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The surprising variation in the number of Hox clusters and genome structure in ray-finned fish lineages reflects the history of duplications and subsequent lineage-specific gene loss. However, there are few studies on whether Hox clusters in the early generations of hybrid lineages show more significant variation due to the continuous genomic oscillation caused by distant hybridization. We sequenced and analyzed Hox gene clusters from a crucian carp-like homodiploid fish (NCRC) lineage (a new hybrid lineage derived from common carp (Cyprinus carpio) (♀) ​× ​blunt snout bream (Megalobrama amblycephala) (♂)). In the NCRC lineage, we reconstructed seven Hox clusters consisting of 48 Hox genes, ten of which were pseudogenes. The number of putative Hox clusters generated in NCRC-F1 was increased greatly by distant hybridization to an average number almost twice that in the maternal parent. This increasing trend continued in the subsequent self-mating generations of NCRC-F1. In contrast, the number of Hox cluster fragments inherited from the original parents gradually decreased as the number of NCRC lineage generations increased. This pattern was also found in the inheritance of recombinant Hox clusters. In terms of base composition, some genetic rules for the inheritance of these Hox clusters between different generations of the NCRC lineage were identified. Furthermore, the newly derived mutated Hox clusters in the NCRC lineage showed phylogenetic relationships that were closer to either crucian carp or silver crucian carp, revealing a clear evolutionary path. This study deepens our understanding of the evolution of Hox genes in the ray-finned fish clade.

The number of olfactory receptor genes (ORs), which are responsible for detecting diverse odor molecules varies extensively among mammals as a result of frequent gene gains and losses that contribute to olfactory specialization. However, how OR expansions/contractions in fish are influenced by habitat and feeding habit and which OR subfamilies are important in each ecological niche is unknown. Here, we report a major OR expansion in a freshwater herbivorous fish, Megalobrama amblycephala, using a highly contiguous, chromosome-level assembly. We evaluate the possible contribution of OR expansion to habitat and feeding specialization by comparing the OR repertoire in 28 phylogenetically and ecologically diverse teleosts. In total, we analyzed > 4,000 ORs including 3,253 intact, 122 truncated, and 913 pseudogenes. The number of intact ORs is highly variable ranging from 20 to 279. We estimate that the most recent common ancestor of Osteichthyes had 62 intact ORs, which declined in most lineages except the freshwater Otophysa clade that has a substantial expansion in subfamily β and ε ORs. Across teleosts, we found a strong association between duplications of β and ε ORs and freshwater habitat. Nearly, all ORs were expressed in the olfactory epithelium (OE) in three tested fish species. Specifically, all the expanded β and ε ORs were highly expressed in OE of M. amblycephala. Together, we provide molecular and functional evidence for how OR repertoires in fish have undergone gain and loss with respect to ecological factors and highlight the role of β and ε OR in freshwater adaptation.

Abstract— Based on the morphological features, four rare alien species in the Kapchagai reservoir were identified: Coregonus peled, Parasalmo mykiss, Megalobrama mantschuricus, and Oreochromis niloticus. The latter two species were recorded for the ichthyofauna of Kazakhstan for the first time. Self-reproducing P. mykiss populations have been known in the Balkhash Basin since the late 1990s, whereas the introduction of C. peled into the Ili basin in 1968−1969 and 1971 did not lead to the naturalization of the species. The reasons for the great diversity of invasive fish species in the Kapchagai reservoir are overviewed.

Background Hybridization, which can quickly merge two or more divergent genomes and form new allopolyploids, is an important technique in fish genetic breeding. However, the merged subgenomes must adjust and coexist with one another in a single nucleus, which may cause subgenome interaction and dominance at the gene expression level and has been observed in some allopolyploid plants. In our previous studies, newly formed allodiploid hybrid fish derived from herbivorous Megalobrama amblycephala (♀) × carnivorous Culter alburnus (♂) had herbivorous characteristic. It is thus interesting to further characterize whether the subgenome interaction and dominance derive dietary adaptation of this hybrid fish. Results Differential expression, homoeolog expression silencing and bias were investigated in the hybrid fish after 70 days of adaptation to carnivorous and herbivorous diets. A total of 2.65 × 108 clean reads (74.06 Gb) from the liver and intestinal transcriptomes were mapped to the two parent genomes based on specific SNPs. A total of 2538 and 4385 differentially expressed homoeologous genes (DEHs) were identified in the liver and intestinal tissues between the two groups of fish, respectively, and these DEHs were highly enriched in fat digestion and carbon metabolism, amino acid metabolism and steroid biosynthesis. Furthermore, subgenome dominance were observed in tissues, with paternal subgenome was more dominant than maternal subgenome. Moreover, subgenome expression dominance controlled functional pathways in metabolism, disease, cellular processes, environment and genetic information processing during the two dietary adaptation processes. In addition, few but sturdy villi in the intestine, significant fat accumulation and a higher concentration of malondialdehyde in the liver were observed in fish fed carnivorous diet compared with fish fed herbivorous diet. Conclusions Our results indicated that diet drives phenotypic and genetic variation, and the asymmetric expression of homoeologous genes (including differential expression, expression silencing and bias) may play key roles in dietary adaptation of hybrid fish. Subgenome expression dominance may contribute to uncovering the mechanistic basis of heterosis and also provide perspectives for fish genetic breeding and application.

