Effects of essential amino acids supplementation in a low-protein diet on growth performance, intestinal health and microbiota of juvenile blotched snakehead (Channa maculata).

Developing a low-protein feed is important for the sustainable advancement of aquaculture. The aim of this study was to investigate the effects of essential amino acid (EAA) supplementation in a low-protein diet on the growth, intestinal health, and microbiota of the juvenile blotched snakehead, Channa maculata in an 8-week trial conducted in a recirculating aquaculture system. Three isoenergetic diets were formulated to include a control group (48.66 % crude protein (CP), HP), a low protein group (42.54 % CP, LP), and a low protein supplementation EAA group (44.44 % CP, LP-AA). The results showed that significantly lower weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER), and feed efficiency ratio (FER) were observed in fish that were fed LP than in the HP and LP-AA groups (P < 0.05). The HP and LP-AA groups exhibited a significant increase in intestinal villus length, villus width, and muscular thickness compared to the LP group (P < 0.05). Additionally, the HP and LP-AA groups demonstrated significantly higher levels of intestinal total antioxidant capacity (T-AOC), catalase (CAT), and superoxide dismutase (SOD) and lower levels of malondialdehyde (MDA) compared to the LP group (P < 0.05). The apoptosis rate of intestinal cells in the LP group was significantly higher than those in the LP and HP groups (P < 0.05). The mRNA expression levels of superoxide dismutase (sod), nuclear factor kappa B p65 subunit (nfκb-p65), heat shock protein 70 (hsp70), and inhibitor of NF-κBα (iκba) in the intestine were significantly higher in the LP group than those in the HP and LP-AA groups (P < 0.05). The 16s RNA analysis indicated that EAA supplementation significantly increased the growth of Desulfovibrio and altered the intestinal microflora. The relative abundances of Firmicutes and Cyanobacteria were positively correlated with antioxidant parameters (CAT and T-AOC), whereas Desulfobacterota was negatively correlated with sod and T-AOC. The genera Bacillus, Bacteroides, and Rothia were associated with the favorable maintenance of gut health. In conclusion, dietary supplementation with EAAs to achieve a balanced amino acid profile could potentially reduce the dietary protein levels from 48.66 % to 44.44 % without adversely affecting the growth and intestinal health of juvenile blotched snakeheads.

Production of all-male non-transgenic zebrafish by conditional primordial germ cell ablation.

Many fish species exhibit remarkable sexual dimorphism, with males possessing numerous advantageous traits for commercial production by aquaculture such as faster growth rate, more efficient food energy utilization for muscle development, and better breeding performance. Several studies have shown that a decrease in the number of primordial germ cells (PGCs) during early development leads predominantly to male progeny. In this study, we developed a method to obtain all-male zebrafish (Danio rerio) by targeted PGC ablation using the nitroreductase/metronidazole (NTR/Mtz) system. Embryos generated by female heterozygous Tg(nanos3:nfsB-mCherry-nanos3 3'UTR) and male wild-types (WTs) were treated with vehicle or Mtz. Compared to vehicle-treated controls, 5.0 and 10.0 mM Mtz treatment for 24 h significantly reduced the number of PGCs and yielded an exclusively male phenotype in adulthood. The gonads of offspring treated with 5.0 mM Mtz exhibited relatively normal morphology and histological characteristics. Furthermore, these males were able to chase females, spawn, and produce viable offspring, while about 20.0% of males treated with 10.0 mM Mtz were unable to produce viable offspring. The 5.0 mM Mtz treatment protocol may thus be suitable for large-scale production of fertile male offspring. Moreover, about half of these males were WT as evidenced by the absence of nfsB gene expression. It may thus be possible to breed an all-male WT fish population by Mtz-mediated PGC ablation.

Characterisation of scavenger receptor class B type 1 in rare minnow (Gobiocypris rarus).

Scavenger receptor class B type 1 (SRB1) is a transmembrane protein belonging to the scavenger receptors (SRs) family and it plays an important role in viral entry. Not much is known on SRB1 in teleost fish. Grass carp reovirus (GCRV) cause huge economic losses in grass carp industry. In this study, rare minnow (Gobiocypris rarus) was used as a model fish to investigate the mechanism of GCRV infection, which is sensitive to GCRV. The structure of SRB1 gene in G. rarus (GrSRB1) was cloned and elucidated. GrSRB1 is composed of 13 exons and 12 introns, and its full-length cDNA is 2296 bp in length, with 1521 bp open reading frame (ORF) that encodes a 506 amino acid protein. The GrSRB1 protein is predicted to contain a typical CD36 domain and two transmembrane regions. In G. rarus, GrSRB1 is expressed strongly in the liver (L), intestines (I), brain (B) and muscle (M), while it is expressed poorly in the heart (H), middle kidney (MK), head kidney (HK) and gills (G). After infection with GCRV, GrSRB1 expression was up-regulated in main immune tissues during the early infection period. Moreover, co-immunoprecipitation assays revealed that GrSRB1 could interact with the outer capsid protein of GCRV (VP5 and VP7). These results suggest that GrSRB1 could be a receptor for GCRV. We have managed to characterize the GrSRB1 gene and provide evidence for its potential functions for GCRV entry into host cells.

