Long-term exposure to 17β-estradiol of the Manila clam (Ruditapes philippinarum): Shifts of gender ratio and changes of gene expression

Estrogen is widely distributed in the aquatic environment, causing feminization, changes of gender ratio and reduced fecundity in aquatic organisms. 17β-estradiol is a potent estrogen with strong estrogenic activity and pseudo-persistence, which poses significant risks to global aquatic ecosystems. In this study, effects of long-term exposure to 17β-estradiol in the Manila clam (Ruditapes philippinarum) were investigated. The clams were treated with 17β-estradiol (10 μg/L) for 2 months and then subjected to dissection. Gonadal tissue sections were prepared for observation, and the sex ratio was calculated. The results showed that the female male ratio of clams in the control group was 0.93, which was close to 1. Nevertheless, the female male ratio of the 17β-estradiol-treated clams was 1.39, which was nearly 50 % higher than that in the control group. Furthermore, for clams in the control group, no hermaphrodites were observed. However, for clams treated with 17β-estradiol, 2 % of them were hermaphroditic, which was higher than that in natural environment (approximately one thousandth). It implies that long-term exposure to 17β-estradiol might affect sexual differentiation as well as sexual reversal in R. Philippinarum, promoting male individuals to reverse into female individuals. Comparative transcriptomics was conducted to identify genes related to sex differentiation in R. Philippinarum. Gene expression profiles of the estrogen-treated females (EF) were compared with those of the estrogen-treated males (EM), and 3751 differentially expressed genes (DEGs) were identified, among which 1512 were up-regulated in females, whereas 2239 were up-regulated in males. Meanwhile, the transcriptome profiles of females (DF) and males (DM) in the control group were compared, and 1718 differentially expressed genes (DEGs) were identified, of which 601 were upregulated in females and 1117 were upregulated in males. GO (gene ontology) and KEGG (Kyoto encyclopedia of genes and genomes) analyses showed that the above mentioned DEGs identified in both the estrogen treatment group and the control group were enriched in the ubiquitination pathway, ribosome, phagosome, and cytochrome P450 metabolic pathways. It is noteworthy that some of the gender-related DEGs identified in the comparison combination of DF vs DM were enriched in ubiquitination. In conculsion, the results indicate that exogenous estradiol treatment might influence sex differentiation in R. Philippinarum, enriching the knowledge on molecular mechanisms underlying sex differentiation in mollusks.