*Article* **FOXO-like Gene Is Involved in the Regulation of 20E Pathway through mTOR in** *Eriocheir sinensis*

**Jiaming Li 1,†, Yuhan Ma 1,†, Zhichao Yang 1, Fengchi Wang 1, Jialin Li 1, Yusheng Jiang 1, Dazuo Yang 1,2, Qilin Yi 1,2,\* and Shu Huang 1,\***


**Abstract:** The Forkhead Box O (FOXO) gene plays a key role in various biological processes, such as growth, metabolism, development, immunity and longevity. Molting is an essential process for crustacean growth, which is mainly regulated by 20-hydroxyecdysone (20E) and molt-inhibiting hormone (MIH). Although the role of FOXO in regulating the immune response of crustaceans is well documented, its involvement in controlling crustacean molting remains unclear. In this study, a FOXO-like gene (designed as *Es*FOXO-like) was identified in *Eriocheir sinensis*, and the regulation of the 20E pathway by *Es*FOXO-like was also investigated. The coding sequence of *Es*FOXO-like was 852 bp, which consisted of 283 amino acids including a conserved Forkhead (FH) domain. *Es*FOXO-like shared high similarity with FOXO genes from other crustaceans, and the mRNA expression levels of the *Es*FOXO-like gene were highest in the hepatopancreas and lowest in the hemocytes. However, transcription and protein expression of the *Es*FOXO-like gene were found to be up-regulated only during the pre-molt stage in the hepatopancreas, with lower expression levels observed at the post-molt stage. To explore the role of *Es*FOXO-like in the 20E pathway, *Es*FOXO-like was firstly inhibited by a specific FOXO inhibitor (AS1842856) and then through an *Es*FOXO-like dsRNA injection, respectively, and the results showed that the relative expression levels of *Es*FOXO-like were notably decreased in the hepatopancreas after both the inhibitor and dsRNA treatments. The 20E concentration, the mRNA expression levels of the 20E receptors including the ecdysone receptor (EcR) and the retinoid-X receptor (RXR) and *Es*mTOR transcription in the AS1842856 group or the *Es*FOXO-RNAi group were all significantly higher than that in the control group, while the mRNA expression level of *Es*MIH was significantly decreased after *Es*FOXO-like inhibition. To further investigate whether the *Es*FOXO-like acts through mTOR or not, Rapamycin was administered to inhibit mTOR activity in *Es*FOXO-like inhibited crabs. The results revealed a significant reduction in the concentration of 20E and the expression level of *Es*MIH in the AS1842856 + Rapamycin group compared to the AS1842856 + DMSO group, accompanied by an increase in *Es*EcR and *Es*RXR expression. These findings collectively suggest that *Es*FOXO-like regulates the 20E pathway through mTOR, which offered valuable insights into the understanding of the molting process in crustaceans.

**Keywords:** FOXO; 20-hydroxyecdysone (20E) pathway; mTOR; molting; *Eriocheir sinensis*