Ribonuclease 1 (RNase1) is a vertebrate-specific enzyme that mainly performs digestive activity in herbivorous mammals. Here we used bacterial viability assays to explore its antimicrobial activity in blunt snout bream (Megalobrama amblycephala). The results showed that Ma-RNase1 rapidly killed Gram-negative and Gram-positive bacteria at micromolar concentrations. Ma-RNase1 increased the permeability of bacterial outer and inner membranes, thus reducing the integrity of bacterial cell wall and membrane. Moreover, Ma-RNase1 effectively counteracted the tissue damage and apoptosis caused by Aeromonas hydrophila infection. Quantitative real-time PCR and immunoblot analysis indicated that RNase1 mRNA and protein were up-regulated in the kidney and gut during infection. Furthermore, A. hydrophila infection significantly induced Tnf-α and Il-1β mRNA expression in liver, but not in the RNase1 pre-treatment group. In addition, a significant increase in the expression of immune-related genes (Nf-κb and Tlr4) was found in liver, kidney and gut of A. hydrophila-infected fish, while a decrease in Myd88 and Tlr4 levels was found in liver, spleen, kidney and gut in the group pre-treated with RNase1. Collectively, these data suggest that Ma-RNase1 has antimicrobial function both in vitro and in vivo, and contributes to the protective effect and immune defense of blunt snout bream.

Intermuscular bone (IB) is a hard-boned spicule exist in lower teleost, which brings a lot of detrimental effects on palatability and economic value of blunt snout bream (Megalobrama amblycephala). Masson trichrome staining for ossific IB indicated that some osteoblasts appeared at the edge of the bone matrix and a few osteocytes are present in the center of the mineralized bone matrix. By comparing the orthologous gene families of fish with IBs and without IBs, we screened the key signaling pathways associated with IB formation. Furthermore, the transcriptomic data demonstrated the functional importance of these gene families. The candidate genes involved in chondrocyte development were highly expressed in stage 1 compared with stage 2 and stage 3, suggesting that the development process of IB might mainly involve in intramembranous ossification. Our research reveals the molecular mechanism of IBs formation, and provides molecular evidence for the further study on intermuscular boneless stains.

Morphological traits measurement still plays a key role in description, identification, classification and phylogenetics of species. However, the traditional morphological measurement method is inefficient and often destructive to the studied organism. In this study, we propose and test an X‐ray‐based technique for non‐destructive morphological studies of fish. We use this approach to attempt to discriminate four species from the genus Megalobrama on the basis of their morphometric and meristic traits. The results showed that there were significant differences (p

Background : Maternal effects contribute to adaptive significance for shaping various phenotypes of many traits. Potential implications of maternal effects are the cause of expression diversity, but these effects on mRNA expression and alternative splicing (AS) have not been fully elucidated in hybrid animals. Results: Two reciprocal cross hybrids following hybridization of Megalobrama amblycephala (blunt snout bream, BSB) and Culter alburnus (topmouth culter, TC) were used as a model to investigate maternal effects. In a comparison of BSB- and TC- homoeolog expression from the two reciprocal cross hybrids, we identified 49–348 differentially expressed BSB-homoeologous genes and 54–354 differentially expressed TC-homoeologous genes. 2402, 2959, and 3418 AS events between the two reciprocal cross hybrids were detected in Illumina data of muscle, liver, and gonads, respectively. Moreover, 21,577 (TC-homoeologs) and 30,007 (BSB-homoeologs) AS events were found in the 20,131 homoeologous gene pairs of TBF3 based on PacBio data, while 30,561 (TC-homoeologs) and 30,305 (BSB-homoeologs) AS events were found in BTF3. These results further improve AS prediction at the homoeolog level. The various AS patterns in bmpr2a belonged to the bone morphogenetic protein family were selected as an AS model to investigate expression diversity and their potential effects to body shape traits.Conclusions: The distribution of differentially expressed genes and AS in BSB- and TC-subgenomes exhibited various changes between the two reciprocal cross hybrids, suggesting that maternal effects were the cause of expression diversity. These findings provide a novel insight into mRNA expression changes and AS under maternal effects in lower vertebrates.

Two in vitro trials were conducted to identify a peptide with antioxidant activity and immunoenhancement from cottonseed meal protein hydrolysate (CPH) for fish. Primary hepatocytes of Megalobrama amblycephala were treated with CPH. In experiment 1, CPH significantly increased aspartate aminotransferase (GOT), alanine aminotransferase (GPT), total superoxide dismutase (t-SOD), catalase (CAT), and lysozyme activities, as well as up-regulated SOD, CAT, antimicrobial peptides 1 (Leap 1) and Leap 2 mRNA levels (p 0.05). However, CPH significantly down-regulated the expression of NADPH oxidase-2 (NOX2), Kelch-like-ECH-associated protein 1 (Keap1), NF-E2-related factor 2 (Nrf2) and BTB and CNC homolog 1 (Bach1) mRNA (p 0.05) in fish hepatocytes. Experiment 2 showed that the molecular mass of CPH was distributed mainly in the 700-1024 Da range. Peptide 1 (P1) and P2 significantly decreased GOT and GPT activities in conditioned medium (p 0.05); however, P4 and P6 did not affect GOT and GPT activities (p 0.05). Furthermore, P4 significantly increased hepatocyte GOT, GPT, t-SOD, CAT levels and lysozyme activities (p 0.05), up-regulated SOD, CAT, Leap1 and Leap2 mRNA expression levels, and down-regulated the expression of Nrf2 and NOX2 mRNA (p 0.05) in fish hepatocytes. The above results indicated that CPH and P4 enhanced hepatocyte metabolism, as well as improved antioxidant capacities and innate immunity of blunt snout bream hepatocytes.

