*3.8. The 20E Concentration and EsEcR, EsRXR and EsMIH mRNA Expression Levels in AS1842856 + Rapamycin Group after Inhibiting mTOR*

To investigate the role of *Es*mTOR in the regulation of the 20E pathway through *Es*FOXO-like, the concentration of 20E and the mRNA expression levels of *Es*EcR, *Es*RXR, and *Es*MIH were measured in the crabs following AS1842856 and Rapamycin injections. The results showed that the 20E concentration in serum, as well as the *Es*MIH mRNA expression level in eyestalk, were significantly decreased in the AS1842856 + Rapamycin group, which was 0.81-fold (*p* < 0.05) and 0.23-fold (*p* < 0.05) of that in AS1842856 + DMSO group for the respective components analyzed (Figure 11a,d). On the contrary, the expression levels of *Es*EcR (5.78-fold, *p* < 0.05) and *Es*RXR (3.69-fold, *p* < 0.05) in the hepatopancreas in the AS1842856 + Rapamycin group were significantly higher than that in the AS1842856 + DMSO group (Figure 11b,c).

**Figure 11.** The 20E concentration and mRNA expression levels of *Es*EcR, *Es*RXR and *Es*MIH in AS1842856 + Rapamycin group. (**a**) The 20E concentration in serum in AS1842856- and Rapamycininjected crabs. (**b**–**d**) The relative expression levels of *Es*EcR and *Es*RXR in hepatopancreas and *Es*MIH expression level in eyestalk in AS1842856 + Rapamycin group. The AS1842856 + DMSO group was treated as the control group. Each bar represents the mean ± standard deviation of three independent biological replicates (Supplementary Table S1). Asterisks indicate significant differences (\* *p* < 0.05). The data were analyzed by Student's *t* test.

#### **4. Discussion**

FOXO plays a key regulatory role in many physiological, metabolic and immunoregulatory responses [55–57]. The FOXO gene is conserved from lower yeast to higher humans [58]. So far, there are four FOXO members found in mammals [2], and only one FOXO member has been identified in invertebrates [59]. In this study, a FOXO-like transcription factor (*Es*FOXO-like) was identified and characterized in *E. sinensis*. The ORF of *Es*FOXOlike was 852 bp and encoded a 283 amino acid polypeptide. The *Es*FOXO-like sequence perfectly contains a typical Forkhead (FH) domain [19]. The DBD domain, also named the FH domain, was found to be highly conserved in different species [60,61]. The FH domain has been confirmed to interact with p53 to stabilize it from degradation, which is required to induce apoptosis [62]. Phylogenetic analysis showed that *Es*FOXO-like exhibits high similarity to crustacean FOXO genes and clusters together with them, suggesting that EsFOXO-like is a member of the FOXO family in crustaceans.

In mammals, different FOXO genes had various tissue expression patterns. For instance, FOXO1 and FOXO3α are expressed in multiple tissues, while FOXO4 is predominantly expressed in the kidney and muscle, and FOXO6 exhibits high expression in the liver [9]. Our results showed that the *Es*FOXO-like transcripts were expressed in various tissues, such as hepatopancreas, hemocytes, heart, stomach, gills, muscles, and eyestalks, with the highest level of expression in the hepatopancreas, which is similar to the distribution of FOXO genes in crustaceans [3,19]. The hepatopancreas of crustaceans has been identified to serve as a vital immunologic and metabolic organ [63]. Furthermore, it is considered to be one of the crucial organs involved in the molting process of crustaceans [22,64,65]. Moreover, the function of four different FOXO genes is also varied in vertebrates. For instance, the global loss of FOXO1 could cause the death of embryonic cells [66]. Furthermore, the overall deletion of FOXO3 can affect lymphatic proliferation and extensive organ inflammation [67]. Additionally, the loss of FOXO4 can exacerbate colitis induced by inflammatory stimuli [68]. Lastly, FoxO6 is preferentially enriched in the hippocampus, and the deletion of the FOXO6 gene can lead to impaired memory consolidation that showed a reduced ability to form long-term contextual and object recognition memories in mice [69]. Recently, many studies have demonstrated that FOXO regulates molting and metamorphosis in insects [16]. Although FOXO has been identified in several crustacean species, most studies only focus on its regulatory role in the immune response against bacterial invasions [3,19]. Thus, the potential involvement of FOXO in growth still needs to be further investigated.

