Genome-wide investigation of the DMRT gene family sheds new insight into the regulation of sex differentiation in spotted knifejaw (Oplegnathus punctatus) with fusion chromosomes (Y).

The role of the DMRT family in male sex determination and differentiation is significant, but its regulatory role in spotted knifejaw with Y fusion chromosomes remains unclear. Through genome-wide scanning, transcriptome analysis, qPCR, FISH, and RNA interference (RNAi), we investigated the DMRT family and the dmrt1-based sex regulation network. Seven DMRTs were identified (DMRT1/2 (2a,2b)/6, DMRT4/5, DMRT3), and dmrt gene dispersion among chromosomes is possibly driven by three whole-genome duplications. Transcriptome analysis enriched genes were associated with sex regulation and constructed a network associated with dmrt1. qPCR and FISH results showed the expression dimorphism of sex-related genes in dmrt-related regulatory networks. RNAi experiments indicated a distinct sex regulation mode in spotted knifejaw. Dmrt1 knockdown upregulated male-related genes (sox9a, sox9b, dmrt1, amh, amhr2) and hsd11b2 expression, which is critical for androgen synthesis. Amhr2 is located on the heterozygous chromosome (Y) and is specifically localized in primary spermatocytes, and is extremely upregulated after dmrt1 knockdown which suggested besides the important role of dmrt1 in male differentiation, the amhr2 along with amhr2/amh system, also play important regulatory roles in maintaining high expression of the hsd11b2 and male differentiation. This study aims to further investigate sex regulatory mechanisms in species with fusion chromosomes.

Expression pattern and cellular localization of pepsinogen in early development and induced by different diets in the spotted knifejaw (Oplegnathus punctatus).

To solve the high mortality rate of early-stage larval feed conversion during aquaculture in Oplegnathus punctatus, the investigation of the structural and functional characteristics of the gastric tissue was conducted. Histological results showed that the gastric gland rudiment appeared at 17 dph. The basic structure of the stomach was fully developed between 26 and 35 dph. Two pepsinogen genes, named OpPGA1 and OpPGA2, were identified in the spotted knifejaw genome. qPCR results of developmental period showed that the two genes were low in expression during early development (5 and 15 dph). At 20 dph, the two genes started to show trace expression, and at 30 dph the mRNA expression levels of OpPGA1 and OpPGA2 reached the highest levels. Results of pepsin activity detection during the development period showed lower activity was detected 22 dph, followed by a peak at 30 dph. Under different feeding inductions, OpPGA1 showed the highest expression in the basic diet group and hard-shell group, while the expression level in the phytophagous group remained consistently low. The mRNA expression level of OpPGA2 in the phytophagous group was significantly higher than in other groups. Enzyme activity determination under different feeding inductions showed slightly higher enzyme activity in the basic diet group and crustacean group. The results of in situ hybridization showed that the mRNA of both OpPGA1 and OpPGA2 genes was both expressed in gastric gland cells. These information can contribute to the development of practical feeding methods in terms of digestive physiology for the development of larvae.

Tea polyphenols, astaxanthin, and melittin can significantly enhance the immune response of juvenile spotted knifejaw (Oplegnathus punctatus).