In fish breeding practices, gamete maturity of females is vital to reproductive success. For some species, it is possible to estimate the female maturation status based on abdomen observation, but quite difficult for some species which mature at big size. To screen out the potential biomarker in fish blood relating to female maturation, we employed the approach integrating the UPLC-MS/MS and RNA-seq techniques to investigate the metabolites and genes reflecting the sexual maturation and spawning of female blunt snout bream Megalobrama amblycephala. The study included four groups, 1-year-old immature female individuals, 2-year-old immature female individuals, 2-year-old sexually mature female individuals, and 2-year-old sexually mature female individuals after 24 h of successful spawning. The upregulated metabolites in mature females were involved in "steroid hormone biosynthesis," "metabolic pathways," "glycerophospholipid metabolism," etc. compared with those of immature individuals. As the key intermediate of steroid hormone biosynthesis, 17α-hydroxypregnenolone exhibited the highest level in 2-year-old mature females than in the immature females. Meanwhile, the metabolites (i.e., dodecanoic acid and myristic acid) participating in fatty acid synthesis exhibited much lower levels in the females after spawning than those before spawning. In addition to the metabolites, the genes involved in ovarian steroidogenesis were significantly upregulated in the 2-year-old immature females compared to the 1-year-old immature females, indicating that the ovarian steroidogenesis plays important roles in ovarian development of M. amblycephala at the early stages. The significant upregulation of genes (i.e., itpr1, camk2, and mekk2) involved in the "GnRH signaling pathway" was observed in the mature females compared with the immature females, which indicated that the estrogen levels increased after female maturation in M. amblycephala. Moreover, many genes (e.g., gck, creb1, tf2-9, ryr2, asgr1, and creb1) regulating insulin secretion and thyroid hormone synthesis were significantly downregulated after female spawning. The dynamics of gene expression and metabolites observed in this study provide novel cues for guiding fish practical artificial reproduction.

The olfactory organ is an important chemoreceptor in vertebrates. However, the sexual disparities in gene expression patterns in the olfactory organ in fish remain unstudied. Here, we conducted a transcriptome analysis of the olfactory epithelium (OE) of male and female blunt snout bream (Megalobrama amblycephala) to identify the differences. The histological analysis showed that there were 22 leaf-like olfactory lamellaes on one side of the OE of the adult blunt snout bream. The sensory area of OE is enriched with ciliated receptor cells and microvilli receptor cells. The transcriptome analysis showed that only 10 out of 336 olfactory receptor genes (224 ORs, 5 V1Rs, 55 V2Rs, and 52 TAARs) exhibited significant expression differences between males and females, and most of the differentially expressed genes were related to the immune system. We also validated these results using qPCR: 10 OR genes and 6 immunity-related genes significantly differed between males and females. The FISH analysis results indicated that the ORs were mainly expressed at the edge of the olfactory lamellae. Collectively, our study reveals that gender is not an important factor influencing the expression of olfactory receptors, but the expression of immune genes varies greatly between the genders in blunt snout bream.

Circular RNAs (circRNAs), a novel class of endogenous RNAs, have been recognized to play important roles in the growth of animals. However, the regulatory mechanism of circRNAs on fish muscle growth is still unclear. In this study, we performed whole transcriptome analysis of skeletal muscles from two populations with different growth rates (fast-growing and slow-growing) of blunt snout bream (Megalobrama amblycephala), an important fish species for aquaculture. The selected circRNAs were validated by qPCR and Sanger sequencing. Pairs of circRNA–miRNA–mRNA networks were constructed with the predicted differentially expressed (DE) pairs, which revealed regulatory roles in muscle myogenesis and hypertrophy. As a result, a total of 445 circRNAs were identified, including 42 DE circRNAs between fast-growing (FG) and slow-growing (SG) groups. Many of these DE circRNAs were related with aminoglycan biosynthetic and metabolic processes, cytokinetic processes, and the adherens junction pathway. The functional prediction results showed that novel_circ_0001608 and novel_circ_0002886, competing to bind with dre-miR-153b-5p and dre-miR-124-6-5p, might act as competing endogenous RNAs (ceRNAs) to control MamblycephalaGene14755 (pik3r1) and MamblycephalaGene10444 (apip) level, respectively, thus playing an important regulatory role in muscle growth. Overall, these results will not only help us to further understand the novel RNA transcripts in M. amblycephala, but also provide new clues to investigate the potential mechanism of circRNAs regulating fish growth and muscle development.

