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Abstract The giant freshwater prawn (Macrobrachium rosenbergii) has many advantages in aquaculture, such as fast growth rate, short breeding cycle and good nutrition, which makes it a freshwater shrimp with high economic value. Herein, high-quality chromosome-level genome of both female and male prawns were obtained by combining Illumina paired-end sequencing, PacBio single molecule sequencing technique and High-through chromosome conformation capture (Hi-C) technologies. In ZZ male prawn, a final contig assembly of 3118.58 Mb with a N50 length of 956,237 bp was obtained. In WW female prawn, a final contig assembly of 3333.31 Mb with a N50 length of 1,143,555 bp was obtained. The assembled genome sequences from prawns were anchored to 59 chromosomes. Moreover, the sex chromosomes including W chromosome and Z chromosome were generated in prawn with the length of 36.23 Mb and 27.33 Mb, respectively. The sequence similarity of Z chromosome and W chromosome reached to 74.90%. The high-quality genome resource will be useful for further molecular breeding and functional genomic research of giant freshwater prawns.

The objective of this study is to investigate a viable and effective method for artificially breeding the mandarin fish (Siniperca chuatsi) in the out-of-breeding season. The mature development of the female mandarin fish's gonad was induced through precise control of water temperature. Measurements were taken during the temperature control period, including body weight, body height, ovulation rate, relative fecundity and gonadosomatic index (GSI). Serum hormone levels were assessed, and histological sections of gonadal tissues were tracked. Subsequently, a series of experiments were conducted to establish the optimal protocol for hormone-induced ovulation and artificial fertilization. The findings revealed that manipulation and elevation of the aquaculture water body temperature could effectively induce gonad development and maturation in mandarin fish. In the high temperature group (HT, water temperature maintained at 25 ± 1 ℃), 81.25 % of the experimental fish reached maturity within 20 days. Subsequent hormone injection resulted in successful ovulation, normal fertilization, and subsequent incubation of viable young fish. Furthermore, the outcomes of the ovulation test using hormones demonstrated that the pairing of luteinizing hormone releasing hormone (LHRH-A2) and domperidone (DOM) proved highly effective in inducing ovulation in mature mandarin fish in the out-of-breeding season. The ideal dosage for a single injection of this combined hormone was determined to be 6 μg LHRH-A2 and 4 mg DOM / kg of body weight. Employing this optimal hormone dosage resulted in a fertilization rate of 83.23 %, a ovulation rate of 100 %, a relative fecundity of 93.40 ± 17.59 103 / kg and a hatching rate of 88.85 %. The anticipated outcome of these studies is to enhance the artificial breeding techniques for mandarin fish in commercial aquaculture, allowing for year-round production of fertilized eggs.

Yellowfin seabream (Acanthopagrus latus) is a hermaphroditic fish with male prematurity, which is also an economically important mariculture species in coastal areas of China. Although the production yield has been increasing in recent years, artificial breeding of this species remains imperfect due to the lack understanding of its sex differentiation mechanism. In this study, we conducted transcriptomic sequencing to screen for sex-related genes in different stage of the testis, ovotestis, ovary, hypothalamus, and pituitary tissues of yellowfin seabream. During gonad development (from intersex to mature stage), Gnrh3, Kiss2, and Dbh were expressed in the hypothalamus and Lhb and Fshb were expressed in the pituitary. Sex-related genes such as Hsd11b1, Foxl2l, Amh, and Gsdf were among the top 10 downregulated genes in the intersex testis compared with the mature testis. The expression of Hsd11b, Wnt, and Sox families genes differed significantly in the sex change process among functional mature gonads. In addition, treatment with estradiol resulted in feminizing of the ovotestis. Inhibition of androgens or estrogens promoted ovarian or testicular steps of ovotestis development. In summary, we systematically evaluated gene expression profiles during yellowfin seabream gonad development from intersex to functional male or female. Our results confirmed that regulation by the hypothalamus-pituitary-gonad (HPG) axis and the promotion of sex steroid hormones acted on this process, and they provide a reference for further studies of the involvement of the HPG axis in sex change and of sex induction methods in yellowfin seabream.