Molecular cloning of the MARCH family in grass carp (Ctenopharyngodon idellus) and their response to grass carp reovirus challenge.

Grass carp (Ctenopharyngodon idellus) is an economical aquaculture species in China, and the Grass Carp Reovirus (GCRV) that causes hemorrhagic disease seriously affects the grass carp cultivation industry. Substantial evidence indicates that there is an association between the membrane-associated RING-CH family of E3 ligase (MARCH) family and immune defense in mammals, while functional studies on non-mammalian MARCH proteins are limited. In order to know the characteristics of the MARCH genes in C. idellus, eight MARCH genes (MARCH1, 2, 5, 6, 7, 8, 9 and 11) were cloned and the open reading frames (ORF) were identified in grass carp. All MARCH proteins in grass carp contained an RING-CH domain, which is characteristic of the MARCH protein. The phylogenetic analysis revealed that different MARCH proteins gathered into their separate clusters. All eight members of the MARCH gene family were detected in all tissues sampled, but the relative expression level differed. In addition, the mRNA expression of all the MARCHs was regulated at different levels in the immune organs after a GCRV challenge, and they responded robustly in both the intestine and liver. The mRNA expression of MARCH8, MHC II, TfR, IL1RAP, EGR1, and DUSP1 in the intestine after GCRV infection was analyzed, and the results showed that MARCH8 could negatively regulate TfR, IL1RAP, EGR1, and DUSP1, which signaled via the MAPK or NF-κB-activation pathways that play vital roles in immunity. Our findings identified a novel gene family in C. idellus and provided novel evidence that MARCH genes are inducible and involved in the immune response. Moreover, MARCH8 might function to negatively regulate immune receptors in C. idellus. Therefore, the MARCH might play a vital role in regulating the immune response of C. idellus.

The New Roles of traf6 Gene Involved in the Development of Zebrafish Liver and Gonads.

Traf6, an adaptor protein, exhibits non-conventional E3 ubiquitin ligase activity and was well studied as an important factor in immune systems and cancerogenesis. In mice, the traf6-null caused a perinatal death, so that the underlying pathophysiology of traf6-defeciency is still largely unclear in animals. Here, in the present study, a traf6 knockout zebrafish line (traf6-/-) was generated and could survive until adulthood, providing a unique opportunity to demonstrate the functions of traf6 gene in animals' organogenesis beyond the mouse model. The body of traf6-/- fish was found to be significantly shorter than that of the wildtype (WT). Likewise, a comparative transcriptome analysis showed that 866 transcripts were significantly altered in the traf6-/- liver, mainly involved in the immune system, metabolic pathways, and progesterone-mediated oocyte maturation. Especially, the mRNA expression of the pancreas duodenum homeobox protein 1 (pdx1), glucose-6-phosphatase (g6pcb), and the vitellogenesis genes (vtgs) were significantly decreased in the traf6-/- liver. Subsequently, the glucose was found to be accumulated in the traf6-/- liver tissues, and the meiotic germ cell was barely detected in traf6-/- testis or ovary. The findings of this study firstly implied the pivotal functions of traf6 gene in the liver and gonads' development in fish species.

Molecular Characterization, Expression Pattern, DNA Methylation and Gene Disruption of Figla in Blotched Snakehead (Channa maculata).

Figla is one of the earliest expressed genes in the oocyte during ovarian development. In this study, Figla was characterized in C. maculata, one of the main aquaculture species in China, and designated as CmFigla. The length of CmFigla cDNA was 1303 bp, encoding 197 amino acids that contained a conserved bHLH domain. CmFigla revealed a female-biased expression patterns in the gonads of adult fish, and CmFigla expression was far higher in ovaries than that in testes at all gonadal development stages, especially at 60~180 days post-fertilization (dpf). Furthermore, a noteworthy inverse relationship was observed between CmFigla expression and the methylation of its promoter in the adult gonads. Gonads at 90 dpf were used for in situ hybridization (ISH), and CmFigla transcripts were mainly concentrated in oogonia and the primary oocytes in ovaries, but undetectable in the testes. These results indicated that Figla would play vital roles in the ovarian development in C. maculata. Additionally, the frame-shift mutations of CmFigla were successfully constructed through the CRISPR/Cas9 system, which established a positive foundation for further investigation on the role of Figla in the ovarian development of C. maculata. Our study provides valuable clues for exploring the regulatory mechanism of Figla in the fish ovarian development and maintenance, which would be useful for the sex control and reproduction of fish in aquaculture.