Distant hybridization is an important technique in fish genetic breeding. In this study, based on the establishment of an allodiploid fish lineage (BT, 2n=48, F1–F6) derived from distant hybridization between female Megalobrama amblycephala (BSB, 2n=48) and male Culter alburnus (TC, 2n=48), and the backcross progeny (BTB, 2n=48) derived by backcrossing female F1 of BT to male BSB, an improved hybrid bream (BTBB, 2n=48) was obtained by backcrossing BTB (♀) to BSB (♂). Moreover, the morphological and genetic characteristics of BTBB individuals were investigated; BTBB was similar to BSB in appearance but had a higher body height than BSB. The study results regarding chromosome numbers and DNA content indicated that BTBB is a diploid hybrid fish. The 5S rDNA and Hox gene of BTBB were inherited from the original parents. Gonadal development in BTBB was normal. On the other hand, BTBB had a faster growth rate, higher muscle protein level, and lower muscle carbohydrate level than BSB. Hence, bisexual fertile BTBB is promoted and can be applied as a high-quality fish, and it can also be used as a new fish germplasm resource to develop high-quality fish further. Thus, this study is of great significance for fish genetic breeding.

Distant hybridization can combine whole genomes from parent species and result in changes in the phenotypes and genotypes in hybrids. The characteristics of many hybrid fishes with even number of chromosomes have been reported, but the hybrids with odd number chromosomes are rarely reported. Blunt snout bream (Megalobrama amblycephala, BSB, 2n = 48) and rare gudgeon (Gobiocypris rarus, RG, 2n = 50) belong to two different subfamilies and have quite different biological characteristics. In this study, we obtain the hybrids (BR) derived from the inter-subfamily hybridization of female BSB and male RG. We investigate the fertilization rate, hatching rate, morphological traits, chromosomal numbers, DNA content, growth rates, and 5S rDNA in the BR. The results show that the BR is an allodiploid fish with 49 chromosomes, and all the measurable traits are significantly different (p < 0.05) among BR, BSB, and BR. Interestingly, the upper part of the BR body color is similar to BSB (gray), the lower part of the BR body color is similar to RG (light yellow), and the BR inherits a unique light yellow wide longitudinal band from the RG. Furthermore, the BR has a fast growth rate compared with RG. The 5S rDNA of the BR inherits the specific bands of its parental 5S rDNA respectively and has some mutations, which show obvious recombination, heredity, and variability in BR. This study will be of great significance in fish genetic breeding.

In terms of taxonomic status, common carp ( Cyprinus carpio , Cyprininae) and crucian carp ( Carassius auratus , Cyprininae) are different species; however, in this study, a newborn homodiploid crucian carp-like fish (2 n =100) (2 n NCRC) lineage (F1–F3) was established from the interspecific hybridization of female common carp (2 n =100)×male blunt snout bream ( Megalobrama amblycephala , Cultrinae, 2 n =48). The phenotypes and genotypes of 2 n NCRC differed from those of its parents but were closely related to those of the existing diploid crucian carp. We further sequenced the whole mitochondrial (mt) genomes of the 2 n NCRC lineage from F1 to F3. The paternal mtDNA fragments were stably embedded in the mt-genomes of F1–F3 generations of 2 n NCRC to form chimeric DNA fragments. Along with this chimeric process, numerous base sites of F1–F3 generations of 2 n NCRC underwent mutations. Most of these mutation sites were consistent with the existing diploid crucian carp. Moreover, the mtDNA organization and nucleotide composition of 2 n NCRC were more similar to those of the existing diploid crucian carp than those of the parents. The inheritable chimeric DNA fragments and mutant loci in the mt-genomes of different generations of 2 n NCRC provided important evidence of the mtDNA change process in the newborn lineage derived from hybridization of different species. Our findings demonstrated for the first time that the paternal mtDNA were transmitted into the mt-genomes of homodiploid lineage, which provided new insights into the existence of paternal mtDNA in the mtDNA inheritance.

Cyprinid herpesvirus II (CyHV-2) is highly contagious and pathogenic to Carassius auratus gibelio (gibel carp), causing enormous economic losses in aquaculture in Yancheng city, Jiangsu province, China; however, to date, there is no effective way to protect C. auratus gibelio from CyHV-2 infection. In this study, a recombinant baculovirus vector vaccine, BacCarassius-D4ORFs, containing a fused codon-optimized sequence D4ORFs comprising the ORF72 (region 1–186 nt), ORF66 (region 993–1197 nt), ORF81 (region 603–783 nt) and ORF82 (region 85–186 nt) genes of CyHV-2, driven by a Megalobrama amblycephala β-actin promoter, was constructed. Then, qPCR, Western blotting and immunofluorescence assays showed that the fused gene D4ORFs was successfully delivered and expressed in fish cells or tissues by transduction with BacCarassius-D4ORFs. The fused gene D4ORFs could not be detected by PCR in the C. auratus gibelio injected with BacCarassius-D4ORFs after 7 weeks. Specific antibody against ORF72 could be detected in the serum of vaccinated C. auratus gibelio by injection with BacCarassius-D4ORFs. Furthermore, when C. auratus gibelio were vaccinated with BacCarassius-D4ORFs via the oral or injection route, followed by challenge with CyHV-2, the relative survival rate of immunized C. auratus gibelio reached 59.3% and 80.01%, respectively. These results suggested that BacCarassius-D4ORFs has the potential to be used as a vector-based vaccine for the prevention and treatment of disease caused by CyHV-2 infection.

