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In order to optimize the parameters for reversing masculinization and establish the techniques for sex induction of pseudo-males and creation of all-female fry in largemouth bass (Micropterus salmodies, LMB), 15-day-old LMB (1.00 ± 0.10 cm in length, 0.10 ± 0.01 g in weight) were fed a diet supplemented with either 17α-methyltestosterone (MT) or letrozole (LE) and their combination. The experimental groups were M20 (20 mg/kg MT), L20 (20 mg/kg LE) and M10L10 (10 mg/kg MT and 10 mg/kg LE). The control group, named C, was not feed MT or LE. After 60 days, exogenous hormone in the diets was stopped and the effects of MT and LE on growth, male ratio, and gonadal development in LMB were evaluated. At 12-month-old, blood and gonadal tissue samples were collected to measure sex steroid hormones levels, analyze expression levels of dmrt1 and cyp19a1a genes, as well as examine the gonads morphology. The results showed no significant differences in growth between the experimental groups and the control group after a 60-day feeding period with the formulated diet (p > 0.05). The sex reversal ratio of M20, L20, M10L10 were 95.00%, 80.00%, 76.47%, respectively. The gonadal tissue sections showed that the gonadal structure of masculinized fish morphologic resembled that of control male fish. At 12-month-old, the sex reversal ratio in M20, L20, M10L10 and C groups were 100%, 86.67%, 73.33% and 50.00%, respectively. The testicular of pseudo-male fish in the M20 group exhibited well-developed morphology similarities to that of the control group males. However, the testes of pseudo-male fish in the L20 and M10L10 groups were smaller size Estradiol (E2) levels in the experimental groups was significantly lower than those in the control group females (p < 0.05), while testosterone (T) levels were significantly higher than that of the control group (p < 0.05). Compared to the female fish in the control group, pseudo-male fish from all experimental groups showed significantly upregulated expression of dmrt1 (p < 0.05), and significantly downregulated expression of cyp19a1a (p < 0.05). Pseudo-males selected from group M20 exhibited a significantly higher proportion of female offspring (92.00%) compared to the control group (46.50%). In summary, 20 mg/kg MT was the optimal inducing concentration.

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.

The sex determination system of largemouth bass (Micropterus salmoides, LMB) is XX/XY; however, the underlying molecular mechanisms involved in early sex differentiation, gonadal development, and exogenous hormone-induced sex reversal remain unknown. In this study, LMB at 15 days post-hatching (dph) were fed diets containing 20 mg/kg of 17α-methyltestosterone (17α-MT) or 30 mg/kg of 17β-estradiol (17β-E2) for 60 days, respectively. Serum steroid levels, histological observations of the gonads, and identification of sex-specific markers were employed to screen the gonads of 60-day-old normal female fish (XX-F), normal male fish (XY-M), 17β-E2 induced pseudo-female fish (XY-F), and 17α-MT-induced pseudo-male fish (XX-M) for transcriptome sequencing in order to uncover genes and pathway involved in the process of sexual reversal. The results from histology and serum sex steroid hormone analysis showed that both 17α-MT and 17β-E2 were capable of inducing sex reversal of LMB at 15 dph. Transcriptome results revealed a total of 2,753 genes exhibiting differential expression, and the expression pattern of these genes in the gonads of XX-M or XY-F resembled that of normal females or males. The male sex-biased genes that are upregulated in XX-M and downregulated in XY-F are referred to as key genes for male reversal, while the female sex-biased genes that are upregulated in XY-F and downregulated in XX-M are referred to as key genes for female reversal. Finally, 12 differentially expressed genes (DEGs) related to male sex reversal were screened, including star2, cyp17a, cyp11b1, dmrt1, amh, sox9a, katnal1, spata4, spata6l, spata7, spata18 and foxl3. 2 DEGs (foxl2a and cyp19a1b) were found to be associated with female sex reversal. The changes in these genes collectively influence the direction of sex differentiation of LMB. Among them, star2, dmrt1 and cyp19a1b with significantly altered expression levels may play potentially crucial role in the process of gender reversal. The expression patterns of 21 randomly selected genes were verified using qRT-PCR which confirmed the reliability and accuracy of the RNA-seq results. These findings not only enhance our understanding of the molecular basis underlying sex reversal but also provide crucial data support for future breeding research on unisexual LMB.