The frequent occurrence of diseases seriously hampers the sustainable development of the spotted knifejaw (Oplegnathus punctatus) breeding industry. Our previous genome-wide scan and cross-species comparative genomic analysis revealed that the immune gene family (Toll-like receptors, TLR) members of O. punctatus underwent a significant contraction event (tlr1, tlr2, tlr14, tlr5, and tlr23). To address immune genetic contraction may result in reduced immunity, we investigated whether adding different doses (0, 200, 400, 600, and 800 mg/kg) of immune enhancers (tea polyphenols, astaxanthin, and melittin) to the bait after 30 days of continuous feeding could stimulate the immune response of O. punctatus. We found that the expression of tlr1, tlr14, tlr23 genes in immune organs (spleen and head kidney) was stimulated when tea polyphenols were added at 600 mg/kg. The tlr2 (400 mg/kg), tlr14 (200 mg/kg), tlr5 (200 mg/kg), and tlr23 (200 mg/kg) genes expression of intestine were elevated in the tea polyphenol group. When the addition of astaxanthin is 600 mg/kg, it can effectively stimulate the expression of tlr14 gene in immune organs (liver, spleen and head kidney). In the astaxanthin group, the expression of the genes tlr1 (400 mg/kg), tlr14 (600 mg/kg), tlr5 (400 mg/kg) and tlr23 (400 mg/kg) reached their highest expression in the intestine. Besides, the addition of 400 mg/kg of melittin can effectively induce the expression of tlr genes in the liver, spleen and head kidney, except the tlr5 gene. The tlr-related genes expression in the intestine was not significantly elevated in the melittin group. We hypothesize that the immune enhancers could enhance the immunity of O. punctatus by increasing the expression of tlr genes, and thereby leading to increased resistance to diseases. Meanwhile, our findings further demonstrated that significant increases in weight gain rate (WGR), visceral index (VSI), and feed conversion rate (FCR) were observed at 400 mg/kg, 200 mg/kg and 200 mg/kg of tea polyphenols, astaxanthin and melittin in the diet, respectively. Overall, our study provided valuable insights for future immunity enhancement and viral infection prevention in O. punctatus, as well as offered guidance for the healthy development of the O. punctatus breeding industry.

Genome-Wide Scan Reveals Toll-Like Receptor Contraction Events in Oplegnathidae.

The striped knifejaw (Oplegnathus fasciatus) and spotted knifejaw (Oplegnathus punctatus) are prominent members of the Oplegnathidae family and are rocky reef-loving fishes with high ecological and economic value. However, the frequent occurrence of diseases in these fishes has severely restricted the development of their breeding industry. Toll-like receptors (TLRs) play an important role in resistance to pathogens as part of innate immunity. Genome-wide scans and cross-species comparative analysis revealed 10 TLRs in O. fasciatus (OfTLRs) and only 5 in O. punctatus (OpTLRs). In contrast to those of mammals and other fishes, the TLR family of Oplegnathidae underwent significant contraction events, especially in O. punctatus (only TLR1, TLR2, TLR14, TLR5, and TLR21 were retained). A phylogenetic tree divided the 10 OfTLRs into 5 subfamilies: TLR1, TLR3, TLR5, TLR7, and TLR11. The five OpTLR genes were divided into three different subfamilies: TLR1, TLR5, and TLR11. Quantitative real-time PCR revealed that all OpTLRs were expressed in the examined tissues, especially the immune system-related tissues, such as the spleen, gill, head kidney, and middle kidney. The expression of OpTLRs was high at the early stage of development (5 days posthatching [dph]) and decreased gradually until 30 dph. We speculated that maternal immunity or the developmental function of TLRs played an important protective role in the early stage. However, from 30 to 60 dph, TLR expression was low. At this time, juvenile fish are susceptible to viruses and begin to show TLR self-expression with weak immunity. Artificial immunity enhancement is needed to improve the environmental resistance of juvenile fish. In summary, our results not only provide valuable basic data for future studies of the TLR gene family in Oplegnathidae fish but also lay a solid foundation for Oplegnathidae fish research.

Identification of Male-Specific Molecular Marker and Development of PCR-Based Genetic Sex Identification Technique in Spotted Knifejaw (Oplegnathus punctatus).

Spotted knifejaw (Oplegnathus punctatus) is a marine teleost species that is economically important for aquaculture and marine pasture proliferation and shows obvious bisexual growth dimorphism, but molecular sex markers are currently lacking. A 290 bp (base pair) insertion with two fragments (230 bp and 60 bp) was identified in male individuals of O. punctatus based on whole-genome sequencing scanning and structural variation analyses. The gene annotation results showed that the insertion event occurred in the Igfn1 gene of male O. punctatus. The results of amino acid analysis further showed that the insertion event resulted in the functional variation of Igfn1 in male O. punctatus, and recombination caused the inactivation of Igfn1. According to the male-specific insertion information, we designed a PCR-based genetic amplification technique for rapid sex identification in O. punctatus. The results of agarose gel electrophoresis showed that two DNA fragments of 635 bp and 925 bp were amplified in male O. punctatus, while only a single DNA fragment of 635 bp was amplified in female individuals. The sex of individuals identified by this method was consistent with their known phenotypic sex, which will improve sex identification efficiency. This method provides a new DNA marker for rapid sex identification in O. punctatus, which has great significance and application value in monosex breeding and provides new insights for the study of Igfn1 gene recombination and inactivation in male O. punctatus.