This study evaluated effects of fishmeal replacement by cottonseed meal protein hydrolysate (CPH) on growth, antioxidant and immunity of Megalobrama amblycephala and its resistance to Aeromonas hydrophila. Fish (average weight: 38.66 ± 0.08 g) were divided into five groups and fed with five isonitrogenous (320 g/kg crude protein), isolipidic (70 g/kg crude fat) and isocaloric (17.8 MJ/kg gross energy) diets replacing fishmeal with 0%, 1%, 3%, 5% and 7% CPH, respectively. The control diet contained 60 g/kg fishmeal. Final weight and weight gain of fish fed 5% and 7% CPH were significantly lower than that of fish fed control diet (p

The present study aim to investigate the effects of dietary Gelsemium elegans alkaloids supplementation in Megalobrama amblycephala. A basal diet supplemented with 0, 5, 10, 20 and 40 mg/kg G. elegans alkaloids were fed to M. amblycephala for 12 weeks. The study indicated that dietary 20 mg/kg and 40 mg/kg G. elegans alkaloids supplementation could significantly improve final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio (PER) (P

This study was conducted to evaluate effects of replacing fish meal with cottonseed meal protein hydrolysate (CPH) on amino acid metabolism of Megalobrama amblycephala. Fish (38.66 ± 0.08 g) were randomly divided into five groups and fed five isonitrogenous (320 g kg−1 crude protein) and isocaloric (17.8 MJ kg−1 gross energy) diets replacing fish meal by CPH 0% (CPH 0), 1% (CPH 1), 3% (CPH 3), 5% (CPH 5) and 7% (CPH 7). Dietary 3% CPH had no effect on plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), succinate dehydrogenase (SDH) and xanthine oxidase (XOD) contents and liver SDH and XOD (P > 0.05), but significantly increased hepatic AST and ALT contents (P

Juvenile blunt snout bream Megalobrama amblycephala were reared under three photoperiods, 8 L: 16D, 12 L: 12D and 16 L: 8D (L: light; D: dark) for over 8 weeks. The results showed that growth performance was significantly enhanced by long photoperiod. Contrary to feed conversion ratio, final length, final weight, weight gain and feed intake increased significantly as illumination time increased from 8 h daily to 16 h daily. Low size heterogeneity and whole-body lipid content in fish exposed to long photoperiod were also observed. Both relative mRNA expression level of GH and IGF-I increased as illumination time increased from 8 h to 16 h daily. The lowest value of plasma cortisol was observed at the middle the photophase while the highest value was observed at the transition between the day and night span. Hepatic MDA content significantly increased as illumination time increased from 8 h daily to 16 h daily. The activities of hepatic catalase and glutathione peroxidase were lowest in fish exposed to 16 L: 8D photoperiod and significantly lower than that in fish exposed to 8 L: 16D. These results indicate that photoperiod manipulation may not only improve growth performance but also reduce size heterogeneity. However, prolonged photoperiod could cause chronic stress since plasma cortisol level was higher in the long photoperiod group, leading to an increasing oxidative stress.

Complement factor D (Df) is a serine protease, which can activate the alternative pathway by cleaving complement factor B, and involves in the innate defense against pathogens infection in teleost. In this study, we cloned, characterized the Df gene from blunt snout bream (Megalobrama amblycephala) (Mamdf), and examined its expression pattern and antimicrobial activity. The open reading frame (ORF) of Mamdf was 753 bp, encoding 250 amino acids with a molecular mass of 27.2 kDa. Mamdf consisted of a single serine protease trypsin superfamily domain, 3 substrate binding sites and 3 active sites, but no potential N-glycosylation site. Pairwise alignment showed that Mamdf shared the highest identity (94%) with grass carp (Ctenopharyngodon idellus). Phylogenetic analysis indicated that Mamdf and other vertebrate Df had a common ancestral origin. Mamdf structured with 4 introns and 5 exons. The Mamdf mRNA expressed relatively high at the intestine appearance stage during early development and constitutively expressed in various tissues with the highest expression in the kidney in healthy adults. After challenged with Aeromonas hydrophila, significant changes of Mamdf at both mRNA and protein levels in the kidney, spleen, liver and head-kidney were observed. The recombinant Mamdf protein showed antimicrobial activity against both gram-positive bacteria and gram-negative bacteria. The above results suggested the immune function of Mamdf, and would benefit further detailed Df function research in the immune process in teleost.

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

Megalobrama pellegrini is a cyprinid fish endemic to upper reaches of the Changjiang (Yangtze) River, China, which is also an important economic species in the local area. In recent years, resources of this species have decreased sharply and its conservation has drawn great attention. In the present study, we collected 120 individuals from the Longxi River, a tributary isolated from the main channel of the Changjiang River, where M pellegrini is still relatively abundant. Using two different molecular markers, mitochondrial cytochrome b (cyt b) gene, and nuclear microsatellite (simple sequence repeat, SSR), we analyzed the genetic diversity of this isolated population. The results show that sequence genetic diversity was low (Hd: 0.290 and Pi. 0.000 77 for cyt b gene), while the SSR genetic diversity was high (Ho: 0.824 4±0.147 2, He: 0.823 5±0.145 1). Analysis indicated that this population had experienced a bottleneck, with inbreeding and small effective population size (around 50). Based on SSR data, relatedness analyzing revealed that the 120 samples were grouped into 10 completely independent clusters. It was inferred that the mating system of M pellegrini was polygamy. We suggested that the low genetic diversity could be induced by the overfishing and inbreeding depression. Therefore, we suggested that the urgent conservation measures should be taken to control the overfishing and give better conditions for the fish to grow and spawn, then to restore population size.

