Genome-Wide Identiﬁcation and Expression Proﬁling of Candidate Sex Pheromone Biosynthesis Genes in the Fall Armyworm ( Spodoptera frugiperda )

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Introduction
Female Lepidoptera (moths) release sex pheromones to attract males for mating [1,2]. Most moth sex pheromones consist of two or more compounds combined in precise ratios with species specificity [3,4]. Based on the difference of their chemical structures and biosynthetic features, sex pheromones are classified into type I and type II [5]. Most known sex pheromones are type I, which are usually synthesized in female sex pheromone glands (PGs) situated in the intersegmental membrane between the eighth and ninth abdominal segments [5,6]. These pheromone components are mainly C10-C18 straightchain compounds, containing 0-4 double bonds in different positions. The carbon chain ends have alcohol, ester, or aldehyde functional groups [7,8]. During type I sex pheromone biosynthesis, fatty acid intermediates such as palmitic acid or stearic acid are used as precursors. The double bond is generated by the desaturation system, and a short-chain reaction is carried out by a special β-oxidase system [9,10]. Oxidase, fatty acyl-CoA reductase, and acyl transferase catalyze the formation of functional groups such as esters, aldehydes, and alcohols to form a mixture of sex pheromones with specific component ratios and amounts. Acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), fatty acid transport protein (FATP), acyl-CoA dehydrogenase (ACD), 3-ketoacyl-CoA thiolase (KCT), hydroxyacyl-CoA dehydrogenase (HCD), enoyl-CoA hydratase (ECH), desaturase (DES), fatty acyl-CoA reductase (FAR), acetyl-CoA acetyltransferase (ACT), acyl-CoA-binding protein (ACBP) are the key enzymes in sex pheromone biosynthesis of moths [8,11,12].
In this study, we identified candidate FAW sex pheromone biosynthesis genes using previously published genome data. Then, we sequenced the transcriptome of PGs and ABs (abdomen without PGs) to analyze the expression of these candidate sex pheromone biosynthesis genes. Quantitative RT-PCR (qRT-PCR) was conducted to validate the transcriptome results. Based on the gene identifications, phylogenetic, and tissue-specific expression analyses, several genes were identified as potentially involved in FAW sex pheromone biosynthesis. Our results could provide potential targets for developing environmentally friendly control methods.

Insect Rearing and Tissue Collection
The original population of S. frugiperda was collected from a maize field of Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, China. Larvae were reared with maize leaves in a growth chamber at (25 ± 1) • C, 55% relative humidity, with a 16:8 h (L:D) photoperiod. Adults were fed with 10% honey water. For the transcriptome sequencing and tissue expression study, 20-25 PGs (with the ovipositor) and 10-15 abdomens (without the PGs) were collected from 3 d old virgin female adults at 6-7 h into the scotophase [24,25]. The FAW shows particularly high mating activity at this time. Three biological replicates were conducted.

cDNA Synthesis and Full-Length cDNA Cloning
The cDNA was synthesized using the PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time) (TAKARA, Toyoko, Japan). Four differentially expressed genes (Sfur-DES2, SfurDES5, SfurFAR2, SfurFAR3) were randomly selected to amplify the full-length ORF sequence of these genes by using TransStart FastPfu Fly PCR Supermix (TransGen Biotech, Beijing, China). PCR conditions were: 5 min at 94 • C, followed by 40 cycles of 94 • C for 20 s, 20 s at 52 • C, and 45 s at 72 • C, followed by incubation at 72 • C for 10 min, carried out in a Bio-Rad thermocycler (Bio-Rad DNA Engine Peltier Thermal Cycler, Bio-Rad, Hercules, CA, USA). The primers were listed in Table S2, designed by Primer 5.0 software. The products were gel-purified and ligated into a pEASY-blunt vector (TransGen Biotech, Beijing, China). The ligation products were transformed into Trans T1 competent cells (TransGen Biotech, Beijing, China). All sequencing was performed by Tsingke Biotechnology Co., Ltd. (Beijing, China).

