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Article

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

by
Cheng Qu
1,†,
Zhiwei Kang
2,†,
Biyun Zhang
1,
Yong Fang
3,
Ran Wang
1,
Fengqi Li
1,
Haipeng Zhao
4,* and
Chen Luo
1,*
1
Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
2
School of Life Science, Institute of Life Science and Green Development, Hebei University, Baoding 071002, China
3
Hunan Agricultural Biotechnology Research Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
4
College of Plant Protection, Shandong Agricultural University, Tai'an 271018, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Insects 2022, 13(12), 1078; https://doi.org/10.3390/insects13121078
Submission received: 30 September 2022 / Revised: 16 November 2022 / Accepted: 18 November 2022 / Published: 23 November 2022
(This article belongs to the Special Issue Environmentally-Friendly Pest Control Approaches for Invasive Insects)
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Abstract

:

Simple Summary

The fall armyworm (FAW), Spodoptera frugiperda, is a serious worldwide agricultural pest, threatening food security and crop production. Sex pheromone lures are commonly used in population monitoring and biological control of FAWs. Although the sex pheromone components of the FAW have been successfully identified, there are no reports on the molecular mechanism of FAW sex pheromone biosynthesis. In this study, we identified a total of 99 genes related to the biosynthesis of sex pheromones from the S. frugiperda genome, which belonged to 11 families of genes. Based on gene expression patterns and phylogenetic analysis, several genes had PG-biased expression, indicating that they may play an important role in sex pheromone biosynthesis. These results could lay a solid foundation for understanding the molecular mechanisms of S. frugiperda sex pheromone biosynthesis and provide new targets for developing novel pest control methods based on disrupting sexual communication.

Abstract

Spodoptera frugiperda is an agricultural pest causing substantial damage and losses to commercial crops. Sex pheromones are critical for successful mating in Lepidoptera and have been used for monitoring and control of many pest species. The sex pheromone of S. frugiperda is known, but the genes involved in its biosynthesis have not been identified. We systematically studied 99 candidate sex pheromone genes in the genome of S. frugiperda including 1 acetyl-CoA carboxylase (ACC), 11 fatty acid synthases (FASs), 17 desaturases (DESs), 4 fatty acid transport proteins (FATPs), 29 fatty acyl-CoA reductases (FARs), 17 acetyl-CoA acetyltransferases (ACTs), 5 acyl-CoA dehydrogenase (ACDs), 3 enoyl-CoA hydratases (ECHs), 3 hydroxyacyl-CoA dehydrogenases (HCDs), 6 ethyl-CoA thiolases (KCTs), and 3 acyl-CoA-binding proteins (ACBPs). Based on the comparative transcriptome results, we found 22 candidate sex pheromone biosynthesis genes predominately expressed in pheromone glands (PGs) than abdomens without PGs including SfruFAS4, SfruFATP3, SfruACD5, SfruKCT3, SfruDES2, SfruDES5, SfruDES11, SfruDES13, SfruFAR1, SfruFAR2, SfruFAR3, SfruFAR6, SfruFAR7, SfruFAR8, SfruFAR9, SfruFAR10, SfruFAR11, SfruFAR14, SfruFAR16, SfruFAR29, SfruACT6, and SfruACT10. A combination of phylogenetic and tissue-specific transcriptomic analyses indicated that SfruDES5, SfruDES11, SfruFAR2, SfruFAR3, and SfruFAR9 may be key genes involved in the sex pheromone synthesis of S. frugiperda. Our results could provide a theoretical basis for understanding the molecular mechanisms of sex pheromone biosynthesis in S. frugiperda, and also provide new targets for developing novel pest control methods based on disrupting sexual communication.

1. 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 straight-chain 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].
The fall armyworm (FAW), Spodoptera frugiperda, native to tropical and subtropical America, is an agricultural pest. It has a wide plant host range, strong migratory ability, high reproductive capacity, and invaded sub-Saharan Africa, Asia, and other regions [13,14,15]. In China, the FAW was first found in Yunnan in January 2019. It then spread rapidly to many other food-producing areas in China and threatened food security and crop production [16,17]. Sex pheromone lures are usually used for population monitoring and biological control of FAW [18,19,20,21,22]. The sex pheromone components of FAW usually contain several acetates, including (Z)-9-Tetradeceny1 acetate (Z9-14:OAc) as a major component, (Z)-7-Dodecenyl acetate (Z7-12:OAc), (Z)-9-dodecenyl acetate (Z9-12:OAc), (Z)-11-hexadecell acetate (Z11-16:OAc), and trans-7-Dodecen acetate-1-yl acetate (E7-12:OAc); these acetate esters are all type-I sex pheromones [23,24,25,26,27,28,29]. Although the sex pheromone components of FAW have been successfully identified, there are no reports on the molecular mechanism of FAW sex pheromone biosynthesis.
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.

