC1/2/3/4 genes. Whereas overexpression of FTREPC1/2/3/4 alone does not activate fish IFN promoters, overexpression of FTREPC1 or FTREPC2, rather than FTREPC3 and FTREPC4, significantly impairs intracellular poly(I:C)-triggered activation of fish IFN promoters. Consistently, FTREPC1/2 promote virus replication through negatively regulating IFN response. Our results provide evidence for the involvement of EPC finTRIM proteins in IFN antiviral response and insights into genus- or species-specific regulation of fish innate immune pathways.

Identification of sex-specific markers and revealing of the sex determination system have important implications for sex control breeding in aquaculture. The zig-zag eel (Mastacembelus armatus) mainly occurs in the Yangtze River and the Pearl River in China. An apparent sexual dimorphism in growth is observed, however, the sex determination mechanism is still unclear to date. In this study, two male-specific 2b-RAD tags and two SNPs were identified by 2b-RAD sequencing and verified by PCR amplification. Two female specific deletions with 281 bp and 4938 bp and a male-specific deletion with 156 bp were identified by sequence comparison between X-specific and Y-specific sequences obtained by genome walking from two 2b-RAD tags. Two sets of primers including Y-specific and XY-shared primers were designed based on these identified deletion regions to distinguish the genetic sex of zig-zag eel. The specificity of these primers was confirmed in 81 offspring of one F1 family. Furthermore, two male-specific SNP primers, which specifically amplify one band only in the males, were also designed. These data indicate that zig-zag eel possesses a male heterogametic XY sex determination system. The sex-specific markers presented here will have the potential to play an important role on the production of all-male populations of zig-zag eel for aquaculture in the future.

Unbalanced copper (Cu) homeostasis is associated with neurological development defects and diseases. However, the molecular mechanisms remain elusive. Here, central neural system (CNS) myelin defects and the down-regulated expression of WNT/NOTCH signaling and its down-stream mediator hoxb5b were observed in Cu2+ stressed zebrafish larvae. The loss/knockdown-of-function of hoxb5b phenocopied the myelin and axon defects observed in Cu2+ stressed embryos. Meanwhile, the activation of WNT/NOTCH signaling and ectopic expression of hoxb5b could rescue Cu induced myelin defects. Additionally, fam168b, similar to pou3f1/2, exhibited significant promoter hypermethylation and reduced expression in Cu2+ stressed embryos. The hypermethylated locus in fam168b promoter acted pivotally in its transcription, and the loss/knockdown of fam168b/pou3f1 also induced myelin defects. This study also demonstrated that fam168b/pou3f1 and hoxb5b axis acted in a seesaw manner during fish embryogenesis: Cu induced the down-regulated expression of the WNT&NOTCH-hoxb5b axis through the function of copper transporter cox17, coupled with the promoter methylation of genes fam168b/pou3f1, and its subsequent down-regulated expression through the function of another transporter atp7b, making joint contributions to myelin defects in embryos.

Stem-loop-binding proteins (SLBPs) have been revealed to interact with stem-loop of histones, and oocyte-specific and oocyte-preferential SLBP2 have been identified in vertebrates including Xenopus (S. tropicalis) and B. taurus to play a key role in histone translation regulation, but no oocyte-specific SLBPs have been characterized in fish. Here, we have identified and characterized the first fish oocyte-specific SLBP2 in Carassius gibelio. Its full-length cDNA contains 975 bp ORF encoding 324 amino acids. Firstly, the polyclonal antibody specific to C. gibelio SLBP2 was prepared. Then, RT-PCR analysis and Western blot detection revealed its oocyte-specific and dynamic expression pattern during oogenesis and embryogenesis of C. gibelio. Moreover, in situ hybridization and immunofluorescence localization observed its abundant expression in cortical alveolar stage oocytes and dynamic distribution in different stage oocytes. Altogether, our current data suggest that C. gibelio SLBP2 might play significant role in the early oogenesis and oocyte growth.

Apolipoproteins, the major components of lipoproteins, play physiological roles in lipoprotein metabolism. Contrary to the well-documented effects on plasma lipid, little is known about the function and regulation of Apolipoproteins during embryonic development. Here we have shown that apolipoprotein C1 (apoc1) gene is highly expressed in the yolk syncytial layer, a structure implicated in embryonic and larval nutrition. The apoc1 transcripts are also observed in the deep cell layer at the ventral and lateral region during gastrulation, and in the tail paraxial mesoderm during somitogenesis. By whole-mount in situ hybridization and quantified RT-RCR, we further demonstrate that apoc1 expression is induced by bone morphogenetic proteins (BMPs) signaling, while retinoic acid (RA) signaling suppresses the expression of BMP ligands and inhibits the BMP effect in this process.