Phylogenetic Analysis
Phylogenetic trees were performed for SfruDESs and SfruFARs with their corresponding homologous genes from S. exigua, S. litura, Sesamia inferens, A. pernyi, Ostrinia nubilalis, and H. assulta, as reported previously [8,30,39]. The DES dataset included 17 sequences from S. frugiperda, and 41 from the other six species (12 from S. litura, 10 from S. exigua, 6 from S. inferens, 6 from A. pernyi, 3 from O. nubilalis, and 4 from H. assulta). The FAR dataset included 29 sequences from S. frugiperda, and 48 from six other insects (13 from S. litura, 13 from S. exigua, 3 from S. inferens, 11 from A. pernyi, 7 from O. nubilalis, and 1 from H. assulta) (Table S3). Amino acid sequences were aligned by ClatalW, and phylogenetic trees were constructed by MEGA X using the neighbor-joining method with position correction of distances and 1000 bootstrap replications. The final phylogenetic tree is displayed in the form of a circular tree diagram, and the color of each branch is labeled using FigTree v1.3.1.

Discussion
In moths, the PGs are the most important organ for synthesizing and releasing sex pheromones [35]. Thus, the sex pheromone synthesis genes are usually highly expressed in PGs. In the present study, 99 candidate sex pheromone biosynthesis genes were identified from the genome of S. frugiperda. Among them, 22 genes were expressed at

Discussion
In moths, the PGs are the most important organ for synthesizing and releasing sex pheromones [35]. Thus, the sex pheromone synthesis genes are usually highly expressed in PGs. In the present study, 99 candidate sex pheromone biosynthesis genes were identified from the genome of S. frugiperda. Among them, 22 genes were expressed at significantly higher levels in PGs than in the abdomen, and most of them were key genes involved in the sex pheromone biosynthesis pathway of moths such as DESs and FARs. Consistent with this study, DESs and FARs also showed a trend of high PG expression in S. litura and S. exigua [8,30].
Sex pheromones released by female moths are composed of a mixture of sex pheromone components in specific ratios that show high species specificity [43,44]. Synthesis of a specific sex pheromone mixture requires the coordination of multiple enzymes, such as ACC and FAS. These two enzyme families mainly work upstream of sex pheromone synthesis and are responsible for the synthesis of fatty acid precursors. Initially, ACC carboxylates acetyl-CoA to malonyl-CoA [45], after which FAS synthesizes malonyl-CoA and NAPDH into fatty acids [46,47]. In this study, one ACC gene and 11 FAS genes were identified from the genome of S. frugiperda. Among the 11 FAS genes, SfurFAS4 had the highest expression in PG, suggesting its important role in fatty acid synthesis. As an evolutionarily conserved membrane-bound protein, FATPs can bind fatty acids and transport them to PG cells via the hemolymph for pheromone biosynthesis [12,48]. Four FATP genes were identified from the genome of S. frugiperda, consistent with the number of FATP genes in S. litura and S. exigua [8,30], and there was a high degree of sequence similarity among these three species. However, only SfurFATP3 had abundant expression levels in the PGs based on the FPKM values.
There are several sex pheromone components of S. frugiperda. Except for Z11-16:OAc, containing 16 carbons, Z9-14:OAc, Z7-12:OAc, Z9-12:OAc, and E7-12:OAc are less than 16-carbon chain length unsaturated fatty acid ester derivatives [24,28]. Therefore, the carbon chain shortening reaction plays an important role in this process, and the pathway to generate sex pheromone precursors with different carbon chain lengths is similar to the local β-oxidation pathway of vertebrate peroxisomes, among which ACD, ECH, HCD, and KCT are four key enzymes in the β-oxidation pathway [9]. A total of five ACD genes, three ECH genes, three HCD genes, and six KCT genes were screened in the FAW genome. SfurACD5 and SfurKCT3 had higher expression abundance in PGs than in the abdomen.
DES is a key enzyme in sex pheromone biosynthesis. It removes hydrogen atoms at specific positions and introduces double bonds to form cis-trans isomers [31,49]. DES is classified into ∆5, ∆9 (18 C > 16 C) ∆9 (16 C > 18 C), ∆10, ∆11, ∆12, and ∆14 desaturases according to the position where the double bond is introduced into the catalytic sub-strate [50][51][52]. Because several sex pheromone components of S. frugiperda have different positions, numbers, and configurations of double bonds, DES is crucial for sex pheromone formation. A total of 17 DESs were identified from the FAW genome. Phylogenetic tree analysis showed that the SfruDES5 was clustered with the DES5 of S. litura and S. exigua, and both were clustered in the ∆11 DES branch. Both transcriptome FPKM values and qRT-PCR showed that SfruDES5 was significantly overexpressed in the PGs of S. frugiperda. SfruDES9 and SfruDES11 are clustered with the corresponding DES9 and DES11 of S. litura and S. exigua and belong to the ∆9 (18 C > 16 C) and ∆9 (16 C > 18 C) desaturase groups, respectively, in which SfruDES11 was specifically expressed in PGs. The ∆9 and ∆11 desaturases are important desaturases in Spodoptera. The ∆9 desaturases and ∆11 desaturases can introduce ∆9-double bonds and ∆11-double bonds in precursors [53]. The Z9-14:OAc and Z11-16:OAc are the key components of S. frugiperda sex pheromone, and ∆9 and ∆11 desaturases are key enzymes for introducing the ∆9 and ∆11 double bonds into the pheromone. Therefore, SfurDES5 and SfurDES11 may participate in the desaturation step from saturated to unsaturated acids during sex pheromone synthesis in S. frugiperda.
The precursor substance forms an intermediate product with a specific length and double bond position after the desaturation reaction and chain shortening reaction. It then needs to be catalyzed by reductases to form alcohols. During this process, FAR is responsible for converting unsaturated fatty acids into the corresponding alcohols [11,54]. In our study, a total of 29 FAR genes were identified from the genome; among the 29 FARs of S. frugiperda, 12 FARs were specifically highly expressed in the PGs. The phylogenetic tree showed that SfruFAR3 was clustered with FAR3 of S. litura and S. exigua, belonging to the PgFAR subfamily of Spodoptera, and had a high expression abundance. This indicated that this gene may play an important role in the synthesis of precursor alcohols. ACT can catalyze the formation of esters from alcohols [55,56]. Since there are only esters in the S. frugiperda sex pheromone, ACT genes play a key role in sex pheromone biosynthesis. A total of 17 ACT genes were identified in the FAW genome, among which the SfurACT6 and SfurACT10 were highly expressed in the PGs. These two genes may play a role in the process of converting alcohol to esters. Furthermore, two ACTs were unplaced in the chromosome, which might be caused by the genome quality. In Bombyx mori, ACBP functions as acyl-CoA or cell deposition [12]. We identified three ACBPs from the S. frugiperda genome.