2. Materials and Methods

2.1. 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.

2.2. RNA Isolation, cDNA Library Construction, Illumina Sequencing

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer instructions. The quality and concentration of RNA samples were checked using a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the RNA integrity was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Davis, CA, USA).
Oligo (dT) magnetic beads were used to purify mRNA from total RNA, and the mRNA was randomly interrupted in the NEB fragmentation buffer. Fragmented mRNA were used as templates to synthesize the first strand of cDNA using a random hexamer, followed by synthesis of the second strand, cDNA, by adding RNaseH, dNTPs, and DNA polymerase I. After end-repair, poly-A tailing, and ligation of adapters, the cDNA was purified by an AMPure XP system (Beckman Coulter, Beverly, MA, USA), and PCR amplification was performed. The constructed library was quality-tested with an Agilent 2100 Bioanalyzer (Agilent Technologies, Davis, CA, USA).
An Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) was used for sequencing. The raw reads were processed to remove low-quality reads, poly-N reads, and adapter reads to obtain the clean reads. Q20, Q30, and GC content were used to assess the sequencing quality. HISAT2 v.2.0.4 software was used to assemble and compare clean data and obtain annotations in the reference genome of S. frugiperda (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/011/064/685/GCF_011064685.1_ZJU_Sfru_1.0/, (accessed on 24 July 2020)). The RNA sequence data was uploaded to the NCBI platform (BioProject Accession: PRJNA885519). Fragments per kilobase of exon per million mapped reads (FPKM) were used to evaluate the expression level.

2.3. Identification and Analysis of Sex Pheromone Biosynthesis-Related Genes

The S. frugiperda genome database was obtained from NCBI (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/011/064/685/GCF_011064685.1_ZJU_Sfru_1.0/, (accessed on 24 July 2020)). The amino acid sequences of sex pheromone biosynthesis genes (ACCs, FASs, FATPs, ACDs, ECHs, HCDs, KCTs, DESs, FARs, ACTs, ACBPs) in Spodoptera exigua, Spodoptera litura, Helicoverpa armigera, Helicoverpa assulta, Helicoverpa zea, Agrotis segetum, Agrotis ipsilon, Heliothis virescens were used as query sequences (Table S1) for local BLASTP search (E-value cutoff of <1 × 10−5 and identity >30%) against the S. frugiperda genome database [8,30,31,32,33,34,35,36,37,38]. The incomplete sequences and duplicated genes were removed to obtain initial genes. All of these potential sex pheromone biosynthesis genes were then verified by BLASTP in NCBI with the E-value < 1 × 10−5 and identity >30%. The location of these genes was generated based on the genome annotation of S. frugiperda (BioProject Accession: PRJNA590312) using TBtools software (v1.0987671).

2.4. 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 (SfurDES2, 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).

2.5. 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.

2.6. Quantitative RT-PCR and Data Analysis

Four differentially expressed genes (SfurDES2, SfurDES5, SfurFAR2, SfurFAR3) were verified by qRT-PCR. Primers for qRT-PCR were designed by NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed on 24 July 2020)). The 10-fold dilution series of cDNA from the PGs of S. frugiperda was used for a standard curve. The corresponding qRT-PCR efficiencies (E) were counted using the equation: E = (10 [−1/slope] − 1) × 100 [40] (Table S4). EF1α and RPS18 were selected as internal reference genes [41]. The qRT-PCR was performed on the ABI PRISM 7500 qRT-PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex TaqTM II (TaKaRa, Toyoko, Japan) under the following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 34 s. Relative quantification was performed using the 2−ΔΔCT method [42].

3. Results

3.1. Identification and Localization of the Candidate Sex Pheromone Biosynthesis Genes in the S. frugiperda Genome

We identified 99 genes encoding pheromone biosynthesis from 11 gene families, including 1 ACC, 11 FASs, 4 FATPs, 5 ACDs, 3 ECHs, 3 HCDs, 6 KCTs, 17 DESs, 29 FARs, 17 ACTs, and 3 ACBPs (Table 1). To clarify the position of sex pheromone biosynthesis genes in chromosomes, chromosome location analysis of these genes was carried out. The results showed that except for two ACTs that were unplaced, the rest of the candidate sex pheromone biosynthesis genes were distributed across 23 chromosomes (Figure 1). ACC was located on chromosome 29. All of the KCT and HCD genes were distributed on only one chromosome (KCTs: chromosome 8 and HCDs: chromosome 16) (Figure 1). Four FATPs were found on chromosomes 18 (SfruFATP1, SfruFATP2, and SfruFATP4) and 24 (SfruFATP3) (Figure 1). Both of ECHs and ACBPs were located on three chromosomes (ECHs: chromosomes 6, 16, and 24; ACBPs: chromosomes 8, 9, and 28) (Figure 1). Seven FASs were clustered together on chromosome 20, followed by chromosomes 10 (SfruFAS2 and SfruFAS3), 7 (SfruFAS1), and 17 (SfruFAS4) (Figure 1). Five ACDs were independently distributed on five chromosomes (1: SfruACD4, 2: SfruACD5, 6: SfruACD2, 20: SfruACD1, and 22: SfruACD22) (Figure 1). Thirty FARs were also located on five chromosomes (1, 7, 10, 29, and 30) (Figure 1). DESs and ACTs were widely distributed on more than five chromosomes (DESs: chromosomes 2, 7, 11, 12, 13, 22, and 29; ACTs: chromosomes 1, 2, 4, 6, 10, 15, 17, 21, 22 and 26) (Figure 1).