The CITED3 protein is a non-DNA-binding transcriptional co-regulator involved in the regulation of various transcriptional responses against hypoxia stress. Here, we characterized two paralogs Cited3 genes (Cited3a and Cited3b) from blunt snout bream (Megalobrama amblycephala), which is a hypoxia-sensitive species. Both genes have an open reading frame of 756 and 723 bp; encoded a protein of 251 amino acid and 240 amino acid, respectively; and they shared a sequence identity of 67%. In adult fish, both Cited3a and Cited3b mRNAs were highly expressed in kidney tissues. In contrast, they were detected in the skin, muscle, and gonad at extraordinarily low levels. During embryogenesis, both Cited3a and Cited3b mRNAs were maternally deposited in eggs and fluctuated from the zygote to the 44-hpf (hours post-fertilization) larvae. Whole-mount in situ hybridization demonstrated that both Cited3a and Cited3b mRNAs were transcribed in the brain, gut, and tailbud at 12 hpf, and at the brain and gut at 24 hpf, and at the brain at 36 hpf embryos. Hypoxic treatment led to upregulated expression of the Cited3 genes during embryogenesis. Under hypoxia, both Cited3a and Cited3b genes in the kidney and brain and Cited3a genes in the liver were significantly upregulated. These results suggest that hypoxia was associated with increases in mRNA levels for both Cited3a (kidney, brain, liver) and Cited3b (kidney and liver).

In this study, we used artificial insemination to generate hybrid groups of fish [MC‐F₁(MA♀×CA♂) and MC‐F₂(MC‐F₁♀×♂)] by intergeneric crosses of Megalobrama amblycephala (MA) and Culter alburnus (CA); sequential backcrosses [CAM‐B₁ (CA♀×MC‐F₁♂) and MCC‐B₁ (MC‐F₁♀×CA♂)] were also performed. All these hybrids showed high rates of fertilization, hatching and survival (p > 0.05). For genetic traits, compared with those of the M. amblycephala and C. alburnus parental lines (Table 1), the fertilization rate, hatching rate and 7‐day survival rate of MC‐F1(MA♀×CA♂), MC‐F2(MC‐F1♀×♂), CAM‐B1 (CA♀×MC‐F1♂) and MCC‐B1 (MC‐F1♀×CA♂) by artificial insemination exhibited similar high rates (p > 0.05). The morphology of the four hybrids MC‐F₁/F₂, CAM‐B₁ and MCC‐B₁ were intermediate between those of their parents. Compared with their parents of MA and CA, weight gain rate (WG), specific growth rate (SGR) and protein efficiency ratio (PER) of hybrids MC‐F₁/F₂, CAM‐B₁ and MCC‐B₁ were significantly (p

As a critical regulator of gene expression, let-7 family miRNAs have been reported to be involved in multiple physiological processes. In this study, in order to elucidate the putative regulatory effect of let-7 miRNAs during fish gonadal development and to identify which member is crucial for this regulation, the expression of ten members including let-7a/b/c/d/e/f/g/h/i/j were quantified in ovary, pituitary, and brain tissues during the different ovarian developmental stages of blunt snout bream Megalobrama amblycephala. According to the data from analysis of expression patterns, let-7a showed the highest expression value in almost all the tested ovaries, pituitaries, and brains, with let-7b and let-7d moderately expressed, following by other let-7 miRNAs. In terms of the differential expression levels of ten let-7 miRNAs at each developmental stage, the results showed that let-7a/b/d/f/h expression gradually increased during the ovary development from stage I to V and dropped significantly at the phase VI in ovary tissues. However, the expression of let-7a/b/e/f in pituitary increased during the ovary development from stage I to IV and declined at stage V. Among all the let-7 miRNAs, let-7a/b/d had the highest expression and their expression patterns were consistent with the gonad development of M. amblycephala. Furthermore, the mostly predicted target genes of let-7 miRNAs are significantly enriched in signaling pathways closely related to gonadal development through KEGG enrichment analysis. These results indicate that let-7 miRNA members, especially let-7a/b/d, may play important roles in the regulation of ovary development in M. amblycephala through negatively regulating expression of their target genes.

Interferon regulatory factor (Irf) family represents one of the most important transcription factor families, with multiple biological roles. In this study, we characterized five Irf family members (irf4a, irf4b, irf6, irf8 and irf10) in Megalobrama amblycephala at the cDNA and (predicted) amino acid levels, analyzed them phylogenetically, and developed gene-specific primers for qPCR analysis. All five irfs were constitutively expressed in all examined tissues, but their transcription was significantly higher in lymphoid organs and tissues, such as kidney, spleen and intestine. Exceptions were irf8, which was expressed at a high level in heart and brain tissues, and irf6, expressed at low levels in most tissues. After a bacterial immune challenge with Aeromonas hydrophila, the expression of irfs in liver was up-regulated: mairf4a 8.12-fold, mairf4b 29.9-fold, mairf6 1.38-fold and mairf10 1.65-fold (mairf8 was an exception: 0.07-fold). In spleen, kidney, intestine and gills, transcript levels of studied irfs increased only at specific time-points. The results suggested that irfs are involved in the immune response to bacterial infection in M. amblycephala, which will help elucidate the biological functions of irfs in the immune system of teleost fish.

