| Southern catfish (Silurus meridionalis, Chen), which is endemic to China and widely distributed in the Yangtze River basin, is a special economic fish in China. Sexual differentiation of southern catfish took place at around7dah (day after hatching). Meiosis is initiated at about55dah and135dah in germ cells of ovary and testis, respectively. The artificial reared fries were all female, while the sex ratio of the feral catfish was1:1. Previous studies from our group have demonstrated that the feminization of the Southern catfish by artificial propagation may be attributed to the feeding of annelids(Limnodilus spp.) through studying of water, photoperiodicity, hatching temperature, rearing temperature after hatching, artificial insemination and feed. How many days did annelids need to induce feminization? Why annelids could cause feminization of Southern catfish? What is the mechanism of feminization? Meiotic initiation affects sex determination of fish. Therefore, whether meiotic initiation could be influenced by annelids? Methods and results based on above questions as follow:To determine the treatment length needed for feminization, the experimental fish were divided into five groups, fed with living Limnodilus spp. for0(control),5,10,30and90days, respectively. According to the previous studies on the morphology and microstructure of Southern catfish, the gender of gonads was identified. The results showed that fish fed with living annelids for5days only displayed73%female, while fish fed with living annelids for10,30and90days were all females. The control fish displayed a1:1sex ratio. Even feeding of annelids for ten days is sufficient for feminization of Southern catfish. Therefore, feminization of catfish fry was resulted from feeding of annelids during the early development stage.Why annelids could cause feminization of Southern catfish? In this study, annelids were collected from three different streams of the Jialing River in Chongqing. After sample pretreatment, EDCs in annelids were detected by HPLC. The results showed that the extraction of annelids contained EDCs, including Bisphenol A (BPA), Diethylstilbestrol (DES),4-tert-octylphenol (4-t-OP) and4-nonylphenol (4-NP), which were further confirmed by LC-MS (Liquid chromatography-mass spectrometry). All of the identified EDCs were detected in the three samples collected from different streams receiving different effluents of Jialing River. Moreover, average content of NP is the highest in the three samples. Therefore, feeding of annelids from the three sample sites resulted in all female.In order to verified annelids on feminization of Southern catfish, different food and time treatment were applied to catfish:1. To confirm that feminization was caused by EDCs in annelids, the experimental fish were divided into two groups, one fed with heat inactivated annelids, the other with commercial diet containing EDCs cocktail.2. The last group was fed with commercial fish diets as control.Gonads of90dah (days after hatching) Southern catfish were dissected and genders of these fish were preliminarily judged by the gonadal shape. The gonads were fixed in Bouin’s solution respectively and then embeded in paraffin and sectioned at5μm thickness. Statistical analysis of sex ratio was performed by microscopic observations of the sections. According to the previous studies on the morphology and microstructure of Southern catfish, the gender of gonads was identified. The results showed that fish fed with heat inactivated annelids resulted in100%female. In addition, diets containing EDCs cocktail also led to all females when checking histologically at90dah. Whereas the control fish displayed a1:1sex ratio.Therefore, complete feminization of catfish was caused by EDCs in annelids.Genes, such as Cyp19a1a, Foxl2, Dmrt1, etc., which were known to play important roles in sex determination and differentiation in teleosts, were found to be expressed in the1-year gonads according to transcriptome analyses, indicating that these genes play important roles not only in early sex determination and differentiation, but also in the later development and maintenance of gonads. Real-time PCR were performed to determine the expression levels of Sfl, Foxl2, Dmrtl and Cypl9ala in gonads from annelids fed fish, female and male control fish. Foxl2and Cypl9ala exhibited significantly higher expression levels in female control gonads; while Sf1and Dmrt1exhibited significantly higher expression levels in male control gonads both at30dah and90dah. In annelids fed gonads, the expression levels of the four genes were similar to those of control female gonads both at30dah and90dah. To determine whether ECDs influence the estrogen, androgen and vitellogenin production, we collected blood samples from the annelids fed and control fish, then measured their serum E2(estradiol-17β),11-KT (11-ketotestosterone) and vitellogenin levels. High E2(5.4ng/mL), vitellogenin (5.7μg/μL) and low11-KT (0.12ng/mL) levels, similar to those of the control female fish (4.3ng/mL and0.17ng/mL), were detected in samples from annelids fed fish; while low E2(0.18ng/mL) and high11-KT (0.89ng/mL) levels were detected in samples from control male fish. Therefore, EDCs could significantly affect expression of key genes involved in sex differentiation in catfish. Up-regulation of Foxl2and Cyp19a1a and down-regulation of Dmrtl could result in up-regulated aromatase expression and estrogen production, which in turn lead to the feminization of catfish.Stra8mRNA expression analysis for different meiotic stages of ovaries and testes were conducted via Real-time PCR. The results showed that the relative expression of Stra8mRNA were at the highest level in ovaries at50dah and testes at130dah, corresponding to the premeiotic stage of germ cells, and at lower levels in ovaries at40and60dah and testes at110and150dah. To identify the localization of Stra8/Stra8in gonads, in situ hybridization and immunohistochemistry were performed using ovaries at40,50and60dah and testes at110,130and150dah. The results showed that specific signals of Stra8/Stra8were observed in germ cells in gonads of both sexes. Weak mRNA and protein signals were detected in40dah ovaries and110dah testes. Strong signals were found in premeiotic germ cells in50dah ovaries and130dah testes. The signals became weaker in the60dah ovary and150dah testes. In addition, RA could upregulate the expression of Stra8. Hence, these results suggest that Stra8might participate in meiotic initiation in teleosts. Moreover, RA signal pathway might also play important roles in meiotic initiation in teleosts. Expression of Stra8in ovary, testis and gonad from annelids fed catfish were detected by immunohistochemistry. The results showed that strong signals were detected in ovary and gonad from annelids fed catfish. While few signal was detected in the testis. Therefore, EDCs might be control meiotic initiation through affecting the expression of Stra8.In summary, our data showed that feeding of annelids for ten days is sufficient for feminization of southern catfish. It is the EDCs in annelids resulted in the feminization through up-regulation of the endogenous estrogen level and altering meiotic initiation. This is the first report showing that feeding of any living organism for such short time resulted in complete feminization of a vertebrate. |
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