Effects of Myostatin b Knockout on Offspring Body Length and Skeleton in Yellow Catfish (Pelteobagrus fulvidraco).

Based on obtaining mstnb gene knockout in Pelteobagrus fulvidraco, a study on the effect of the mstn gene on skeletal morphology and growth was performed by comparing the number and length of the vertebrae of mutant and wild-type fish in a sibling group of P. fulvidraco, combined with the differences in cells at the level of vertebral skeletal tissue. It was found that mstnb gene knockdown resulted in a reduction in the number of vertebrae, the length, and the intervertebral distance in P. fulvidraco, and these changes may be the underlying cause of the shorter body length in mutant P. fulvidraco. Further, histological comparison of the same sites in the mstn mutant and wild groups of P. fulvidraco also revealed that the number and density of osteocytes were greater in mstnb knockout P. fulvidraco than in wild-type P. fulvidraco. Our results demonstrated that when using genome editing technology to breed new lines, the effects of knockout need to be analyzed comprehensively and may have some unexpected effects due to insufficient study of the function of certain genes.

Characterization, expression and CpG methylation analysis of Dmrt1 and its response to steroid hormone in blotched snakehead (Channa maculata).

Dmrt1 is an important transcriptional regulator that plays critical role in male gonadogenesis, testicular differentiation and development. In this study, Dmrt1 was cloned from blotched snakehead (Channa maculata), which is designated as CmDmrt1. CmDmrt1 encoded a putative protein with 293 amino acids and presented an extremely conserved DM domain. It was nearly expressed in the gonads, and the expression was more than 15 times higher in the testis than in the ovary. 1851 bp promoter sequence of CmDmrt1 was characterized and the methylation levels of the CpG sites were analyzed to detect sex-related differences. A significant negative correlation between CmDmrt1 expression and CpG methylation level of its promoter was found in the testis and ovary. During gonadal development, CmDmrt1 transcription displayed strong male-biased expression patterns, increased with the maturation of testis and reached the peak at 195 days after hatching (dah), which indicates a significant role of Dmrt1 in spermatogenesis. Steroid treatment could influence CmDmrt1 expression, and long-term 17ß-estradiol (E2) treatment could induce the male-to-female secondary sex reversal (SSR), which resulted in the differentiated testis transformed to ovary or ovotestis. Meanwhile, CmDmrt1 expression was down-regulated to fairly low level in the ovary of the SSR XY fish, which was similar to that in normal XX females ovary. Our research illustrates that Dmrt1 is linked to testis differentiation and spermatogenesis in blotched snakehead, providing information for functional studies on sex differentiation and gonadal development of C. maculata, and scientific basis for the production practice of all-male snakehead breeding.

Cloning and expression analysis of GATA1 gene in Carassius auratus red var.

BACKGROUND: GATA1 is a key transcription factor in the GATA family, and promotes the differentiation and maturation of red blood cell, which is essential for normal hematopoiesis. RESULTS: Our results showed that the cDNA sequence of GATA1 was 2730 bp long encoding 443 amino acids. qRT-PCR analysis demonstrated that GATA1 had the highest expression in testis (T), followed by pituitary (P) and spleen (S). GATA1 gene expression in C. auratus red var. embryo from the neuroblast stage (N) to the embryo hatching (H) changes continuously; and the gene expression levels of nonylphenol (NP)-treated and those of control embryos were significantly different. Moreover, Methylation levels of GATA1 gene in NP-treated embryos were higher than those in control embryos, indicating that NP affected GATA1 methylation. CONCLUSIONS: Our study provides cues for further studying the roles of GATA1 gene in fish development, and suggested a potential molecular mechanism by which NP leads to abnormal development of fish embryos.

Chromosome-level genome assemblies of Channa argusandChanna maculata and comparative analysis of their temperature adaptability.