Most of the freshwater aquaculture fish species in the world, especially cyprinid fishes, contain intermuscular bones (IBs). IBs are often small and sharp, and they consequently limit how the fish can be processed, cause injury or trauma if lodged in consumers' throat or mouth and therefore affect the appeal of the fish to many consumers. In order to assess the possibility of decreasing IBs counts through selective breeding, this study evaluated the heritability of the number of IBs in blunt snout bream (Megalobrama amblycephala), which is one major aquaculture species in China. A total of 758 offsprings from 69 parents were genotyped at nine polymorphic microsatellite loci, and then 672 offspring from 213 full-sib families (33 female and 25 male breeders) were used for genetic parameter estimates. IBs were carefully teased from different poached sections of the flesh and counted. Heritability was estimated using an animal model. The results showed that IB number ranged between 84 and 146 per individual in the test population, with an average of 118 IBs. Coefficient of variation in IB number (CV) in ventral sections (13.63%) was substantially higher in comparison to dorsal sections (6.60%). The genetic correlation of IB number between the two sections was significantly positive and high (0.93) (P

The barbel chub (Squaliobarbus curriculus) is a kind of small size commercial fish species that is widely spread in Asia and has shown significant resistance to disease. In this study, the full-length cDNA sequences of Toll-like receptor (TLR) 7 and 8 from S. curriculus, designated as ScTLR7 and ScTLR8, were cloned, and their differences in the structure and the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation were investigated. The full-length 3715 base pair (bp) cDNA of ScTLR7 contained a complete open reading frame of 3162 bp and encoded a putative polypeptide of 1053 amino acid residues. The full-length 4624 base pair (bp) cDNA of ScTLR8 contained a complete open reading frame of 3072 bp and encoded a putative polypeptide of 1023 amino acid residues. ScTLR7 and ScTLR8 consisted of N-terminal signal peptide, leucine-rich repeats (LRRs), and Toll/IL-1 Receptors domain. LRR motifs of ScTLR7 and ScTLR8 bend into horseshoe-like solenoid structure, while the number of LRRs between the two genes is different. Phylogenetic analysis showed that both the ScTLR7 and ScTLR8 were closely clustered with Ctenopharyngodon idellus and Megalobrama amblycephala. Quantitative real-time polymerase chain reaction analysis showed that the expression levels of ScTLR7 in S. curriculus were most abundant in the brain followed by the spleen and heart, and the lowest in the intestine. The highest expression level of ScTLR8 was observed in spleen and the lowest in the liver. After LPS stimulation, the relative expression levels of both ScTLR7 and ScTLR8 exhibited an overall trend of up-regulation. The expression levels of type I-IFN showed an overall trend of down-regulation at time points of 12, 24, 72 and 168 h compared to that of 6 h after LPS stimulation. Compared to 6 h post GCRV infection, the transcription level of ScTLR7 was up-regulated from 12 to 168 h, and transcription levels of ScTLR8, MyD88, and type I-IFN were firstly up-regulated from 12 to 72 h, and then down-regulated from 72 to 168 h. Correlation analysis showed that expression level of ScTLR7 in the spleen was significantly positively correlated with that of MyD88 (Pearson correlation coefficient: 0.909, P: 0.033), and a significantly positive correlation was also observed between expression levels of MyD88 and type I IFN (Pearson correlation coefficient: 0.962, P: 0.009), after GCRV stimulation. These results indicate that ScTLR7 and ScTLR8 may play important roles in the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation and trigger different downstream immune signal pathways.

Autotetraploid fish (4n = 200, abbreviated as 4nRR), which reach sexual maturity at 1 year of age, were derived from the whole genome duplication of red crucian carp Carassius auratus red var. (RCC; 2n = 100) and possess four sets of chromosomes from RCC. The histological features of the gonads showed that the RCC and 4nRR both possessed normal gonadal structure and could arrive at maturation. To understand the expression characteristics of genes related to reproductive development in the autotetraploid fish, we analysed the nucleotide sequence and expression characteristics of the gnrh2, gthb and gthr genes, which are the pivotal genes of the hypothalamic-pituitary-gonadal (HPG) axis. We found that the gnrh2, gthb and gthr genes in 4nRR share remarkable homology with RCC, but there were obvious differences in expression levels between 4nRR and RCC. These results demonstrate that autotetraploidization can lead to gene expression changes. This study provides insights into the molecular mechanism underlying the reproductive development of autotetraploid fish and is expected to be of great significance for subsequent research on polyploidization.

