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Members of the transforming growth factor β (TGF-β) signaling pathway regulate diverse cellular biological processes in embryonic and tissue development. We took mandarin fish (Siniperca chuatsi) as the research object to identify all members of the TGF-β signaling pathway, measure their expression pattern in the key period post hatching, and further explore their possible role in the process of sex regulation. Herein, we identified eighty-three TGF-β signaling pathway members and located them on chromosomes based on the genome of mandarin fish. TGF-β signaling pathway members were highly conserved since each TGF-β subfamily clustered with orthologs from other species. Transcriptome analysis, qRT-PCR and in situ hybridization demonstrated that most mandarin fish TGF-β signaling pathway members presented stage-specific and/or sex-dimorphic expression during gonadal development, and different members of the TGF-β signaling pathway participated in different stages of gonadal development. Taken together, our results provide new insight into the role of TGF-β signaling pathway members in the sex regulation of mandarin fish., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Background: Spermatogenesis is an intricate process regulated by a finely organized network. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish, but the regulatory mechanism of its spermatogenesis is not well-understood. In the present study, transcriptome sequencing of the male germ cells isolated from orange-spotted grouper was performed to explore the molecular mechanism underlying spermatogenesis., Results: In this study, the orange-spotted grouper was induced to change sex from female to male by 17alpha-methyltestosterone (MT) implantation. During the spermatogenesis, male germ cells (spermatogonia, spermatocytes, spermatids, and spermatozoa) were isolated by laser capture microdissection. Transcriptomic analysis for the isolated cells was performed. A total of 244,984,338 clean reads were generated from four cDNA libraries. Real-time PCR results of 13 genes related to sex differentiation and hormone metabolism indicated that transcriptome data are reliable. RNA-seq data showed that the female-related genes and genes involved in hormone metabolism were highly expressed in spermatogonia and spermatozoa, suggesting that these genes participate in the spermatogenesis. Interestingly, the expression of zbtb family genes showed significantly changes in the RNA-seq data, and their expression patterns were further examined during spermatogenesis. The analysis of cellular localization of Eczbtb40 and the co-localization of Eczbtb40 and Eccyp17a1 in different gonadal stages suggested that Eczbtb40 might interact with Eccyp17a1 during spermatogenesis., Conclusions: Our study, for the first time, investigated the transcriptome of the male germ cells from orange-spotted grouper, and identified functional genes, GO terms, and KEGG pathways involved in spermatogenesis. Furthermore, Eczbtb40 was first characterized and its role during spermatogenesis was predicted. These data will contribute to future studies on the molecular mechanism of spermatogenesis in teleosts.

Background: Interspecies hybridization is widely used to achieve heterosis or hybrid vigor, which has been observed and harnessed by breeders for centuries. Natural allopolyploid hybrids generally exhibit more superior heterosis than both the diploid progenies and their parental species. However, polyploid formation processes have been long ignored, the genetic basis of heterosis in polyploids remains elusive., Results: In the present study, triploid hybrids had been demonstrated to contain two sets of chromosomes from mother species and one set from father species. Cellular polyploidization process in the embryos had been traced. The triploid hybrids might be formed by failure formation of the second polarized genome during the second meiosis stage. Four spindle centers were observed in anaphase stage of the first cell division. Three spindle centers were observed in side of cell plate after the first cell division. The 5S rDNA genes of four types of groupers were cloned and analyzed. The diploid and triploid hybrids had been proved to contain the tandem chimera structures which were recombined by maternal and paternal monomer units. The results indicated that genome re-fusion had occurred in the hybrid progenies. To further elucidate the genetic patterns of diploid and triploid hybrids, fluorescence chromosome location had been carried out, maternal 5S gene (M-386) were used as the probe. The triploid hybrids contained fewer fluorescence loci numbers than the maternal species. The results indicated that participation of paternal 5S gene in the triploid hybrid genome had degraded the match rates of M-386 probe., Conclusions: Our study is the first to investigate the cellular formation processes of natural allopolyploids in hybrid fish, the cellular polyploidization process may be caused by failure formation of the second polarized genome during the meiosis, and our results will provide the molecular basis of hybrid vigor in interspecies hybridization.