BACKGROUND: Channa argus and Channa maculata are the main cultured species of the snakehead fish family, Channidae. The relationship between them is close enough that they can mate; however, their temperature adaptability is quite different. RESULTS: In this study, we sequenced and assembled the whole genomes of C. argus and C. maculata and obtained chromosome-level genome assemblies of 630.39 and 618.82 Mb, respectively. Contig N50 was 13.20 and 21.73 Mb, and scaffold N50 was 27.66 and 28.37 Mb, with 28,054 and 24,115 coding genes annotated for C. argus and C. maculata, respectively. Our analyses showed that C. argus and C. maculata have 24 and 21 chromosomes, respectively. Three pairs of chromosomes in C. argus correspond to 3 chromosomes in C. maculata, suggesting that 3 chromosomal fusion events occurred in C. maculata. Comparative analysis of their gene families showed that some immune-related genes were unique or expandable to C. maculata, such as genes related to herpes simplex infection. Analysis of the transcriptome differences related to temperature adaptation revealed that the brain and liver of C. argus rapidly produced more differentially expressed genes than C. maculata. Genes in the FoxO signalling pathway were significantly enriched in C. argus during the cooling process (P < 0.05), and the expression of 3 transcription factor genes in this pathway was significantly different between C. argus and C. maculata (P < 0.01). CONCLUSIONS: C. maculata may have higher resistance to certain diseases, whereas C. argus has a faster and stronger response to low-temperature stress and thus has better adaptability to a low-temperature environment. This study provides a high-quality genome research platform for follow-up studies of Channidae and provides important clues regarding differences in the low-temperature adaptations of fish.

De novo screening of disease-resistant genes from the chromosome-level genome of rare minnow using CRISPR-cas9 random mutation.

BACKGROUND: Mutants are important for the discovery of functional genes and creation of germplasm resources. Mutant acquisition depends on the efficiency of mutation technology and screening methods. CRISPR-Cas9 technology is an efficient gene editing technology mainly used for editing a few genes or target sites, which has not been applied for the construction of random mutant libraries and for the de novo discovery of functional genes. RESULTS: In this study, we first sequenced and assembled the chromosome-level genome of wild-type rare minnow (Gobiocypris rarus) as a susceptible model of hemorrhagic disease, obtained a 956.05 Mb genome sequence, assembled the sequence into 25 chromosomes, and annotated 26,861 protein-coding genes. Thereafter, CRISPR-Cas9 technology was applied to randomly mutate the whole genome of rare minnow with the conserved bases (TATAWAW and ATG) of the promoter and coding regions as the target sites. The survival rate of hemorrhagic disease in the rare minnow gradually increased from 0% (the entire wild-type population died after infection) to 38.24% (F3 generation). Finally, 7 susceptible genes were identified via genome comparative analysis and cell-level verification based on the rare minnow genome. CONCLUSIONS: The results provided the genomic resources for wild-type rare minnow, and confirmed that the random mutation system designed using CRISPR-Cas9 technology in this study is simple and efficient and is suitable for the de novo discovery of functional genes and creation of a germplasm resource related to qualitative traits.

Comparative transcriptome analysis on four types of gonadal tissues of blotched snakehead (Channa maculata).

Blotched snakehead (Channa maculata) is an economically important freshwater fish in China, of which males grow much faster than females. To illuminate the molecular mechanism of sex differentiation and gonad development, RNA-Sequencing was performed to identify sex-related genes and pathway in gonads of 6-month-old normal XX females (XX-F), normal XY males (XY-M), XY sex reversal females (XY-F) and YY super-males (YY-M). The analysis showed that many differentially expressed genes (DEGs) had similar expression patterns in XY-F and XX-F, which were different from XY-M and YY-M. qRT-PCR indicated that Amh, Dmrt1, and Sox9 had relatively high expression in testes of XY-M and YY-M. Taking Amh as an example, there was a relative fold change of 1.0 in XX-F, 2.1 fold change in XY-F, 36.1 fold change in XY-M, and 26.0 fold change in YY-M. Cyp19a1a, Figla, and Foxl2 were highly expressive in ovaries of XX-F and XY-F. Taking Figla as an example, there was a relative fold change of 557 in XX-F, 304.5 fold change in XY-F, 5.6 fold change in XY-M, and 4.4 fold change in YY-M. KEGG analysis revealed many DEGs distributed in pathways related to sex differentiation, steroid hormone synthesis and growth, etc. Significant variation and trends in relative expression levels tested by qRT-PCR were consistent with those recorded by RNA-Sequencing. This is the first time that transcriptome of snakehead has been investigated systematically and in an integrated way. Large quantities of candidate genes involved in sex differentiation, gonad development and growth dimorphism were identified. The study provides useful resources for understanding sex differentiation and growth dimorphism, potentially assisting mono-sex production of snakehead in aquaculture.

Expression profile and estrogenic regulation of Amh during gonadal sex differentiation in northern snakehead (Channa argus).