Yellowfin tuna (Thunnus albacares) is a valuable pelagic migratory fish with potential for aquaculture. Despite this, there is limited understanding of the biological and physiological characteristics of this species, particularly regarding sex differences in growth performance. The liver, a crucial organ for digestion and metabolism, plays a significant role in regulating fish growth. This study aimed to compare liver enzyme activities and transcriptome profiles between female and male yellowfin tuna to uncover the molecular mechanisms underlying difference between the sexes. The results revealed that female yellowfin tuna exhibited higher amylase and lipid metabolism enzyme activities, while male yellowfin tuna showed higher glucose-6-phosphate dehydrogenase and antioxidant enzyme activities. Additionally, through Illumina sequencing technology, the study generated 37.74 Gb of clean data and identified 36,482 unique genes (UniGenes) in the liver transcriptome. A total of 2542 differentially expressed genes were found, with enriched Gene Ontology terms and pathways related to metabolic processes, particularly lipid metabolism and transport. These findings suggest that female yellowfin tuna have superior digestive enzyme activities and lipid metabolism, while male yellowfin tuna excel in sugar metabolism, ATP production, and antioxidant defense. This study provides valuable insights into sex differences in yellowfin tuna and could aid in advancing full-cycle aquaculture practices for this species.

The blotched snakehead Channa maculata is an important economical freshwater species in East Asia. However, there has been relatively little research conducted on the correlation between gender and gut microbes. In this study, 36 of 1000 blotched snakeheads were randomly selected for growth performance measurement and gut microbiota high-throughput sequencing. Results showed that microbial diversity, composition, and metabolic functions were altered by gender and growth performance except the microbial network. In our study, Proteobacteria were the most abundant phylum, with Fusobacteria showing enrichment in males and Bacteroidetes in females. Notably, phylum Deinococcus-Thermus was identified as a significant biomarker. The Cetobacterium was the most abundant genus-level taxon. Furthermore, gut microbes specializing in the production of gut-healthy substances, such as coenzymes and vitamins, were identified as biomarkers in the fast-growing group. Our investigation highlighted the impact of gender on the composition and abundance of gut microbial biomarkers in both males and females, thereby influencing differential growth performance through the modulation of specific metabolic functions.

Low-density lipoprotein receptor (LDLR) superfamily is an ancient gene family composed of different genes encoding membrane receptors involved in ligand binding and endocytosis. These receptors typically consist of class A ligand-binding repeats (LBRs), epidermal growth factor precursor-like (EGF) domain, class b YWXD motifs, transmembrane (TM) domain, and cytoplasmic (CP) domain. Low-density lipoprotein receptor related protein 13 (Lrp13), a vitellogenin receptor with a unique domain structure, belonging to the LDLR superfamily, would play an important role in vitellogenesis in teleosts. In this study, the full-length cDNA of Ec-lrp13 was isolated from the ovary of orange-spotted grouper (Epinephelus coioides), an economically important aquaculture species in China. The open reading frame (ORF) of Ec-lrp13 is 3831 bp in length, encoding 1276 amino acids. The Ec-Lrp13 includes several typical domains of the LDLR superfamily and contains the specific structure of the Lrp13 protein. Ec-lrp13 is predominantly expressed in ovary. During ovarian development, Ec-lrp13 was highly expressed in stage II and III ovaries but its mRNA level was significantly decreased in stage IV ovary. In situ hybridization (ISH) analysis revealed that Ec-lrp13 mRNA signals were obviously observed in the ooplasm of PG and EPV oocytes, but decreased in the LPV oocytes, and weakly found in the VG oocytes. Immunohistochemistry (IHC) detection showed that Ec-Lrp13 protein signals were only observed in the LPV and VG oocytes. Our data provides preliminary evidence that Lrp13 may be involved in the Vtg uptake during ovarian development of orange-spotted grouper.