High-fat diets are associated with fatty liver and aberrant hepatic lipid metabolism, and glycyrrhetinic acid (GA) has been shown to exert a beneficial effect on lipolysis and fat deposition in fish. In the present study, we evaluated the effect of GA on the growth performance and expression of hepatic lipid transport related genes in blunt snout bream ( Megalobrama amblycephala) fed a high fat diet. Two hundred and sixteen fish (average body weight: 45.57 g ± 0.98 g) were fed three experimental diets (6% fat/L6 group, control, 11% fat/L11 group, and 11% fat with 0.3 mg kg −1 GA/L11GA group) for 8 weeks. Compared to the control group, the weight gain and specific growth rate of high-fat fed group at the end of the trialwere significantly improved ( P P > .05). Dietary supplementation with 0.3 mg kg −1 GA significantly decreased the hepatosomatic index, viscera/body ratio, and intraperitoneal fat ratio ( P P P > .05). In conclusion, GA significantly rescued high-fat diet induced hepatic lipid accumulation and metabolism dysfunction in M. amblycephala by stimulating hepatic fatty acid transport and β-oxidation. Dietary GA may be used as a promising supplement to alleviate high-fat diet induced side effects on M. amblycephala.

Background: Autotetraploid fish (4n = 200, RRRR) (abbreviated as 4nRR) are derived from whole genome duplication of red crucian carp (2n = 100, RR) (abbreviated as RCC). rDNA is often used to study molecular evolution of repeated sequences because it has high copy rate and special conserved coding regions in genomes. In this study, we determined the sequences (5S, ITS1-5.8S-ITS2 region), structure, methylation level (NTS and IGS), and expression level (5S and 18S) of 5S and 45S rRNA genes in 4nRR and RCC in order to elucidate the effects of autotetraploidization on ribosomal DNA (rDNA) in fish. Results: Results showed that there was high sequence similarity of 5S, 5.8S and ITS1 region between 4nRR and RCC. This study also identified two different types of ITS2 region in 4nRR and predicted the secondary structure. It turns out that both secondary structures are functional. Compared with the diploid ancestor of RCC, there was no significant difference in NTS (5S) methylation level, but the expression level of 5S rRNA was lower in 4nRR, indicating that methylation had little effect on the expression level in 4nRR. IGS (45S) was hypermethylated in 4nRR compared to RCC, but the expression of 18S gene were no significantly different from that in RCC, indicating that methylation regulation affected gene expression in 4nRR. Conclusion: These results demonstrate the effects of related structure and expression of autotetraploidization on rDNA. In addition, this study provides reference for studying the effect of autopolyploid on the evolution of species.

p65 is one of the important subunits of the inflammation-related transcription factor NF-κB. In the present study, we cloned and identified the p65 from Megalobrama amblycephala (Mnp65) by homologous cloning and RACE technique. The full-length Mnp65 cDNA consisted of 2331 bp, and included one open reading frame encoding a 604-amino acid putative protein. The protein sequence included a DNA binding motif, a well conserved N-terminal Rel-homology domain (RHD), and a C-terminal IG-like plexins transcription (IPT). Mnp65 was closely related with the other p65 proteins of Cypriniformes and clearly distinct from that of Perciformes and Salmoniformes in terms of sequence homology. Mnp65 homodimer may interact with IκBα in the IPT domain based on the predicted 3D structure of IκBα/Mnp65 complex. Mnp65 was ubiquitously expressed in M. amblycephala tissues, and the highest levels were detected in muscle and liver. Intragastric infection with Aeromonas hydrophila caused respiratory burst and cytokine storm from 8 h to 48 h, showing significantly higher level of respiratory burst activities and significantly high cytokines levels, such as TNF-α, IL-1β, IL-6, IL-8 etc., compared to 0 h. In addition, the bacterial challenge downregulated the IkBα, and upregulated Mnp65 and TNF-α in the liver. IkBα-Mnp65 was regulated by the negative feedback of cytokine storm, to increase IkBα and decrease Mnp65. Then cytokine storm was relieved at 96 h. Finally, severe intestinal inflammation was observed from 24 h to 48 h after infection, characterized by extensive villous necrosis, epithelial hyperplasia and lymphocyte infiltration, all of which were relieved at 96 h. Taken together, Mnp65 plays a crucial role in the physiological response of teleost fish to bacterial infection.

Fish has poor utilization capacity for glucose metabolism. The possible reasons are related to the core regulatory elements of glucose metabolism: transport proteins. Studies on the species and functions of Sglt1 in aquatic animals are scarce, therefore further studies are needed. In this study, the full length of blunt snout bream (Megalobrama amblycephala) sglt1 (Masglt1) was 2965 bp including 5′-UTR region of 168 bp and a 3′-UTR region of 820 bp. Masglt1 have a highest sequence homology in Cypriniformes fish. MaSglt1 protein was identified as a transmembrane protein with 14 α-helix structures locating plasma membrane by the methods of predicted tertiary structure and immunohistochemical staining. MaSglt1 protein has a hollow channel forms which could be specifically coupled with two Na+ ions to recognize glucose and carry out transmembrane transport. High sglt1 mRNA was found in the intestine and kidney. The mRNA levels of intestinal sglt1 had a positive correlation with dietary starch levels at 3 h after feeding, and the mRNA was significantly higher than that at 24 h, however, the mRNA levels of renal sglt1 presented results opposite to those of intestinal sglt1. The mRNA levels of renal sglt1 had a positive correlation with dietary starch levels at 24 h after feeding, and the expression was significantly higher than that at 3 h. These results confirmed that Masglt11 was mainly found in the intestine and kidney and was located in the cell membrane, playing a role in glucose homeostasis.