*2.8. Statistical Analysis*

Data were firstly checked for normality of distribution and homogeneity of variances using the Shapiro–Wilk and Levene's tests, respectively. The Mann–Whitney U test was also used for the non-normally distributed data. The Student's *t* test and one-way ANOVA analysis were used for the normally distributed data. A Turkey multiple comparison test was also used after the one-way ANOVA analysis. Finally, the data were graphed by Origin 8.1. Significant differences were accepted at *p* < 0.05, and the data were shown as mean ± SD (N = 3) (\* *p* < 0.05 and \*\* *p* < 0.01).

#### **3. Results**

#### *3.1. Sequence and Phylogenetic Analysis of EsFOXO-like Gene*

The ORF sequence of *Es*FOXO-like was found to be 852 bp long and encoded a polypeptide consisting of 283 amino acids (Figure 1a). Notably, the *Es*FOXO-like protein was found to contain a Forkhead (FH) domain between amino acids 140 and 208 (Figure 1b), which bears similar protein kinase A (PKA) phosphorylation modification sites. The *Es*FOXO-like protein has also been found to have a glycogen synthase kinase 3 (GSK3) phosphorylation modification site and a PKA phosphorylation modification site outside the FH domain (Figure 2). The deduced amino acid sequence of *Es*FOXO-like shared high

similarity with those FOXOs from other invertebrates species (Figure 2), such as *Penaeus japonicus* (MW080526.1, 83.51%), *Penaeus monodon* (XP\_037792098.1, 88.37%), *Portunus trituberculatus* (MPC 39137.1, 88.55%), *Penaeus vannamei* (XP\_027228067.1, 75.00%), *Clupea harengus* (XP\_031428678.1, 83.33%) and *Salvelinus alpinus* (XP\_023824765.1, 85.45%) (Table 2).

**Figure 1.** Sequence features and domain architecture of *Es*FOXO-like. (**a**) Nucleotide sequence and deduced amino acid sequence of *Es*FOXO-like. The number of amino acids and nucleotides are listed on the left of the sequence. The start codon (ATG) and stop codon (TAG) are marked in red. The FH domain is marked in yellow. (**b**) SMART analysis of *Es*FOXO-like. The *Es*FOXO-like protein domain contained two low complexity regions (pink boxes) and a conserved FH domain (yellow polygon).

**Figure 2.** Multiple sequence alignment of the FOXOs from both vertebrates and invertebrates. The amino acids shaded in dark are identical residues between all analyzed sequences, and relatively conserved amino acids are shown in gray. The FH domain is marked in the yellow box. Sequence information of the FOXO proteins is listed in Table 2. PKA: protein kinase A; GSK3: glycogen synthase kinase 3.


**Table 2.** The FOXO genes used in the alignment of *Es*FOXO-like.


**Table 2.** *Cont.*

In the phylogenetic tree of FOXOs, all the selected FOXO members were divided into invertebrate and vertebrate branches. *Es*FOXO-like was firstly clustered with FOXO from *Penaeus japonicus*, and shared a closer evolutionary relationship with FOXO from *Portunus trituberculatus*, *Penaeus vannamei* and *Penaeus monodon*, enabling categorization into the invertebrate FOXO branch. The other FOXOs from *Clupea harengus*, *Salvelinus alpinus*, *Orcinus orca*, *Orys dammah* and *Homo sapiens* were clustered into the vertebrate clade (Figure 3).

**Figure 3.** Phylogenetic relationship of FOXO sequences. The phylogenetic tree of FOXO gene was constructed based on the amino acid sequences of *Es*FOXO-like and another twenty FOXOs (Table 2) using maximum likelihood method implemented in the Mega 11.0 software. The bootstrap values of the branches were obtained by testing the tree 1000 times and are shown as percentage numbers.

#### *3.2. The Distribution of EsFOXO-like Gene in Different Tissues*

The *Es*FOXO-like gene mRNA transcripts were detected in different tissues, including the hepatopancreas, hemocytes, stomach, heart, gill, muscle and eyestalk. The highest levels of *Es*FOXO-like mRNA transcript were detected in the hepatopancreas (33.65-fold more than that in hemocytes, *p* < 0.05). The recorded mRNA relative expression levels of *Es*FOXO-like in the heart, stomach, muscles, gills, and eyestalks were 4.06-fold, 1.92-fold, 2.86-fold, 3.12-fold and 6.58-fold of that in hemocytes (*p* < 0.05), respectively (Figure 4).

**Figure 4.** The mRNA expression level of *Es*FOXO-like in different tissues. The data were analyzed statistically using one-way ANOVA analysis. Each bar represents the mean ± standard deviation of three independent biological replicates (Supplementary Table S1). Different lowercase letters indicate significant differences (*p* < 0.05).