The estrogen-related receptors (ERRs), a group of nuclear receptors, are the first orphan receptor to be identified. In this study, six errs (errα, errβ, errγ, errγA, errγB, errδ) were first cloned from the gonads of mandarin fish (Siniperca chuatsi). Gene cloning results showed the open reading frames (ORF) of both errα, errβ, errγ, errγA, errγB, errδ were 1302 bp, 1434 bp, 1323 bp, 1566 bp and 1308 bp, coding a 432, 476, 439, 520, 434 and 337 amino acid (AA) peptide, respectively. Tissue distribution analysis showed that errα was relatively highly expressed in heart and muscle both in male and female and errγA was highly expressed in brain, stomach and muscle while the errγB was extremely highly expressed in heart and liver both in male and female. The errβ expressed relatively highly in medulla oblongata and gonad of male and female and kidney of male, while errγ expressed relatively highly in stomach of male and female. Gonad expression pattern revealed that errα, errγ, errγA and errγB were all detected from 5 to 180 day after hatching (dah) in both males and females, and errδ began to express after 60 dah. Moreover, errγ, errγA and errγB were highly expressed in 5–10 dah while errα and errδ were mainly expressed in 60–180 dah. Furthermore, luciferase assay showed that errγ could response to estrogen stimulation by modulating estrogen-signaling pathway in mandarin fish.

The Jumonji family (Jmj family) is a large family of proteins that contain the JmjC domain. Various members of this family are lysine (K)-specific demethylases, which remove methylation from the lysines of histones, and thus, are known as Jmj-KDMs. In this study, all Jmj-KDMs were identified in the orange-spotted grouper Epinephelus coioides and compared with those from Homo sapiens, Danio rerio, Takifugu rubripes, Oreochromis niloticus, and Xenopus tropicalis. The comparison revealed the teleost-specific loss of jmjd1a and the teleost-specific expansion of kdm5b, jmjd3, jmjd2a, and kdm6a in E. coioides. Analysis of the temporal expression pattern of Jmj-KDM genes in E. coioides enabled their division into different types according to their sex-related expression patterns. The kdm6 subfamily genes kdm6aa and jmjd3 (kdm6bb), which are also teleost specific, were selected for further analysis. Quantitative PCR analysis revealed the significantly higher expression of kdm6bb in bisexual-phase gonads and testes compared with ovary. Histological expression pattern analysis based on in situ hybridization (ISH) showed the high expression of kdm6aa in primary-growth stage oocytes, cortical-alveolus stage oocytes and granulosa cells, whereas kdm6aa and jmjd3 were highly expressed in Sertoli cells and spermatogonium. These results suggest that Jmj-KDMs, especially the kdm6 subfamily genes kdm6aa and jmjd3, are involved in sex-related processes in E. coioides.

G Protein-coupled receptor 3 (Gpr3) plays a critical role in maintaining oocyte meiotic arrest in mammals. To ascertain the function of Gpr3 in oocyte maturation in fish, the Gpr3 cDNA was cloned from orange-spotted grouper (Epinephelus coioides). The open reading frame of cloned sequence was 993 bp in length, encoding 330 amino acids. Gpr3 was primarily expressed in brain regions and ovary. During oocyte maturation induced by human chorionic gonadotropin (hCG), Gpr3 expression levels were dramatically elevated in follicular cell layers. Functional analysis showed that Gpr3 could constitutively active the cAMP/PKA signaling pathway, and hCG treatment activated Gpr3 promoter activities in cultured eukaryotic cells expressing luteinizing hormone receptors (LHRs). Finally, the role of Gpr3 in oocyte maturation was studied. Sphingosine 1-phosphate (S1P), an agonist of Gpr3, had no effect on oocyte maturation in vivo, suggesting that Gpr3 may be not involved in oocyte maturation in orange-spotted grouper.

