Various bacterial diseases have caused great economic losses to the high-density and intensive aquaculture industry; however, the pathogenic mechanism underlying the large-scale challenged to caused ...by many bacteria remain unclear, making the prevention and treatment of these diseases difficult. In the present study, we isolated a bacterial strain from
Cyprinus
carpio
having a typical bacterial disease and named it Cc2021. Through subsequent morphological observations, a regression challenge, biochemical identification, and 16S rRNA gene sequence analysis, we determined Cc2021 to be
Plesiomonas shigelloides
. Subsequently, we comprehensively investigated the pathogenicity of
P. shigelloides
in
C. carpio
through a regression challenge and assessed the underlying the pathogenic mechanism. Mortality results revealed that
P. shigelloides
is highly pathogenic and infects various tissues throughout the body, resulting in edema of the liver, spleen, and body and head kidneys. Histopathological analysis revealed obvious inflammation, bleeding, and necrosis in the intestine, spleen, and head kidney. The body’s immune tissues actively produce complement C3, superoxide dismutase, and lysozyme after a challenge to resist bacterial invasion. With regard to the underlying pathogenesis of
P. shigelloides
, comparative transcriptome analysis revealed 876 upregulated genes and 828 downregulated genes in the intestine of
C. carpio
after the challenge. Analysis of differentially expressed unigenes revealed the involvement of major immune pathways, particularly the TNF signaling pathway, interleukin (IL)-17 signaling pathway, and Toll-like receptor signaling pathway. The present study provides new valuable information on the immune system and defense mechanisms of
P. shigelloides
.
Due to sexual dimorphism in the growth of certain cultured fish species, the production of monosex fishes is desirable for the aquaculture industry. Nowadays, the most widely practiced technique ...available for the mass production of monosex fish populations is sex steroid-induced sex reversal. Here, a novel strategy for the successful production of all-female (AF) common carp (Cyprinus carpio L.), to take advantage of the sexual dimorphism in growth documented in this species, has been developed using genetic engineering via single gene-targeting manipulation without any exogenous hormone treatments. Male and female heterozygous cyp17a1-deficient common carp were first obtained using the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) technique. An all-male phenotype for homozygous cyp17a1-deficient carp, regardless of the individuals’ sex-determination genotypes (XY or XX), has been observed. A male-specific DNA marker newly identified in our laboratory was used to screen the neomale carp population with the XX genotype from the cyp17a1-deficient carp. These neomale carp develop a normal testis structure with normal spermatogenesis and sperm capacity. The neomale common carp were then mated with wild-type (WT) females (cyp17a1+/+ XX genotype) using artificial fertilization. All the AF offspring sample fish from the neomale-WT female mating were confirmed as having the cyp17a1+/− XX genotype, and normal development of gonads to ovaries was observed in 100.00% of this group at eight months post-fertilization (mpf). A total of 1000 carp fingerlings, 500 from the WT male and female and 500 from the neomale and WT female mating, were mixed and reared in the same pond. The average body weight of cyp17a1+/− XX females was higher by 6.60% (8 mpf) and 32.66% (12 mpf) than that of the control common carp. Our study demonstrates the first successful production of a monosex teleost population with the advantages of sexual dimorphism in growth using genetic manipulation targeting a single locus.
Ovarian growth is a critical phase of oogenesis that involves several events such as steroidogenesis, lipid deposition, and vitellogenesis. Although many studies have been conducted on fish ovarian ...growth and development, the molecular mechanisms that contribute to steroidogenesis and lipid deposition in common carp, the world's third largest freshwater aquaculture fish, remain elusive. In this study, transcriptome analyses were performed on ovaries from Yellow River carp at three different developmental stages: primary growth stage (PG), pre-vitellogenesis stage (PV), and mid-vitellogenesis stage (MV). A total of 22,511 genes were detected, and of these 122, 253, and 278 were differentially expressed genes (DEGs) when comparing PG to PV, PV to MV, and PG to MV stages, respectively. Gene ontology (GO) and KEGG enrichment analysis showed that the DEGs were mainly involved in steroidogenesis (e.g., fshr, cyp19a1a, hsd3b and foxl2b), lipid deposition (e.g., lpl, slc27a6, fabp4, acsl1a, agpat3, plpp3 and dgat1b), and vitellogenesis (e.g., lrp13, plb1, dgkaa and pik3r1). We also identified many signalling pathways involved in ovarian growth, including steroid hormone biosynthesis, the phospholipase D signalling pathway, glycerophospholipid metabolism, the Notch signalling pathway, the TGF-βsignalling pathway, and the Wnt signalling pathway. qPCR results of 9 representative genes related to steroidogenesis and lipid deposition verified the reliability of the generated RNA-seq data. Finally, a potential model for the formation of lipid droplets and yolks in the ovaries of common carp was proposed. Overall, the data from this study will contribute to further research on the molecular regulatory mechanisms of ovarian growth and development in common carp and other fish species.