Molting is a typical biological trait that directly determines the behavior and physiological processes in arthropods [70–73]. Generally, the molting cycle of crustaceans has been divided into three main stages: inter-molt, pre-molt and ecdysis [21,22]. We found that *Es*FOXO-like mRNA expression was different at each molting stage, with the highest level at pre-molt and the lowest level at post-molt. In *H.armigera*, the FOXO protein in the fat body was up-regulated during the fifth-larvae molting stage and metamorphosis when compared to the feeding stage. This difference indicated that the expression of FOXO also increased during molting and metamorphosis [16]. In insects, 20E and juvenile hormone are mutually antagonistic and jointly regulate molting processes. Usually, the juvenile hormone (JH) concentration decreases sharply in the juvenile developmental stage, and the increase in 20E concentration causes pupation or adult morphogenesis [74]. It has been discovered that FOXO plays a role in regulating the degradation of juvenile hormone (JH) to control the growth of *B. mori* [11]. Additionally, in *Tribolium castaneum*, it has been observed that 20E up-regulates FOXO expression, promoting its nuclear migration, where FOXO subsequently regulates protease factors [75]. In *H. armigera*, 20E also up-regulates FOXO expression which, in turn, induces the expression of Carboxypeptidase A to regulate the final proteolysis step during the molting process [16]. In our study, it was observed that the mRNA transcripts and protein expression of *Es*FOXO-like reached their highest levels during the pre-molt stage, suggesting its potential involvement in the molting process.

Although FOXO has been demonstrated to play a key role in the regulation of metamorphosis and growth of insects, the involvement of FOXO in the regulation of molting in crustaceans is unclear. The role of *Es*FOXO-like in regulating crab molting was explored in our research by evaluating the 20E concentration and expression levels of moltingrelated genes after AS1842856 [44] and *Es*FOXO-like dsRNA injections. In this study, the *Es*FOXO-like mRNA expression was significantly decreased after injections of AS1842856 or *Es*FOXO-like dsRNA. In mammals, AS1842856, which served as a FOXO1 inhibitor, could inhibit the protein activity of FOXO1 but had no effect on the mRNA transcription of FOXO1 [44,45,76]. It has also been demonstrated that treatment with AS1842856 displayed similar effects to FOXO1 knockdown in pancreatic progenitors [76]. Until now, the potential role of AS1842856 on FOXO genes has not been reported in crustaceans. In this study, our results showed that the mRNA and protein expression levels of EsFOXO-like were both significantly decreased after AS1842856 injection. This observation might be related to the differences in metabolism between crustaceans and mammals. Similarly, in the study of 3T3-L1 preadipocytes cells, it has also been found that the level of total FOXO1 protein was reduced after AS1842856 treatment [77]. Additional investigation revealed a significant increase in 20E concentration after the inhibition of *Es*FOXO-like. In addition, our study showed that when *Es*FOXO-like was inhibited, the *Es*EcR and *Es*RXR expressions were significantly up-regulated, while the *Es*MIH expression was significantly down-regulated. In crustaceans, molting is promoted by 20E through its dependence on EcR and RXR, which leads to the activation of molting-related genes [78]. EcR and RXR proteins bind together to form a complex, which then interacts with the 20E hormone to create an active trimer. This trimer is responsible for regulating the growth and molting process in crustaceans [79]. On the other hand, the MIH hormone has been shown to have a negative effect on the synthesis of 20E [23]. Although the studies on the regulation of FOXO on EcR, RXR and MIH expressions have not been reported in crustaceans, it has been studied in insects. For example, it has been found that FOXO could directly interact with Ultraspiracle (Usp), the ecdysone receptor, and FoxO/Usp complexes suppress 20E biosynthesis in *Drosophila* [80]. Additionally, JH regulated the growth rate via inhibition of the 20E pathway, and it is also dependent on FOXO [81]. While it has been reported that FOXO inhibition does not affect the expression levels of EcRB1 and USP1, it could significantly block the molting of larvae in *H. armigera* [16]. These results collectively suggested that FOXO was closely related to the physiological process of molting in *E. sinensis* by inhibiting the 20E signal. However, whether FOXO affects the 20E signal through related genes or signaling pathways remains to be further explored.