3.2. Phylogenetic Analyses of DESs and FARs

To assign putative functions, two phylogenetic trees of DESs and FARs were constructed using protein sequences from S. frugiperda, S. exigua, S. litura, S. inferens, A. pernyi, O. nubilalis, and H. assulta. The DESs phylogenetic trees showed that all three identified SfruDESs from the S. frugiperda genome were clustered in three different clades of Lepidoptera desaturases: Δ11 desaturase (SfruDES5), Δ9 desaturase (18 C > 16 C) (SfruDES9), and Δ9 desaturase (16 C > 18 C) (SfruDES11) (Figure 2). In the FARs phylogenetic tree, four SfruFARs (SfruFAR2, SfruFAR3, SfruFAR9, and SfruFAR17) were clustered within the Lepidoptera pgFAR group, which is a clade with functionally investigated FARs (Figure 3).

3.3. Expression Profile of Sex Pheromone Biosynthesis Genes

To further screen the candidate sex pheromone biosynthesis genes in S. frugiperda, we performed a transcriptome analysis for the genes expressed in female PGs and ABs (Table S5). Based on the transcriptome results (Table S6), we analyzed the expression patterns of all of the candidate sex pheromone biosynthesis genes (Figure 4). None of the genes in ACC, ECH, HCD, and ACBP were significantly higher expressed in the PGs (Figure 4). Only one gene in FAS (SfruFAS4), FATP (SfruFATP3), ACD (SfruACD5), and KCT (SfruKCT3) had significantly higher expression levels in pheromone glands (PGs) than abdomens (ABs) (Figure 4). Five DESs (SfruDES2, SfruDES5, SfruDES11, and SfruDES13), twelve FARs (SfruFAR1, SfruFAR2, SfruFAR3, SfruFAR6, SfruFAR7, SfruFAR8, SfruFAR9, SfruFAR10, SfruFAR11, SfruFAR14, SfruFAR16, and SfruFAR29) and two ACTs (SfruACT6 and SfruACT10) were predominately expressed in the PGs of S. frugiperda (Figure 4). SfruFAR3 was specifically highly expressed in PGs (Figure 4I). Among the predominately expressed genes in the PGs, the FPKM of SfruKCT3, SfruFAR8, SfruFAR9, SfruFAR11, SfruFAR16, SfruFAR29, and SfruACT10 were below 10, while the FPKM of SfruFAS4, SfruACD5, SfruDES5, SfruDES11, SfruFAR3, SfruFAR10, SfruFAR14, and SfruACT6 exceeded 100 (Figure 4). The qRT-PCR validation experiments of SfruDES2, SfruDES5, SfruFAR2, and SfruFAR3 confirmed that all of these four genes were predominately expressed in the PGs (Figure 5).

4. 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 substrate [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 Δ9 (18 C > 16 C) and Δ9 (16 C > 18 C) desaturase groups, respectively, in which SfruDES11 was specifically expressed in PGs. The Δ9 and Δ11 desaturases are important desaturases in Spodoptera. The Δ9 desaturases and Δ11 desaturases can introduce Δ9-double bonds and Δ11-double bonds in precursors [53]. The Z9-14:OAc and Z11-16:OAc are the key components of S. frugiperda sex pheromone, and Δ9 and Δ11 desaturases are key enzymes for introducing the Δ9 and Δ11 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.
According to the sex pheromone components of S. frugiperda, we speculated the S. frugiperda sex pheromone biosynthesis pathway. Firstly, ACC catalyzes acetyl-CoA to malonyl-CoA, which is followed by FAS to produce the most saturated palmitic acid (16: CoA). The 16: CoA was desaturated by Δ11 desaturase (Z11/E11) to produce Z11-16: CoA/E11-16: CoA. After the desaturase-induced formation of a double bond, specific β-oxidation enzymes shorten the chains to Z9-14: CoA, Z7-12: CoA, or E7-12: CoA. In addition, the 16: CoA was shortened by β-oxidation enzymes to 12: CoA. Then, 12: CoA generated Z9-12: CoA under Δ9 desaturase (Z9). These acyl-CoA precursors were further reduced by FARs to form corresponding fatty alcohols. Finally, pheromone components were produced after the oxidation by ACTs (Figure S1).