The common carp (
) accounts for approximately 10% of the annual freshwater aquaculture production and is an ideal model to study cyprinidae reproduction. Female common carp grow faster than the ...males; therefore, related research presents an opportunity with high application value. Although we have a detailed understanding of common carp's early gonadal differentiation process, information about genome-wide gene expression, regulation, and underlying molecular mechanisms during this process remain limited. Here, time-course data comprising six key stages during testicular differentiation and maturation were investigated to further understand the molecular mechanisms underlying the testicular development in cyprinid species. After integrating these time-series data sets, common carp genome, including 98,345 novel transcripts and 3,071 novel genes were re-annotated and precisely updated. Gene co-expression network analysis revealed that the ubiquitin-mediated proteolysis pathway was essential for metabolism during testicular differentiation in the endocrine system of
. Functional enrichment analyses indicated that genes mainly related to amino acid metabolism and steroid hormone synthesis were relatively highly expressed at the testicular undifferentiation stages, whereas genes associated with cell cycle and meiosis were expressed from the beginning of testicular differentiation until maturation. The dynamics of alternative splicing events demonstrated that exon skipping accounted for majority of the alternative splicing events in the testis and the brain during gonad development. Notably, several potential male-specific genes (
and
) and brain-specific genes (
,
, and
, etc.) were identified. Importantly, we traversed beyond the level of transcription to test for stage- and gonad-specific alternative splicing patterns between the brain and testis. This study is the first to describe a comprehensive landscape of alternative splicing events and gene expression patterns during gonadogenesis in common carp. This work is extremely valuable to elucidate the mechanisms underlying gonadal differentiation in Cyprinidae as well as other fish species.
Many economically important fish have a long sexual maturation cycle, with the first sexual maturation time exceeding four years, which poses a long-term challenge for the cultivation of excellent ...varieties of these fish. Gonadal development and reproductive timing of fish are closely related to changes in environmental temperature. This study used the common carp as an economic fish model and set up three aquaculture modes for a one-month old common carp population: constant temperature test group (CTTG), normal temperature control group (NTCG), and variable temperature control group (VTCG). A long-term (approximately 11 months), temperature-controlled aquaculture experiment was conducted. The results of growth traits and gonadal development tests showed that at 10 months, the body length and weight of the CTTG were significantly higher than those of the other two groups. At seven months, the gonadosomatic index (GSI) value of CTTG was also significantly higher than that of the other two groups; At 10 months, the serum estradiol (E2) concentration of CTTG was significantly higher than that of the other two groups, while the testosterone (T) levels of VTCG and CTTG were significantly higher than those of NTCG, and only the 11-ketotestosterone (11-KT) concentration of VTCG was significantly higher than that of the other two groups. Simultaneously, qPCR analysis of sex hormone regulatory genes revealed that the expression of Cyp19a1a in CTTG ovaries was significantly higher than that in NTCG and VTCG ovaries, whereas the expression of Cyp11b and Hsd11b2 in VTCG ovaries was significantly higher than that in NTCG and CTTG ovaries. At 12 months, artificial reproduction revealed that only fish from the CTTG (66.67%) successfully laid eggs and fertilized normally. The results indicate that long-term constant temperature cultivation at 28 °C starting from the juvenile stage is not only beneficial for the body growth of common carp, but also promotes their gonadal development. This may be due to the conversion of T into E2 by Cyp19a1a in the common carp, ultimately facilitating the functional development of gonadal tissue under hypothalamus-pituitary-gonadal (HPG) axis regulation, normal egg laying, and fertilization. This study reduced the normal first sexual maturation time of common carp by at least 50% and provides a reference for the rapid cultivation of excellent varieties of other economically important fishes.
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•Common carp has a high yield and is an ideal model for the Cyprinidae family.•Constant temperature aquaculture promote the gonadal development of common carp.•This method reduced the sexual maturation time of common carp by at least 50%.•This method can be directly used for rapid cultivation of excellent varieties.
The common carp, a gonochoristic teleost fish with an XX/XY sex-determining system, provides an excellent model for studying cyprinidae gonadal sex differentiation. The present study aims at ...investigating the detailed process of early gonadal sex differentiation of common carp by combining the expression patterns of sex related genes (cyp19a1a, foxl2, amh, dmrt1, sox9b, sycp3, dmc1) with gonadal histological changes using mono-sex female and male Yellow River carp. H&E staining, immunohistochemistry and immunofluorescence of gonads indicated that the ovaries and testis of common carp were both directly differentiated from undifferentiated gonads. Oogonia and spermatogonia were first observed at 40 dph (days post hatching) and 70 dph in female and male gonads, respectively. Female related genes cyp19a1a and foxl2 and male related genes amh, dmrt1 and sox9b increased significantly in 30–40 dph female, and male gonads, respectively. These suggest that the critical timing of sex determination of common carp takes place between 30 and 40 dph or possibly earlier. Meiotic marker gene dmc1 and sycp3 increased expression significantly in 55 dph female gonads while sycp3 increased expression significantly in 100 dph male gonads. Oocytes in meiosis and spermatocytes were first observed in 55 dph female and 100 dph male gonads, respectively. These indicate that the initiation time of gonadal sex differentiation is 50–55 dph in female common carp and 90–100 dph in male common carp. To our knowledge, this is the first study investigating the detailed process of early gonadal sex differentiation of common carp by combining the expression patterns of sex related genes with gonadal histological changes. We have identified the critical window of sex determination and the initiation times of oogenesis and spermatogenesis in the Yellow River carp. These results provide researchers with valuable information to possibly reveal the mechanisms of sex determination and differentiation of common carp.