Mandarin fish (Siniperca chuatsi) is one of the most important commercial fish in aquaculture in the world. As females grow faster than males, the cultivation of all-female mandarin fish has great economic value and commercial prospects. However, little is known about the molecular mechanism of the early gonad development and sex reversal of mandarin fish. In this study, histological methods and transcriptome sequencing techniques were used to study the early gonadal development and gene expression pattern of genetically female (XX) mandarin fish during MT-induced masculinization. The histological results showed that the labile period of sex differentiation of XX mandarin fish was before 36 dpf and MT treatment before this period could induce complete sex reversal. The transcriptome results showed that the expression levels of a few female-related genes were down-regulated by MT during the morphological differentiation stage to cytological differentiation stage of XX gonad, such as cyp19a1a, foxl2, foxl3, hsd17b1, figla, gdf9, bmp15 and β-catenin, suggesting that the changes of these genes may determine the direction of sex differentiation in mandarin fish. In addition, MT also activated the expression of some male-related genes, such as dmrt1, hsd17b3, hsd11b2, cyp11a1, cyp11b, amh, amhr2, sox9 and gsdf. Some of these genes were activated during spermatogenesis stage, suggesting that the up-regulation of these male-related genes may be the result of sex differentiation rather than the cause. The qRT-PCR results of eight randomly selected differentially expressed genes were basically consistent with the transcriptome results, which proved that the transcriptome data were reliable. These results indicated that the expression of genes related to estrogen synthesis in early development stage may play a decisive role in the direction of sex differentiation in mandarin fish.

Abstract 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.

Dmrt gene family plays a very critical role in sex determination and differentiation in animals. In this study, dmrt1, dmrt2 and dmrt3 were first cloned from the gonads of mandarin fish (Siniperca chuatsi). The complete open reading frame sequences of dmrt1, dmrt2 and dmrt3 were 891, 1581 and 1419 bp, encoding 296, 526 and 473 amino acids. Multiple sequence alignments revealed that dmrt proteins were highly conserved in teleosts, especially in DNA-binding domain. Tissue distribution analysis showed that dmrt1 expressed mainly in testis, dmrt2 expressed in ovary and dmrt3 expressed in heart, muscle and gonad. Gonad expression pattern revealed dmrt1 mainly expressed in testis after 15 day after hatching, while dmrt2 mainly expressed after 60 dah. After 17α-methyltestosterone treatment, expression levels of dmrt1 and dmrt3 increased significantly, while dmrt2 expression was repressed. Furthermore, luciferase assay showed dmrt1 could activate gsdf-promotor and inhibit cyp19a1a-promotor alone in vitro. These results suggest that the dmrt1 play a most important role in testis differentiation/development and dmrt2 is critical in ovary development in mandarin fish.

Steroid hormones are essential for the normal reproductive activity in females and males, such as early sex differentiation, gonad development and sex reversal, which works by binding with the steroid receptors. In the present study, the full-length of estrogen receptors (ERα, ERβ1 and ERβ2) and androgen receptors (ARα and ARβ) cDNAs were isolated from the mandarin fish (Siniperca chuatsi). Real time quantitative PCR (RT-qPCR) revealed that ERα and ERβ2 expressed mainly in the liver of females and males, and ERβ1 mostly distributed in ovary and testis. Additionally, the high expressions of ARα and ARβ in both females and males were detected in gonads and livers respectively. During the early sex differentiation stage, the expressions of ERs and ARs in female were higher than in male during 5–30 day after hatching (dah), and reached the peak level at 30 dah. In males, the expressions of ERβ1, ERβ2, and ARβ increased largely during 30 dah to 60 dah, and revealed significantly higher levels than in females during 60–180 dah. In female, the expression of ERβ2 in ovary increased sharply at 180 dah. Moreover, the result of gonadal histology revealed that the spermatocytes were observed at 45 dah and the spermatozoon could be found largely at 60 dah in males and oil droplets appeared for the first time in the oocytes at 180 dah. The expression levels of three ERs and ARβ were up-regulated and the ARα was down-regulated during female to male sex reversal induced by 17α-methyltestosterone (MT). Furthermore, after MT treatment for 30 and 35 days, the expressions of ERβ1, ERβ2 and ARβ were significantly up-regulated. These results suggest that ERβ1, ERβ2 and ARβ may play critical roles in spermatogenesis and masculinization, while ERβ2 and ERβ1 may be essential for vitellogenesis and ovary development in female.