Obscure pufferfish (Takifugu obscurus) is an important aquaculture species in China, and sex control breeding is extremely attractive in this species because the testes of male pufferfish are more popular and expensive. However, the sex-specific marker remains unknown and reliable genetic sex identification method has not been established, which has hampered the development of monosexual aquaculture. In this study, the sex-linked amhr2 SNP site of obscure pufferfish was firstly identified by sequencing and high-resolution melting (HRM) analysis. The males exhibit heterozygous genotype (C/G), while females have homozygous genotype (C/C), which is identical to its closely-related species (T. rubripes, T. poecilonotus and T. pardalis). Then, two pairs of male-specific primers were designed based on the sex-specific SNP site and a high-throughput sexing method was subsequently developed with benefits including high specificity and superb sensitivity. Particularly, this method has greatly reduced analysis time and costs compared to the previously established HRM analysis. These results will be beneficial to develop the sex control breeding in obscure pufferfish.

Mammalian mitochondrial antiviral signaling protein (MAVS) is an essential adapter involved in retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR)-triggered interferon (IFN) antiviral immunity. Fish MAVS homologues have been identified in several fish species; however, the signaling pathway involving fish MAVS-mediated IFN response remains to be investigated. In the present study, we report identification of a fish MAVS orthologue from crucian carp Carassius auratus blastulae embryonic (CAB) cells and its function role in fish RLR signaling. Crucian carp MAVS is constitutively expressed in CAB cells and is not transcriptionally induced by cytosolic poly (I:C) and IFN. Overexpression of crucian carp MAVS results in activation of fish IFN promoter and ISRE-containing promoter as well as transcriptional expression of IFN and ISGs including PKR and Mx1, which is impaired by functional blockade of signaling molecules TBK1 and IRF3/7. Either cytosolic poly (I:C)-induced or RIG-I-induced IFN response is attenuated by functional blockade of crucian carp MAVS. These results together indicate that fish MAVS contributes to IFN antiviral immunity downstream of cytosolic poly (I:C) and RIG-I and upstream of TBK1 and IRF3/7. Moreover, we provide evidence that apart from crucian carp MAVS, crucian carp MITA is also involved in cytosolic poly (I:C)- and RIG-I-induced IFN response.

Mammalian viperin is a typical interferon (IFN)-induced antiviral protein. Fish have viperin homologs; however, little is known about the expression regulation of fish viperins. In this study, we report the expression regulation and antiviral function of a fish viperin from crucian carp Carassius auratus during IFN response. Crucian carp viperin is induced at mRNA and protein levels by fish IFNs and IFN stimuli such as poly(I:C). Consistently, this gene promoter contains multiple transcription factor binding sites including IFN-stimulated response elements (ISRE) and IFN gamma activation sequences (GAS), and is activated by two types of fish IFNs and also by the intracellular and extracellular poly(I:C). Activation of crucian carp viperin promoter by the intracellular poly(I:C) is mediated by retinoic acid-inducing gene I (RIG-I)-like receptors (RLR)-triggered IFN signaling pathway, which is further verified by the findings that each signaling molecule of RLR pathway is able to induce the expression of crucian carp viperin at mRNA and protein levels. Finally, overexpression of crucian carp viperin in cultured fish cells confers significant protection against infection of grass carp reovirus (GCRV). These data suggest that similar to mammalian homologs, crucian carp viperin exerts a conserved function through RLR-triggered IFN signaling pathway.

A series of MHC alleles (including 26 class IA, 27 class IIA, and 17 class IIB) were identified from Chinese giant salamander Andrias davidianus (Anda-MHC). These genes are similar to classical MHC molecules in terms of characteristic domains, functional residues, deduced tertiary structures and genetic diversity. The majority of variation between alleles is found in the putative peptide-binding region (PBR), which is driven by positive Darwinian selection. The coexistence of two isoforms in MHC IA, IIA, and IIB alleles are shown: one full-length transcript and one novel splice variant. Despite lake of the external domains, these variants exhibit similar subcellular localization with the full-length transcripts. Moreover, the expression of MHC isoforms are up-regulated upon in vivo and in vitro stimulation with Andrias davidianus ranavirus (ADRV), suggesting their potential roles in the immune response. The results provide insights into understanding MHC variation and function in this ancient and endangered urodele amphibian.

Vertebrate interferon (IFN) response defenses against viral infection through the induction of hundreds of IFN-stimulated genes (ISGs). Most ISGs are conserved across vertebrates; however, little is known about the species-specific ISGs. In this study, we reported that grass carp reovirus (GCRV)-induced gene 1 (Gig1), previously screened as a virus-induced gene from UV-inactivated GCRV-infected crucian carp (Carassius auratus) blastulae embryonic (CAB) cells, was a typical fish ISG, which was significantly induced by intracellular poly(I:C) through retinoic acid-inducible gene I (RIG-I)-like receptors-triggered IFN signaling pathway. Transient or stable overexpression of Gig1 prevented GCRV replication efficiently in cultured fish cells. Strikingly, Gig1 homologs were found exclusively in fish species forming a novel gene family. These results illustrate that there exists a Gig1 gene family unique to fish species and the founding gene mediates a novel fish IFN antiviral pathway.