•We successfully produced mono-sex female and male Yellow River carp.•The critical window time of sex determination of common carp is at 30-40 dph or even earlier.•Gonadal sex differentiation initiation is at 50-55 dph and 90-100 dph in females and males, respectively.•cyp19a1a and amh may be used as molecular markers for sex identification in common carp, even before sex differentiation.
Common carp is the fourth most cultivated fish species in the world. Sexually dimorphic growth of common carp makes its sex differentiation an important research topic. The main objective of this ...study was to investigate the role of germ cells in sex differentiation of common carp. We generated the germ cell-deficient common carp through knocking down dead end gene with a morpholino antisense oligonucleotide during the embryogenesis. In the common carp without germ cells, we found about half of the gonads (type I gonads) contain granulosa cells and theca cells, which were the same with somatic cells we observed in the wild-type ovaries. The remaining half of the gonads (type II gonads) contain Sertoli cells and Leydig cells, which were the same with somatic cells in the wild-type testes. Furthermore, the expression of sex differentiation related genes was detected in the gonads without germ cells. It was found cyp19a1a was mainly expressed in the type I gonads, and the cyp19a1a mRNA level is much higher in each type I gonad than in the type II gonad. While dmrt1 was mainly expressed in the type II gonads, and the dmrt1 mRNA level is much higher in each type II gonad than in the type I gonad. Our comprehensive analysis showed that the gonadal structure and expression of genes related with sex differentiation were sexually dimorphic in the germ cell-deficient carp. These findings are valuable for discovering the underlying mechanism of common carp sex differentiation which is crucial for cultivating monosexual carp for aquaculture.
•Germ cell-deficient common carp model was generated through knocking down dnd gene.•Common carp without germ cells displayed sexual dimorphism of gonadal structure.•Expression of cyp19a1a and dmrt1 genes was sexually dimorphic in germ cell-deficient gonads.•Gonadal differentiation is germ cell independent in common carp.
MicroRNAs (miRNAs) are essential for cellular proliferation, differentiation, and apoptosis. However, currently, their functions in fish germ cell proliferation and differentiation are poorly ...defined. In this study, we found that miR-153b-3p was predominantly expressed in the brain, pituitary, hypothalamus, and testes in the common carp (Cyprinus carpio). miR-153b-3p was primarily expressed in spermatogonia and some somatic cells in the testis. Over-expressing miR-153b-3p in vivo using the agomir method promoted male germ cell proliferation. Silencing miR-153b-3p in vivo using the antagomir method repressed male germ cell proliferation. A dual-luciferase reporter assay suggested that amh was a direct target of miR-153b-3p. In conclusion, miR-153b-3p plays roles in spermatogenesis by regulating male germ cell proliferation and differentiation by targeting amh in common carp.
•miR-153b-3p was localized in spermatogonia in adult testis in common carp.•miR-153b-3p was identified firstly to regulate spermatogenesis in common carp.•miR-153b-3p regulates spermatogenesis via targeting amh in common carp.
The common carp, Cyprinus carpio, is the third most cultivated freshwater species worldwide, but is also considered an invasive species. The Trojan Y chromosome strategy is one of the most promising ...methods to eradicate this invasive species. However, obtaining fertile YY supermale (MYY) and YY physiological female (FYY) common carp was thought to be very difficult. The present study focused on the production of androgenetic MYY and XY physiological female (FXY) common carp. We first optimized the conditions for artificially induced androgenesis. The results indicated that the optimum ultraviolet (UV) exposure time was 4 min at an irradiation distance of 26 cm and the optimum initiation time with heat shock (40 ± 0.5 C for 2 min) was 30 min after fertilization. Then, we produced androgenetic MYY Yellow River carp with only paternal inheritance, which were viable and identified by paternity testing and test crossing. Finally, we successfully produced FXY Yellow River carp by feeding 60‐d‐old MXY with commercially available feed mixed with 17β‐estradiol (200 mg/kg) and Flutamide (200 mg/kg) for 3 mo. In conclusion, by combining artificially induced androgenesis with an artificially induced sex reversal technique, we could cost‐effectively produce MYY and FXY common carp.
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