The tomato hind, Cephalopholis sonnerati, is a bottom-dwelling coral reef fish, which is widely distributed in the Indo-Pacific and Red Sea. C. sonnerati also features complex social structures and behaviour mechanisms. Here, we present a high-quality, chromosome-level genome assembly for C. sonnerati that was derived using PacBio sequencing and Hi-C technologies. A 1043.66 Mb genome with an N50 length of 2.49 Mb was assembled, produced containing 795 contigs assembled into 24 chromosomes. Overall, 97.2% of the complete BUSCOs were identified in the genome. A total of 26,130 protein-coding genes were predicted, of which 94.26% were functionally annotated. Evolutionary analysis revealed that C. sonnerati diverged from its common ancestor with E. lanceolatus and E. akaara approximately 41.7 million years ago. In addition, comparative genome analyses indicated that the expanded gene families were highly enriched in the sensory system. Finally, we found the tissue-specific expression of 8108 genes. We found that these tissue-specific genes were highly enriched in the brain. In brief, the high-quality, chromosome-level reference genome will provide a valuable genome resource for studies of the genetic conservation, resistance breeding, and evolution of C. sonnerati.

Rigid polyurethane foam (RPUF) was an organic porous material, which was applied in many fields for excellent thermal insulation and mechanical properties, especially in building insulation. However, the poor fire performance significantly suppresses its further application. In this work, aluminum diethylphosphinate (ADP) combined with expanded graphite (EG) to form a synergistic flame retarded system, which was introduced to fabricate flame retarded rigid polyurethane foam composites (FR-RPUF) by one-step water-blown method. Furthermore, thermal insulation, thermal stability, fire performance, and decomposition products of RPUF and FR-RPUF composites were systematically investigated. It was found that FR-RPUF composites possessed LOI of 25.9 vol% with V-1 rating in UL-94 test when 10 php of ADP and 20 php of EG were added, which were better than RPUF composites with ADP or EG added alone. MCC test showed that RPUF/ADP24/EG6 had the lowest PHRR value of 159.85 W/g, which was 52.01 W/g lower than that of pure RPUF. Gas phase products investigation implied that the combination of ADP and EG could decrease toxic and combustible gases intensities, thus significantly enhancing fire safety of FR-RPUF composites. SEM test indicated that ADP and EG promoted the formation of dense and continuous char residue, which significantly inhibited heat and substance transfer in combustion, thus significantly enhancing fire performance of FR-RPUF composites.

Sex-specific markers are extremely important for unisexual breeding and understanding of sex determination mechanisms. Spotted mandarin fish (Siniperca scherzeri) and big eye mandarin fish (Siniperca kneri) are economically important freshwater fish in China. Here, for the first time, we successfully identified three male-specific markers in the two species. Through genome sequencing and bioinformatic analysis of three males, three females, one male mixed pool and one female mixed pool of spotted mandarin fish, we screened 27 potential male-specific sequences. After PCR validation, three male-specific markers were further identified. Moreover, these markers could also be used to identify genetic sex of spotted mandarin fish from different populations with 100 % accuracy. In addition, these markers were also successfully used in the identification of genetic sex of big eye mandarin fish. Notably, the sequences of male-specific markers were highly similar in the two species and the corresponding sequence of one marker was highly similar to partial genome sequence of amhy gene in mandarin fish (Siniperca chuatsi). In all, the verified male-specific markers suggested XY sex determination system existed in spotted mandarin fish and big eye mandarin fish and would play an important role in future work of sex control breeding.