BACKGROUND: Anti-Müllerian hormone (Amh) plays a critical role in both early sex determination and later gonad development in vertebrate species. However, it remains unknown in northern snakehead (Channa argus), which is economically important freshwater fish with sexual dimorphism. OBJECTIVE: This study aimed to identify the expression profiles and estrogenic regulation of CaAmh during gonadal sex differentiation in C. argus. METHODS: The cDNA and genomic DNA sequences of CaAmh were identified by PCR and RACE techniques. The expression patterns of CaAmh were detected by qRT-PCR during the gonadal sex differentiation and after 17α-ethinyloestradiol (EE2) treatments. RESULTS: CaAmh is composed of seven exons and six introns, and its full-length cDNA is 2413 bp in length, with 1635 bp open reading frame (ORF) that encodes a 544 amino acid protein. Tissues expression patterns revealed that CaAmh display the highest expression in testis of XY males (40.36 folds, p < 0.01). The spatio-temporal expression patterns during gonadal sex differentiation indicated that CaAmh expression differed between XX females and XY males at 30 day after hatching (dah), and reached to the peak (36.03 folds, p < 0.01) at 90 dah in XY gonads. However, CaAmh expression in XX gonads remained low throughout the sampling period. Furthermore, CaAmh expression in the gonads (ovaries) of the sex-reversed XY fish (XY-F) by the administration of estrogen EE2 was downregulated to low level, similar to that in ovaries of normal XX females (XX-F). CONCLUSIONS: These results show that Amh plays a critical role in testicular differentiation of C. argus and it is apparently modulated by estrogens in this species.

The DNA methylation level is associated with the superior growth of the hybrid fry in snakehead fish (Channa argus × Channa maculata).

Hybrid vigour, or heterosis, refers to the increased productivity and growth rate of hybrid offsprings relative to the parents. Various heterosis have been well exploited in fish for fisheries. However, the molecular mechanisms underlying heterosis are largely unknown in fish. In this study, two inbred and hybrid lines between the northern snakehead (NS, Channa argus) and blotched snakehead (BS, Channa maculata) were generated. The analysis on various growth traits, including body length, head length, and body height, showed that hybrid fry obviously exhibited a spontaneous growth heterosis over the inbred. Moreover, the methylation-sensitive amplification polymorphism (MSAP) analysis revealed that the DNA methylation levels were negatively related to the body growth in all fry. Especially, the DNA methylation levels in the hybrid fry were significantly lower than those in the inbred. Additionally, qRT-PCR showed that the snakehead fish Dnmt3a mRNA was initially detectable in embryos at 12 hpf and gradually increased as developing. Intriguingly, the level of Dnmt3a mRNA expression was found to be closely correlated to the DNA methylation level in embryos/fry. The results of this study firstly demonstrated the correlations between growth heterosis, DNA methylation level and Dnmt3a mRNA expression in fish fry. The findings of this study implied that the hybrids' heterosis formation is probably accompanied by DNA methylation alterations and modulated by Dnmt3a gene in fish. This study would provide new clues for further investigations on mechanisms behind heterosis formation in fish hybrid.

An NGS-based approach for the identification of sex-specific markers in snakehead (Channa argus).

We described a next generation sequencing (NGS)-based approach to identify sex-specific markers and subsequently determine whether a species has male or female heterogamety. To test the accuracy of this technique, we examined the snakehead (Channa argus), which is economically important freshwater fish in China. Males grow faster than females, and there is significant interest in developing methods to skew breeding towards all-males to increase biomass yields. NGS was conducted on DNAs of individual female and male, the male reads were spitted into 60 bp K-mers and aligned to the female reference genome assembled by female reads, unaligned male K-mers-60 were kept in next filter process. Meanwhile, DNA sample of 48 females was pooled and sequenced, this data was further used to filter out the previous unaligned male K-mers-60. Hence, numbers of candidate Y chromosome-specific sequences were screened out, their sex-specificity were validated in wild snakeheads through PCR amplification. Finally, three Y chromosome-specific fragments (Contig-275834, Contig-359642, and Contig-418354) were identified, and specific primers were obtained to distinguish the sex of snakehead. Additionally, a pair of primers of Contig-275834 (275834X/Y-F and 275834X/Y-R) was exploited to distinguish XX females, XY males, and YY super-males, whose amplification products of different lengths were produced for different sexes. Therefore, our work demonstrated the ability of NGS data in identification of sex-specific markers, and the pipeline adopted in our study could be applied in any species of sex differentiation. Furthermore, the sex-specific markers have tremendous potential for improving the efficiency of all-male breeding practices in snakehead.