We present the complete mitochondrial genome of the Trachinotus ovatus in this study. The mitochondrial genome is 16,563 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and composition of T. ovatus mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 29.03% of A, 28.86% of C, 26.23% of T and 15.88% of G. With the exception of ND6 and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. Two copies of tandem repeat sequence (56 bp) was observed in the 5' end of the control region.

Wnt4 (Wingless-type MMTV integration site family member 4) has been demonstrated to play critical roles in ovarian development in mammals, but its function in fish reproduction is still unclear. In the present study, two full-length wnt4 cDNA sequences (named wnt4a and wnt4b ) were cloned from the orange-spotted grouper ( Epinephelus coioides ). Amino acid alignment analysis showed that both orange-spotted grouper Wnt4s proteins had the typical characteristics of the Wnt family. RT-PCR revealed that both wnt4a and wnt4b were highly expressed in the ovaries of the orange-spotted grouper. Temporal expression profiles of both wnt4 genes during embryonic and ovarian development were examined. The expressions of wnt4a and wnt4b genes were first detected at the embryonic morula stage, but the gens showed different expression patterns. During ovarian development, high expression of wnt4a was observed in the ovarian lumen formation and gonium proliferation stage, while wnt4b exhibited strong expression in the early developmental stage of oocytes. Taken together, the present study indicates that the two wnt4 genes are involved in the regulation of ovarian development in the orange-spotted grouper. [ABSTRACT FROM AUTHOR]

We present the complete mitochondrial genome ofEpinephelus coioidesin this study. The mitochondrial genome is 16,458 bp in length, containing 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. All of them are encoded on the heavy strand except ND6 and 8 tRNA genes on the light strand. The nucleotide compositions of the light strand are 28.88% of G, 28.75% of T, 26.58% of A and 15.80% of C. All the protein-coding genes share ATG initiation codon except ATP6 with CTG and COXI with GTG, and four types of inferred termination codons are T (ND2, COXII, ND3, ND4 and Cytb), TA (COXIII), TAA (ND1, ATP8, ND4L, ND5, ATP6 and COXI) and TAG (ND6). There are 10 intergenic spacers, 5 gene overlaps and no tandem repeat sequence. Gene arrangement and distribution are consistent with the typical vertebrates. [ABSTRACT FROM PUBLISHER]

The complete mitochondrial genome of theEpinephelus tukulawas presented in this study. The mitochondrial genome is 16,503 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and composition ofEpinephelus tukulamitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 28.37% of A, 29.00% of C, 26.36% of T and 16.26% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and seven tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM AUTHOR]

In this study, we have sequenced the complete mitochondrial genome of theParastromateus niger. The mitochondrial genome is 16,561 bp long and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and composition ofParastromateus nigermitochondrial genome is similar to that of most other vertebrates. The nucleotide compositions of the light strand are 28.23% of A, 29.51% of C, 26.01% of T and 16.16% of G. With the exception of five tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of the hybrid grouper Epinephelus coioides ♀ × Epinephelus akaara ♂ is presented in this study. The mitochondrial genome is 16,458bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The gene order and composition of the hybrid grouper mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand in descending order is 28.87% of G, 28.26% of T, 26.58% of A and 15.79% of C. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. The phylogenetic analysis by maximum-likelihood (ML) method showed that the hybrid grouper has the closer relationship to the hybrid grouper Epinephelus coioides ♀ × Epinephelus lanceolatus ♂. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of the hybrid grouperEpinehelus moara ♀×Epinephelus lanceolatus ♂is presented in this study. The mitochondrial genome is 16,418 bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The gene order and composition of the hybrid grouper mitochondrial genome were similar to that of most other vertebrates. The nucleotide compositions of the light strand in descending order was 28.86% of G, 28.61% of T, 26.46% of A and 16.08% of C. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. The phylogenetic analysis by maximum-likelihood (ML) method shows that the hybrid grouper has closer relationship toEpinehelus moaraandEpinehelus bruneus. [ABSTRACT FROM PUBLISHER]