Beta-hydroxysteroid dehydrogenases (β-HSDs) are a group of steroidogenic enzymes that are involved in steroid biosynthesis and metabolism, and play a crucial role in mammalian physiology and development, including sex determination and differentiation. In the present study, a genome-wide analysis identified the numbers of β-hsd genes in orange-spotted grouper (Epinephelus coioides) (19), human (Homo sapiens) (22), mouse (Mus musculus) (24), chicken (Gallus gallus) (16), xenopus (Xenopus tropicalis) (24), coelacanth (Latimeria chalumnae) (17), spotted gar (Lepisosteus oculatus) (14), zebrafish (Danio rerio) (19), fugu (Takifugu rubripes) (19), tilapia (Oreochromis niloticus) (19), medaka (Oryzias latipes) (19), stickleback (Gasterosteus aculeatus) (17) and common carp (Cyprinus carpio) (27) samples. A comparative analysis revealed that the number of β-hsd genes in teleost fish was no greater than in tetrapods due to gene loss followed by a teleost-specific whole-genome duplication event. Based on transcriptome data from grouper brain and gonad samples during sex reversal, six β-hsd genes had relatively high expression levels in the brain, indicating that these genes may be required for neurogenesis or the maintenance of specific biological processes in the brain. In the gonad, two and eight β-hsd genes were up- and downregulated, respectively, indicating their important roles in sex reversal. Our results demonstrated that β-hsd genes may be involved in the sex reversal of grouper by regulating the synthesis and metabolism of sex steroid hormones.

The tunnel muck has a great potential to be used as a recyclable engineering material in transit and other civil work projects instead of being deposited as construction waste. In this work, the geopolymer is generated by alkali-activated fly ashes, which act as cementitious materials to strengthen the tunnel muck. The tunnel muck has to be dehydrated, grinded, and screened before being treated by alkali-activated fly ash. The effect of the mass ratio between fly ash and tunnel muck (Mfa/Mtm), the mass ratio between Na2SiO3 solution and NaOH solution (MNa2SiO3/MNaOH), the ratio between liquid and solid (Mliquid/Msolid), and molarity of NaOH on the strength of geopolymer were systematically studied by conducting the uniaxial compression experiments. The experimental results indicate that the liquid-to-solid ratio is the most important parameter to the geopolymer strength after the alkali-activated fly ash treatment. On the contrary, molarity of NaOH is less effective on the geopolymer strength. Moreover, the optimum scheme is concluded according to the experimental results as follows: the mass ratio between tunnel muck and fly ash, the mass ratio between Na2SiO3 solution and NaOH solution, the ratio between liquid and solid, and molarity of NaOH are 1 : 2, 1.8, 0.18, and 10 mol/L, respectively. Meanwhile, the SEM images indicate that flocculence from the active substance in fly ash is a crucial component as the cementing material.

In this study, we injected cortisol into the protogynous orange-spotted grouper (Epinephelus coioides) to investigate the role of this hormone in sex change. Following injection, we evaluated gonadal changes, serum levels of steroid hormones, and sex-related gene expression during the processes of cortisol-induced sex change and cortisol withdrawal in the orange-spotted grouper. Cortisol treatment caused the degeneration of oocytes and induced sex change in a dose-dependent manner. Over the long-term, we observed a significant increase in serum 11-ketotestosterone (11-KT) levels in all cortisol-treated groups, although levels of 17β-estradiol did not change significantly. Consistent with the elevation of serum 11-KT levels, the expression of genes related to testicular development was also significantly up-regulated in the cortisol-treated groups. Based on our results, we propose that cortisol may trigger masculinization by inducing the synthesis of 11-KT and by directly activating the expression of sex-related genes. Furthermore, we found that cortisol-induced sex change was not permanent and could be reversed after the withdrawal of cortisol treatment.