We previously showed that a fish interferon (IFN) regulatory factor 9 (IRF9) homologue, crucian carp Carassius auratus IRF9, displays constitutively nuclear localization and involvement in fish IFN-dependent JAK-STAT signaling; however, little is known about the expression regulation of fish IRF9. Here, we characterized the expression of zebrafish IRF9 by promoter analysis. Zebrafish IRF9 gene promoter contained several putative transcription factor binding sites, including one ISRE (IFN-stimulated response element), one GAS (IFN gamma activation sequence) and three GATEs (IFN gamma activated transcriptional element, GATE1/2/3). Further sequence analyses revealed that GAS and GATE motifs existed in all promoters of IRF9 from mammals and fishes. Luciferase assays confirmed that zebrafish IRF9 promoter could be activated by zebrafish IFN phi s and zebrafish IFN gamma 2, as well as transcription factors IRF3, IRF7, and combination of IRF9 and STAT2. Treatment of recombinant crucian carp IFN protein or overexpression of zebrafish IFN gamma 2 both led to significant increase in crucian carp IRF9 mRNA and protein in cultured fish cells. Comparison of IFN-stimulated promoter activity revealed much more significant induction of zebrafish IRF9 by zebrafish IFN gamma 2 than by zebrafish IFN phi s. Mutation analyses showed that the putative GAS and GATE3 contributed to zebrafish IFN gamma 2-triggered IRF9 expression, whereas the putative ISRE and the other two GATEs were not functional for induction of zebrafish IRF9. These results together indicated that the expression property of IRF9 might be conserved from fish to mammals and that some not yet identified mechanisms could exist in IRF9 gene transcription regulation in response to IFNs. (C) 2013 Elsevier Ltd. All rights reserved.

Carassius auratus complex is believed to originate from East Eurasia and consist of diploid and triploid forms. Diploid form reproduces sexually, whereas triploid form possesses mixture modes of unisexual gynogenesis and sexual reproduction, which makes it a unique case to study evolutionary issues among vertebrates. In this study, we identified 337 triploid individuals from 386 specimens of Carassius auratus complex sampled from 4 different sites of Xingkai Lake and Suifen River on the northeast Asia transboundary areas of Russia and China, and found that triploids were ubiquitous, whereas diploids existed only in SII site of Suifen River. Triploid males were detected in all surveyed sites, and an unusually high triploid male incidence (23%) was found in the Chinese reach of Suifen River. Then, nuclear and cytoplasmic markers were used to analyze their genetic diversity and phylogenetic relationship. A total of 61 distinct tf alleles and 35 mtDNA CR haplotypes were revealed. Higher genetic diversity and divergence were confirmed in triploids than in diploids, and identical genetic background between triploid males and females was demonstrated. Moreover, evolutionary implications and roles of triploid males were suggested in population proliferation and diversity creation of the triploid form.

The nanos gene family was essential for germ line development in diverse organisms. In the present study, the full-length cDNA of a nanos1 homologue in A. sinensis, Asnanos1, was isolated and characterized. The cDNA sequence of Asnanos1 was 1489 base pairs (bp) in length and encoded a peptide of 228 amino acid residues. Multiple sequence alignment showed that the zinc-finger motifs of Nanos1 were highly conserved in vertebrates. By RT-PCR analysis, Asnanos1 mRNAs were ubiquitously detected in all tissues examined except for the fat, including liver, spleen, heart, ovary, kidney, muscle, intestines, pituitary, hypothalamus, telencephalon, midbrain, cerebellum, and medulla oblongata. Moreover, a specific polyclonal antibody was prepared from the in vitro expressed partial AsNanos1 protein. Western blot analysis revealed that the tissue expression pattern of AsNanos1 was not completely coincided with that of its mRNAs, which was not found in fat, muscle and intestines. Additionally, by immunofluoresence localization, it was observed that AsNanos1 protein was in the cytoplasm of primary oocytes and spermatocytes. The presented results indicated that the expression pattern of Asnanos1 was differential conservation and divergence among diverse species.

Mammalian interferon (IFN) regulatory factor 9 (IRF-9) has long been recognized as the DNA sequence recognition subunit of IFN-stimulated gene factor 3 (ISGF3) complex, which is critical for type I IFN to induce the expression of IFN-stimulated genes (ISGs) against viral infection. Recent studies have shown that fish IFN exerts antiviral effects by induction of a number of ISGs and also of itself; however, little is known about the role of fish IRF9 in IFN signaling. Here we identify a fish IRF9 orthologue (CaIRF9) from IFN-producing cell line, crucian carp Carassius auratus blastulae embryonic (CAB) cells. Analysis of subcellular distribution of CaIRF9-green fluorescent protein indicates that CaIRF9 is constitutively present in the nucleus, which is driven by two nuclear localization signals (NLS), one locating within DNA-binding domain (DBD) of CaIRF9 and the other immediately behind DBD, although human IRF9 contains only one NLS analogous to the former of CaIRF9. Overexpression of CaIRF9 together with CaSTAT2 not only activates ISRE-containing promoter but also upregulates the expression of fish ISGs. Strikingly, CaIRF9 together with CaSTAT2 also exhibits an ability to activate crucian carp IFN promoter, and blockade of cellular CaIRF9 attenuates IFN itself-induced activation of crucian carp IFN promoter. Taken together, these data suggest that crucian carp IFN induces the expression of ISGs and also of itself possibly by the JAK-STAT signaling pathway that is conserved from fish to mammals.