Genome-wide identification, characterization and expression analysis of the BMP family associated with beak-like teeth in Oplegnathus.

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta (TGF-ß) family, are critical for the control of developmental processes such as dorsal-ventral axis formation, somite and tooth formation, skeletal development, and limb formation. Despite Oplegnathus having typical healing beak-like teeth and tooth development showing a trend from discrete to healing, the potential role of BMPs in the development of the beak-like teeth is incompletely understood. In the present study, 19 and 16 BMP genes were found in O. fasciatus and O. punctatus, respectively, and divided into the BMP2/4/16, BMP5/6/7/8, BMP9/10, BMP12/13/14, BMP3/15 and BMP11 subfamilies. Similar TGFb and TGF_ß gene domains and conserved protein motifs were found in the same subfamily; furthermore, two common tandem repeat genes (BMP9 and BMP3a-1) were identified in both Oplegnathus fasciatus and Oplegnathus punctatus. Selection pressure analysis revealed 13 amino acid sites in the transmembrane region of BMP3, BMP7, and BMP9 proteins of O. fasciatus and O. punctatus, which may be related to the diversity and functional differentiation of genes within the BMP family. The qPCR-based developmental/temporal expression patterns of BMPs showed a trend of high expression at 30 days past hatching (dph), which exactly corresponds to the ossification period of the bones and beak-like teeth in Oplegnathus. Tissue-specific expression was found for the BMP4 gene, which was upregulated in the epithelial and mesenchymal tissues of the beak-like teeth, suggesting that it also plays a regulatory role in the development of the beak-like teeth in O. punctatus. Our investigation not only provides a scientific basis for comprehensively understanding the BMP gene family but also helps screen the key genes responsible for beak-like tooth healing in O. punctatus and sheds light on the developmental regulatory mechanism.

Identification of Male-Specific Molecular Markers by Recombination of RhoGEF10 Gene in Spotted Knifejaw (Oplegnathus punctatus).

The spotted knifejaw (Oplegnathus punctatus) is a marine economic fish with high ecological value, food value, and fishing value, and its growth has obvious sex dimorphism. The rapid identification of its sex is beneficial to the development of sex determination and breeding. In this study, the method of comparative genomics and PCR amplification was used to further establish a rapid detection method for the recombinant RhoGEF10 gene in O. punctatus, which can quickly, accurately, and efficiently identify the sex of the O. punctatus to be tested. The homologous comparison results of male and female individuals showed that the DNA fragment length of the RhoGEF10 gene on the X1 chromosome was 326 bp, and the DNA fragment length on the Y chromosome was 879 bp. Therefore, it can be concluded that there is an insert fragment of 553 bp on the Y chromosome. PCR amplification results showed that the two DNA fragments of 879 bp and 326 bp were amplified in the Y chromosome and X1 chromosome of the male O. punctatus (X1X2Y), respectively, and the 879 bp fragment was a unique marker fragment of the recombinant RhoGEF10 gene; The female O. punctatus (X1X1X2X2) only a single DNA fragment of 326 bp was amplified. At the same time, the inserted fragment of the male individual resulted in partial inactivation of the RhoGEF10 protein, which in turn resulted in a slowing of peripheral nerve conduction velocity and thinning of the myelin sheath in male O. punctatus. The method shortens the time for accurate identification of the O. punctatus RhoGEF10 gene recombination and improves the detection efficiency. It is of great significance and application value in the research of nerve conduction and myelin development, male and female sex identification, the preparation of high male seedlings, and family selection based on the RhoGEF10 gene in the O. punctatus.

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.

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.