Multiple studies have shown that mTOR, a key regulator of cellular metabolism, plays a role in controlling the synthesis of 20E [30–32]. Research has shown that the activation of Y-organ ecdysteroidogenesis is dependent on the activity of mTOR and that mTORC1 is primarily responsible for driving the increase in 20E concentration [48,82]. Therefore, in order to explore the mechanism of FOXO regulation in the 20E pathway in crabs, we first measured the *Es*mTOR mRNA expression level after FOXO inhibition. The results showed that *Es*mTOR transcription was significantly increased in the AS1842856-injected group and the *Es*FOXO-like-RNAi group. In mammals, the PI3K–AKT–FOXO–mTOR pathway has been verified to be a regulator of metabolism and somatic growth [83]. It has also been found that loss of FOXO function resulted in precocious differentiation in tissues with high mTOR activity on nutrient restriction in *Drosophila* [84]. Moreover, the cells that lost the function of FOXO have higher mTOR activity and are more sensitive to 20E response, possibly due to a similar inhibitory action of FOXO on the 20E receptor complex [84]. Moreover, Rapamycin, an effective and specific mTOR activity inhibitor, was injected into crabs to explore the function of mTOR in the FOXO regulation of the 20E pathway in this study [85]. Injections of AS1842856 and Rapamycin resulted in reduced expression of the *Es*mTOR transcript, and the AS1842856 + Rapamycin group exhibited a significantly lower concentration of 20E compared to the AS1842856 + DMSO group. Similarly, in *Manduca sexta*, Rapamycin resulted in delayed larval molting and reduced 20E production [86]. In

*B. mori*it, it has been found that rapamycin could also inhibit 20E synthesis and secretion [32]. Although accumulating evidence has suggested that mTOR could stimulate 20E synthesis and inhibit the expression levels of MIH signaling-related genes [29,30]. 20E is transduced via binding of an isomeric dimer complex consisting of EcR and RXR [25], but the impact of mTOR on EcR and RXR is unknown. Notably, mTOR signaling plays an essential role in the regulation of growth, body size and aging of arthropods. It has also been confirmed that the insulin receptor (InR) and phosphoinositide 3-kinase (PI3K) genes, involved in growth and development progress, showed progressively increasing mRNA levels in rapamycin-treated crabs [29]. Similarly, the InR and PI3K could promote EcR and USP (RXR in crustaceans) expression in insects [87]. Therefore, we speculate that the increase in *Es*EcR and *Es*RXR expression observed in the AS1842856- + Rapamycin-inhibited group in this study might be due to up-regulation of the insulin and PI3K pathway. Furthermore, 20E and its receptors have been confirmed to negatively affect MIH expression [27,28]. Thus, the decrease of *Es*MIH expression also observed in the AS1842856 + Rapamycin inhibited group might be due to the increased expression of *Es*EcR and *Es*RXR. It might be also associated with the sampling time after rapamycin injection. For instance, in *Gecarcinus lateralis*, rapamycin did not block or reverse the decrease in MIH signaling genes until the seventh day after rapamycin injection [29]. Overall, these results suggest that FOXO could regulate the 20E pathway through mTOR in mitten crabs.

#### **5. Conclusions**

In summary, a Forkhead Box O-like (named *Es*FOXO-like) was identified in *E. sinensis*, with the highest expression in the hepatopancreas at pre-molt stage and lowest expression at post-molt stage. It was also found that the 20E and *Es*MIH mRNA expressions were both significantly down-regulated after *Es*FOXO-like inhibition, while the expression of *Es*EcR and *Es*RXR showed the opposite expression pattern. Taken together, these results suggest that *Es*FOXO-like has a negative effect on 20E signaling by inhibiting mTOR. Molting is an essential process for the growth of crustaceans, and FOXO served as a key transcription factor. Its role in regulating molting had not yet been studied in crustaceans. Our current results reveal the inhibitory function of FOXO in the molting of crustaceans. Future research should be performed on the regulatory mechanisms involved in the promotion of molting and growth by inhibiting FOXO.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jmse11061225/s1, Table S1: The original data for analysis in this manuscript.

**Author Contributions:** Conceptualization, J.L. (Jiaming Li) and S.H.; methodology, J.L. (Jiaming Li) and Y.M.; software, Z.Y. and F.W.; validation, J.L. (Jialin Li) and Y.M.; formal analysis, J.L. and F.W.; investigation, J.L. (Jialin Li) and Z.Y.; writing—original draft preparation, J.L. (Jiaming Li) and Y.M.; writing—review and editing, Y.J., D.Y., Q.Y. and S.H.; supervision, Q.Y. and S.H.; project administration, S.H.; funding acquisition, Q.Y. and S.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by research grants from Educational Department of Liaoning Province (20220078), Natural Resources Department of Liaoning Province (20220001-18) and Educational Department of Liaoning Province (S202210158002X).

**Institutional Review Board Statement:** The animal study protocol conformed with the Animal Care Committee of Dalian Ocean University.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article. All the data are available from the corresponding author upon request.

**Acknowledgments:** We are grateful to all the members for their help and support in this experiment.

**Conflicts of Interest:** The authors declare no conflict of interests.