The complete mitochondrial genome of theEpinephelus awoarawas presented in this study. The mitochondrial genome is 16 798-bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region. The gene order and composition ofEpinephelus awoaramitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand in descending order is 27.35% of A, 16.53% of C, 28.44% of T, and 27.69% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. The phylogenetic analysis by maximum-likelihood (ML) method shows that theEpinephelus awoarawas closer toEpinephelus fasciatomaculosusin the phylogenetic relationship. [ABSTRACT FROM AUTHOR]

We presented the complete mitochondrial genome of theEpinephelus corallicolain this study. The mitochondrial genome is 16,647 bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The gene order and composition ofEpinephelus corallicolamitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 26.60% of A, 15.89% of C, 28.66% of T and 28.85% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of the hybrid grouper (Epinephelus coioides♀ × Epinephelus lanceolatus♂) was presented in this study. The mitochondrial genome is 16,743 bp long and consists of 13 protein-coding genes, two rRNA genes, 22tRNAgenes, and a control region. The gene order and the composition of the hybrid grouper mitochondrial genome were similar to that of most other vertebrates. The nucleotide compositions of the light strand are 26.56% of A, 15.79% of C, 28.74% of T, and 28.91% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of theEpinephelus fuscoguttatuswas presented in this study. The mitochondrial genome is 16,648 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a control region. The gene order and the composition ofE. fuscoguttatusmitochondrial genome were similar to that of most other vertebrates. The nucleotide compositions of the light strand are 26.91% of A, 15.58% of C, 29.19% of T, and 28.32% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM AUTHOR]

We presented the complete mitochondrial genome of theEpinephelus moarain this study. The mitochondrial genome is 16,696 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and composition ofEpinephelus tukulamitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 26.45% of A, 16.09% of C, 28.60% of T and 28.86% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM PUBLISHER]

The complete mitochondrial genome of theEpinephelus akaarawas presented in this study. The mitochondrial genome is 16,743 bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The gene order and composition ofEpinephelus akaaramitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 27.31% of A, 16.20% of C, 28.68% of T and 27.81% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM PUBLISHER]

The complete mitochondrial genome of theEpinephelus lanceolatuswas presented in this study. The mitochondrial genome is 16,743 bp long and consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. The gene order and composition ofE. lanceolatusmitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 26.55% of A, 15.02% of C, 29.67% of T and 28.76% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM PUBLISHER]

The complete mitochondrial genome of theDrepane punctatawas presented in our study. The mitochondrial genome is 16,397 bp long and consists of 13 protein-coding genes, two rRNA genes, 16 tRNA genes and a control region. The gene order and composition ofD. punctatamitochondrial genome was different from that of most other vertebrates. The nucleotide compositions of the light strand are 24.56% of A, 16.02% of C, 27.81% of T and 31.61% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and five tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM PUBLISHER]

The complete mitochondrial genome of theRhabdosargus sarbawas presented in our study. The mitochondrial genome is 16,644 bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The gene order and composition ofR. sarbamitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 27.01% of A, 17.96% of C, 26.02% of T and 29.01% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. [ABSTRACT FROM PUBLISHER]

