**1. Introduction**

The Forkhead Box O (FOXO) protein, which belongs to the Forkhead transcription factor family, was initially identified as a significant downstream molecular target of the insulin pathway [1,2]. FOXO is known to participate in numerous physiological responses that determine development, metabolism, cell cycle, apoptosis and longevity [3,4]. The

**Citation:** Li, J.; Ma, Y.; Yang, Z.; Wang, F.; Li, J.; Jiang, Y.; Yang, D.; Yi, Q.; Huang, S. FOXO-like Gene Is Involved in the Regulation of 20E Pathway through mTOR in *Eriocheir sinensis*. *J. Mar. Sci. Eng.* **2023**, *11*, 1225. https://doi.org/10.3390/ jmse11061225

Academic Editor: Nguyen Hong Nguyen

Received: 26 April 2023 Revised: 9 June 2023 Accepted: 12 June 2023 Published: 14 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

FOXO gene is evolutionarily conserved with a typical Forkhead (FH) DNA domain, which consists of three α-vehicles, three β-sands and two wing-like loops [5,6]. Until now, four FOXO homologous genes, including FOXO1, FOXO3α, FOXO4 and FOXO6, were identified in higher animals [7,8]. The distribution characteristics of the four FOXO genes are different as they are involved in the regulation of various biological processes [9]. However, only one FOXO gene was identified in invertebrates [10].

In recent studies of insects, the FOXO gene has been demonstrated to be involved in the regulation of development and growth [11]. For instance, FOXO overexpression in the adipose body could prolong the lifespan of *Drosophila melanogaster* [12]. Additionally, in *Bactrocera doralis*, the weight of the larvae body was significantly increased after FOXO inhibition [13]. Moreover, in *Blattella germanica*, *Bg*FOXO had an inhibitory effect on juvenile hormone (JH) biosynthesis in the case of nutrient deficiency [14]. The activation of FOXO through inhibition of insulin signaling led to reproductive diapause and, ultimately, resulted in the cessation of JH production in *Culex pipiens* [15]. In addition, it has been demonstrated that FOXO is involved in the metamorphosis of insects. Knocking down the expression of FOXO in *Helicoverpa armigera* larvae led to a molting failure [16]. Similarly, *Tribolium castaneum* larvae with silenced FOXO genes exhibited a significant delay in pupation [17]. Ecdysone, also known as 20-hydroxyecdysone (20E), has been found to stimulate FOXO transcription factor activity, resulting in increased expression of acid lipase-1 and subsequent promotion of fat decomposition in *Bombyx mori* [18]. In *H. armigera*, it has been observed that 20E can activate FOXO to facilitate protein hydrolysis during the molting cycle [16]. In larvae of *Tribolium castaneum*, RNA interference of the FOXO gene also led to delayed pupation, reduced levels of 20E and decreased expression of both the prothymotropic hormone (PTTH) and the spook (spo) gene, which are crucial for ecdysone biosynthesis [17]. Studies on the physiological role of FOXO in crustaceans are mainly focused on the immune response. It has been reported that the expression of FOXO was significantly decreased in the intestine of crabs after hepatopancreatic necrosis disease (HPND) stimulation [19]. In the Chinese mitten crab, FOXO has also been found to have a positive impact on the expression levels of genes coding antimicrobial peptides (AMPs) [19]. In *Marsupenaeus japonicas*, FOXO was discovered to upregulate the expression of AMPs via the IMD pathway as well as enhance the phagocytosis of hemocytes against pathogenic bacteria [1,3]. Despite the known involvement of FOXO in various physiological processes in crustaceans, it remained unclear whether the gene plays a role in the regulation of molting.

Molting is an important biological process closely related to the growth and development of crustaceans [20]. The molting cycle could be divided into three vital stages including pre-molt, post-molt and inter-molt [21,22]. As the primary component of ecdysteroids, 20-hydroxyecdysone (20E) plays a crucial role in mediating the changes that occur during the molting process [23]. The signals mediated by 20E are transduced via the binding of an isomeric dimer complex consisting of an Ecdysone receptor (EcR) and a Retinoid-X receptor (RXR) [24–26]. In addition, 20E and molt-inhibiting hormone (MIH) are mutually antagonistic and jointly regulate the molting process [27,28]. Recent studies demonstrated that the mammalian target of rapamycin (mTOR) is essential for the production of ecdysteroids in arthropods [29]. The mTOR gene has been shown to stimulate the synthesis of 20E and simultaneously downregulate the expression of molting inhibiting hormone (MIH) signaling genes [30]. Inhibition of mTOR expression by Rapamycin has been shown to impair ecdysteroid secretion in the prothoracic gland of insects [31,32]. Moreover, it is worth noting that FOXO could block rapamycin complex 1 (mTORC1) signal transduction in mammals, and the inactivation of FOXO alleviated mTORC1 inhibition [33]. However, whether FOXO regulates 20E synthesis and expression of molting-related genes through mTOR still remains unknown. In insects, it has been discovered that mTOR can enhance 20E production by regulating the size of the prothoracic and the molting glands [34–36]. Here, we speculated that FOXO might activate the ecdysone signaling pathway through mTOR activation, thereby regulating the occurrence of molting.

The Chinese mitten crab, *Eriocheir sinensis*, is one of the most important aquaculture crustaceans in China [37]. So far, the roles of 20E and its receptors, i.e., *Es*EcR, *Es*RXR as well as *Es*MIH, in the regulation of molting have been studied in this species [22,38–40]. It was found that *Es*EcR and *Es*RXR were highly expressed in the hepatopancreas at pre-molt and lowly expressed at post-molt, while *Es*MIH was highly expressed in eyestalk and showed the opposite expression pattern to that of *Es*EcR and *Es*RXR [40]. In this study, a FOXOlike molecule containing an FH domain was identified and characterized in *E. sinensis* (designated as *Es*FOXO-like), with the objectives to (1) examine its expression pattern at three molting stages, (2) investigate the impact of *Es*FOXO-like on the concentration of 20E and the expression levels of genes related to molting (*Es*EcR, *Es*RXR and *Es*MIH) and (3) explore the involvement of mTOR in the regulation of *Es*FOXO-like and its impact on the 20E pathway. Overall, these findings would be helpful for understanding the role of FOXO in the molting of crustaceans.