Heat stress has become a significant issue in Largemouth bass (Micropterus salmoides, LMB). Identifying populations with superior heat tolerance abilities contribute to the breeding of new varieties with stress resistance. In this study, three populations of Northern LMB, including “YouLu No.3″ LMB (YL3), wild Northern LMB (NLMB) and Taiwan LMB (TW), as well as one Florida LMB population (M. floridanus, FLMB) were subjected to chronic heat stress. After being exposed to a temperature of 34 ℃ for 60 days, the FLMB group showed the highest final mean body weight (FBW), survival rate (SR), specific growth rate (SGR), and the lowest feed coefficient ratio (FCR) compared to the three Northern LMB groups. The lowest SR was observed in the TW group, while the lowest FBW and SGR, as well as the highest FCR were observed in the NLMB group. The FLMB group showed the lowest damage to the liver, with small focal infiltrations of inflammatory cells. However, the degree of liver damage was gradually worsened in the NLMB, YL3, and TW groups. The NLMB group had a small number of inflammatory cells, while the TW group had more lipid vacuoles and inflammatory cells. The FLMB group exhibited the highest activities of pepsin and lipase, whereas the NLMB group showed the lowest activity of amylase. Compared to the three Northern LMB groups, the FLMB group exhibited higher expressions of HSP60/70 and HSP90aa1/b1. Additionally, the YL3 group exhibited higher expressions of HSP60 and HSP90aa1/b1 compared to the NLMB and TW groups. In conclusion, our results indicated that the FLMB population had better chronic heat tolerance than the Northern LMB populations. Additionally, the YL3 population exhibited superior heat tolerance compared to the TW and NLMB populations. Our study provides valuable information for the future selective breeding of LMB varieties.

Largemouth bass (Micropterus salmoides L.) is generally considered to comprise two subspecies, Florida bass (M. floridanus) and Northern Largemouth bass (M. salmoides), which have biological characteristic differences because of their geographical distribution. In this study, whole-genome re-sequencing was performed among 10 Florida and 10 Northern largemouth bass, respectively. In total, 999,793 SNPs and 227,797 InDels were finally identified, and 507,401 SNPs (50.75%) and 116,213 InDels (51.01%) were successfully mapped to annotated 18,629 genes and 14,060 genes, respectively. KEGG classification indicated that most of these genes were focused on the pathways including signal transduction, transport and catabolism, and endocrine system. Genetic diversity analysis indicated that Florida largemouth bass had higher genetic diversity than Northern largemouth bass, indicating that the germplasm quality of Northern largemouth bass had markedly reduced in China. To examine the accuracies of the identified markers, 23 SNPs and eight InDels (the insertions or deletions of more than 45 bp) were randomly selected and detected among Florida largemouth bass, Northern largemouth bass, and their F1 hybrids. The detection efficiencies of all the markers were higher than 95%; nineteen SNPs and three InDels could accurately distinguish the two subspecies and their F1 hybrids with 100% efficiencies. Moreover, the three InDel markers could clearly distinguish the two subspecies and their F1 hybrids with a PCR-based agarose gel electrophoresis. In conclusion, our study established a simple PCR-based method for the germplasm identification of largemouth bass, which will be useful in the germplasm protection, management, and hybridization breeding of largemouth bass.

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.

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.

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.

A 40 day experiment was conducted to investigate the effects of different salinities (0, 5 and 10 ppt) on the growth, osmoregulatory and hypothalamic–pituitary–somatotropic (HPS) axis response of juvenile largemouth bass (Micropterus salmoides). At the same time, two extra groups salinities (8 and 9 ppt) were used to explore the threshold of osmoregulation in M. salmoides to salinity stress. The survival were 94.45 %, 98.15 % and 32.00 % in 0, 5, 10 ppt, respectively. According the final body weight, specific growth rate (SGR), feed conversion ratio (FCR), condition factor (K) and chemical composition of muscle, largemouth bass should not be cultured at or above 10 ppt and 5 ppt is better than 0 ppt. The results found that the content of serum prolactin (PRL) and cortisol (COR) were no significant differences between groups of the 0, 5, 8, 9 and 10 ppt. Compared with the control group (0 ppt), the serum thyroxine (T4) content of 8 and 9 ppt groups decreased significantly and the serum triiodothyronine (T3) content all decreased significantly. Serum osmolality increased with increasing salinity. Na+-K+-ATP (NKA) and Ca2+-Mg2+-ATP (CMA) activities in the gills of largemouth bass at 0, 5, 8, 9 and 10 ppt showed tendency of ascend at first and descend at last, among which the activities in 10 ppt were significantly lowest. Gene expression of hepatic growth hormone (GH) at 10 ppt was highest. Meanwhile, insulin-like growth factor-I (IGF-I) expression at salinities of 8, 9 and 10 ppt were significantly higher than those at 0 and 5 ppt. Taken together, the findings of this study provide guidance for the practical rearing of largemouth bass in saline waters and contribute to research on stress physiology.

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.

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