Neurokinin B (NKB) plays important roles in the mammalian reproductive axis by modulating the release of gonadotropin-releasing hormone (GnRH) and gonadotropins. In the present study, the tac3 cDNA was cloned from a hermaphroditic species, the orange-spotted grouper. Sequence analysis showed that the grouper Tac3 precursor encoded two tachykinin peptides, NKB and NKB-related peptide (NKBRP). Expression analysis in different tissues revealed that tac3 mRNA was highly expressed in the brain of the orange-spotted grouper. In situ hybridization further revealed that it was localized in some hypothalamic nuclei associated with reproductive regulation. During ovarian development, an increase of tac3 expression in the hypothalamus was observed at vitellogenesis stage. Intraperitoneal administration of NKB could increase the gnrh1 and lhβ mRNA levels, and enhance the serum estrogen levels, but did not significantly influence lhβ expression in cultured pituitary cells, indicating that NKB does not directly exert its actions on the pituitary gland. However, it was found that NKBRP had no effect on the expression of two gnrhs and two gths in vivo and in vitro . Effects of sex steroids on tac3 expression were further investigated. During the 17-methyltestosterone-induced sex change in the orange-spotted grouper, hypothalamic tac3 expression showed no significant change. Interestingly, ovariectomy greatly stimulated tac3 expression, while the 17β-estradiol treatment reversed this effect. In general, our data highly indicated that NKB signaling could activate the reproductive axis in the orange-spotted grouper. Our study is the first description of the NKB signaling in the hermaphroditic species. [ABSTRACT FROM AUTHOR]

Sex-specific markers are the prerequisite for interpreting the mechanisms of sex determination and sex control breeding in fish. Here, we provided a simple next generation sequencing strategy to identify potential sex-specific sequences in mandarin fish (Siniperca chuatsi) that is a greatly important freshwater fish in China. Through genome assembly, reads mapping, sequencing depth and coverage analysis of sequenced genomes of three males and three females, we identified 212 potential male-specific sequences. After two rounds of verification, a total of eight primer pairs were identified as potential candidate markers to be able to distinguish the genetic sex of mandarin fish. Subsequently, the best three markers were further tested in another four different farmed populations of mandarin fish, and the authenticity was 100%. Moreover, based on these markers, eight neomales were demonstrated to be genetic females and about 1: 1 sex ratio was observed in 215 juvenile offspring from two full-sib families. Notably, a duplicate of anti-Müllerian hormone, a gene participating in sex determination process in fish, was identified from the verified male-specific sequences. Overall, this study provided a simple and effective next generation sequencing method to identify sex-specific sequences and the male-specific molecular markers suggested an XY sex determination system in mandarin fish. • A simple next generation sequencing strategy were used to identify potential sex-specific sequences in mandarin fish. • Eight candidate sex-specific DNA markers were first developed to distinguish the genetic sex of mandarin fish. • A male-specific duplicate of anti-Müllerian hormone were identified in mandarin fish. [ABSTRACT FROM AUTHOR]

Vasa and dazl genes have been reported to play pivotal roles in germ cell development and differentiation both in vertebrates and invertebrates; however, little is known about their functions in germ cell differentiation during gametogenesis and sex reversal in hermaphroditic fish. In the present study, vasa (Ecvasa) and dazl (Ecdazl) cDNA were cloned from orange-spotted grouper (Epinephelus coioides). The full-length cDNA sequences of Ecvasa and Ecdazl were 2162 and 2101 bp, and encoded 646 and 214 amino acid residues, respectively. Reversal transcription PCR showed that Ecvasa and Ecdazl mRNA were highly expressed in the gonads. Further, in situ hybridization revealed that Ecvasa and Ecdazl RNA were dynamically expressed in germ cells at different stages during oogenesis, sex reversal, and spermatogenesis in orange-spotted grouper. Intriguingly, the signals for Ecvasa and Ecdazl mRNA became weaker in oocytes of ovo-testes gonads, indicating that the expression of germ cell genes could be suppressed in oocytes during sex reversal in the orange-spotted grouper. Our study is the first time to describe the expression profiles of vasa and dazl mRNA in germ cells during gametogenesis and sex reversal in the orange-spotted grouper. These findings will provide new insights into understanding the mechanisms through which vasa and dazl regulate germ cell differentiation in hermaphrodite fish species. • Ecvasa and Ecdazl mRNA were highly expressed in gonads. • Expression of germ cell genes could be suppressed in oocytes during sex reversal. • Vasa and dazl can be used as marker genes to trace the germ cells development during sex reversal in orange-spotted grouper. [ABSTRACT FROM AUTHOR]