The spotted scat (Scatophagus argus) is an economically important cultured marine fish that exhibits a typical sexual size dimorphism (SSD). SSD has captivated considerable curiosity for farmed fish production; however, up till now the exact underlying mechanism remains largely unclear. As an important digestive and metabolic organ, the liver plays key roles in the regulation of fish growth. It is necessary to elucidate its significance as a downstream component of the hypothalamic-pituitary-liver axis in the formation of SSD. In this study, the liver physiological differences between the sexes were evaluated in S. argus, and the activity of several digestive and metabolic enzymes were affected by sex. Females had higher amylase, protease, and glucose-6-phosphate dehydrogenase activities, while males exhibited markedly higher hepatic lipase and antioxidant enzymes activities. A comparative transcriptomics was then performed to characterize the responsive genes. Illumina sequencing generated 272.6 million clean reads, which were assembled into 79,115 unigenes. A total of 259 differentially expressed genes were identified and a few growth-controlling genes such as igf1 and igfbp1 exhibited female-biased expression. Further analyses showed that several GO terms and pathways associated with metabolic process, particularly lipid and energy metabolisms, were significantly enriched. The male liver showed a more active mitochondrial energy metabolism, implicating an increased energy expenditure associated with reproduction. Collectively, the female-biased growth dimorphism of S. argus may be partially attributed to sexually dimorphic metabolism in the liver. These findings would facilitate further understanding of the nature of SSD in teleost fish.

Follicle-stimulating hormone (FSH) signaling is considered to be essential for early gametogenesis in teleosts, but its functional roles during sex differentiation are largely unknown. In this study, we investigated the effects of long-term and short-term FSH injection on sex differentiation in the protogynous orange-spotted grouper (Epinephelus coioides). Long-term FSH treatment initially promoted the formation of ovaries but subsequently induced a male fate. The expression of female pathway genes was initially increased but then decreased, whereas the expression of male pathway genes was up-regulated only during long-term FSH treatment. The genes related to the synthesis of sex steroid hormones, as well as serum 11-ketotestosterone and estradiol, were also up-regulated during long-term FSH treatment. Short-term FSH treatment activated genes in the female pathway (especially cyp19a1a) at low doses but caused inhibition at high doses. Genes in the male pathway were up-regulated by high concentrations of FSH over the short term. Finally, we found that low, but not high, concentrations of FSH treatment activated cyp19a1a promoter activities in human embryonic kidney (HEK) 293 cells. Overall, our data suggested that FSH may induce ovarian differentiation or a change to a male sex fate in the protogynous orange-spotted grouper, and that these processes occurred in an FSH concentration-dependent manner.

Sex differentiation in teleost fishes occurs in response to sex determination signals, which induce the gonad to develop as either an ovary or testis. However, sex differentiation mechanisms in fishes are diverse, and information on gonad differentiation in sex changing fishes remains limited. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish that provides an ideal model for investigating gonad differentiation in vertebrates. In this study, Transcriptome data showed that expression levels of amh and amhrII in gonads were increased during sex differentiation. Then we investigated the effect of overexpression anti-Müllerian hormone (Amh) on gonad development in juvenile orange-spotted groupers. Expression levels of female-related genes and serum 17β-estradiol levels were decreased, while expression of male-related genes and serum 11-ketotestosterone levels were increased in fish fed with amh-plasmid. Overexpression of Amh was also promoted the spermatogonia proliferation and induced the development of male gonads in undifferentiated orange-spotted groupers, but that this male tendency was preceded by female differentiation. In summary, these results illustrated that Amh overexpression by amh-plasmid feeding induced male gonad development in undifferentiated groupers.