Conclusions
In conclusion, we identified a total of 99 genes belonging to gene families involved in the biosynthesis of sex pheromones from the S. frugiperda genome. Based on gene expression patterns and phylogenetic analysis, several genes highly expressed in the PGs might play an important role in sex pheromone synthesis. The specific functions of these genes in the process of sex pheromone biosynthesis in S. frugiperda require further study.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/insects13121078/s1. Figure S1: Hypothetical sex pheromone biosynthesis pathways of S. frugiperda; Table S1: Query gene sequences; Table S2: Primers used for RT-PCR and qPCR; Table S3: Sequences for phylogenetic tree; Table S4: Primer amplicon characteristics of 4 genes for qRT-PCR; Table S5: Summary of the transcriptome sequencing data of of Spodoptera frugiperda; Table S6: Fragments per kilobase million (FPKM) for the different samples.
Funding: This work was supported by the Science and Technology Innovation Ability Construction of BAAFs (KJCX20200432); the key research and development program of Hunan Province (China) (2020NK2034); the Shandong Province Modern Agricultural Technology System Peanut Innovation Team, China (SDAIT-04-08); Hebei Natural Science Foundation (C2022201042).

Data Availability Statement:
The transcriptome data that support the findings of this study are openly available in SRA the Genbank SRA database (BioProject ID: PRJNA885519). Other data presented in this study are available in the article and Supplementary Materials.

Conflicts of Interest:
The authors declare no conflict of interest.