The kisspeptin/GPR54 system plays a crucial role in the regulation of the reproductive axis in vertebrates. Male pregnancy and ovoviviparity are special reproductive phenomena among vertebrates. To better understand the neuroendocrine mechanisms of male pregnancy, cDNAs encoding kiss2 and GPR54 were cloned and functionally characterized from the lined seahorse, Hippocampus erectus, an ovoviviparous teleost with male pregnancy. The core mature peptide of seahorse Kiss2 is high conserved among seahorses, but unique among vertebrate Kiss orthologs. In the phylogenic analysis, the seahorse Kiss clustered with the teleost Kiss2 clade. The kiss2 transcripts were shown to be widely expressed in various tissues, notably in the brain and gonad of the seahorse, while GPR54-2 mRNA was expressed exclusively in the brain. In addition, kiss2 mRNA found in male seahorse brain tissue increased significantly at the early pubertal stage, and decreased significantly during pregnancy. Intraperitoneal administration of seahorse Kiss2-10 to sexual mature male seahorses demonstrated to stimulate lutropin β (LHβ) and follitropin β (FSHβ) release and increased serum testosterone levels. In summary, we first identified the kisspeptin/GPR54 system in an ovoviviparous fish with male pregnancy, which might be involved in the regulation of the reproductive functions of pubertal onset, gonadal development, and male pregnancy via regulating the synthesis of both gonadotropic hormone (GTH) and testosterone.

Leptin is an essential hormone for the regulation of energy metabolism and food intake in vertebrate animals. To better understand the physiological roles of leptin in nutrient regulation in paternal ovoviviparous fish (family Syngnathidae), the present study cloned the full-length of leptin-a and leptin receptor (lepr) genes in lined seahorse (Hippocampus erectus). Results showed that there was a 576-bp intron between two exons in leptin-a gene but no leptin-b gene in seahorse. Although the primary amino acid sequence conservation of seahorse leptin-a was very low, the 3-D structure modeling of seahorse leptin-a revealed strong conservation of tertiary structure with other vertebrates. Seahorse leptin-a mRNA was highly expressed in brain, whereas lepr mRNA was mainly expressed in ovary and gill. Interestingly, both leptin-a and lepr mRNA were expressed in the brood pouch of male seahorse, suggesting the leptin system plays a role during the male pregnancy. Physiological experiments showed that the expression of hepatic leptin-a and lepr mRNA in unfed seahorses was significantly higher than that in those fed 100%, as well as 60%, of their food during the fasting stage, showing that seahorse might initiate the leptin system to regulate its energy metabolism while starving. Moreover, the expression of leptin-a in the brood pouch of pregnant seahorse was significantly upregulated compared with non-pregnant seahorse, whereas the expression of lepr was downregulated, suggesting that the leptin system might be involved in the male pregnancy. In conclusion, the leptin system plays a role in the energy metabolism and food intake, and might provide new insights into molecular regulation of male pregnancy in seahorse.

Giant groupers, the largest grouper type in the world, are of economic importance in marine aquaculture for their rapid growth. At the same time, bacterial and viral diseases have become the main threats to the grouper industry. Here, we report a high-quality genome of a giant grouper sequenced by an Illumina HiSeq X-Ten and PacBio Bioscience Sequel platform. A total of 254 putative antimicrobial peptide (AMP) genes were identified, which can be divided into 34 classes according to the annotation of the Antimicrobial Peptides Database (APD3). Their locations in pseudochromosomes were also determined. Thrombin-, lectin-, and scolopendin-derived putative AMPs were the three largest parts. In addition, expressions of putative AMPs were measured by our transcriptome data. Two putative AMP genes (gapdh1 and gapdh2) were involved in glycolysis, which had extremely high expression levels in giant grouper muscle. As it has been reported that AMPs inhibit the growth of a broad spectrum of microbes and participate in regulating innate and adaptive immune responses, genome sequencing of this study provides a comprehensive cataloging of putative AMPs of groupers, supporting antimicrobial research and aquaculture therapy. These genomic resources will be beneficial to further molecular breeding of this economically important fish.

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 ♂.