5. 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.

Author Contributions

Conceptualization, C.L. and H.Z.; methodology, C.Q.; software, Z.K.; validation, Z.K., B.Z., Y.F. and R.W.; formal analysis, C.Q.; investigation, H.Z.; resources, H.Z.; data curation, C.Q. and F.L.; writing—original draft preparation, C.Q. and Z.K.; writing—review and editing, C.L. and H.Z. visualization, R.W.; supervision, C.L.; project administration, C.Q.; Funding acquisition, C.Q. All authors have read and agreed to the published version of the manuscript.

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.

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Figure 1. Localization of the candidate sex pheromone biosynthesis genes in the S. frugiperda genome. According to the annotation file of S. frugiperda, we determined the sex pheromone biosynthesis gene locations in the genome.
Figure 1. Localization of the candidate sex pheromone biosynthesis genes in the S. frugiperda genome. According to the annotation file of S. frugiperda, we determined the sex pheromone biosynthesis gene locations in the genome.
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Insects 13 01078 g002 550
Figure 2. Phylogenetic tree of insect desaturase (DES). The S. frugiperda translated genes are shown in red. Sfru: S. frugiperda, Slit: S. litura, Sexi: S. exigua, Sinf: S. inferens, Aper: A. pernyi, Onub: O. nubilalis, Hass: H. assulta.
Figure 2. Phylogenetic tree of insect desaturase (DES). The S. frugiperda translated genes are shown in red. Sfru: S. frugiperda, Slit: S. litura, Sexi: S. exigua, Sinf: S. inferens, Aper: A. pernyi, Onub: O. nubilalis, Hass: H. assulta.
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Insects 13 01078 g003 550
Figure 3. Phylogenetic tree of insect fatty acid reductase (FAR). The S. frugiperda translated genes are shown in red. Sfru: S. frugiperda, Slit: S. litura, Sexi: S. exigua, Sinf: S. inferens, Aper: A. pernyi, Onub: O. nubilalis, Hass: H. assulta.
Figure 3. Phylogenetic tree of insect fatty acid reductase (FAR). The S. frugiperda translated genes are shown in red. Sfru: S. frugiperda, Slit: S. litura, Sexi: S. exigua, Sinf: S. inferens, Aper: A. pernyi, Onub: O. nubilalis, Hass: H. assulta.
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Figure 4. Expression profiles of the candidate sex pheromone biosynthesis genes in S. frugiperda. (A) ACC; (B): FASs; (C): FATPs; (D): ACDs; (E): ECHs; (F): HCDs; (G): KCTs; (H): DESs; (I); FARs; (J): ACTs; (K): ACBPs.
Figure 4. Expression profiles of the candidate sex pheromone biosynthesis genes in S. frugiperda. (A) ACC; (B): FASs; (C): FATPs; (D): ACDs; (E): ECHs; (F): HCDs; (G): KCTs; (H): DESs; (I); FARs; (J): ACTs; (K): ACBPs.
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Figure 5. Relative expression levels of four sex pheromone biosynthesis genes. PGs: female pheromone glands, ABs: female abdomens without pheromone glands. The relative expression level (PGs VS ABs) is indicated as mean ± SE (n = 3). The asterisk indicates a significant difference between PGs and ABs (p < 0.05, Mann-Whitney U test).
Figure 5. Relative expression levels of four sex pheromone biosynthesis genes. PGs: female pheromone glands, ABs: female abdomens without pheromone glands. The relative expression level (PGs VS ABs) is indicated as mean ± SE (n = 3). The asterisk indicates a significant difference between PGs and ABs (p < 0.05, Mann-Whitney U test).
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Table
Table 1. Genes related to sex pheromone biosynthesis in Spodoptera frugiperda.
Table 1. Genes related to sex pheromone biosynthesis in Spodoptera frugiperda.
Gene NameGene IDORF
(aa)		Gene_chrGene StartGene EndBest BlastX Match
Gene NameAcc.no.SpeciesE ValueIdentity
(%)
Acetyl-CoA carboxylase (ACC)
SfruACC1LOC1182804102385NC_049738.12,772,6522,888,149acetyl-CoA carboxylaseXP_022824105.1Spodoptera litura0.099.20
Fatty acid synthase (FAS)
SfruFAS1LOC1182651221503NC_049716.113,860,53413,873,617fatty acid synthase-likeXP_035448777.1Spodoptera frugiperda0.0100
SfruFAS2LOC1182676012337NC_049719.17,708,8387,745,487fatty acid synthase-likeXP_035437568.1Spodoptera frugiperda0.0100
SfruFAS3LOC1182677412328NC_049719.17,644,0747,683,559fatty acid synthase-likeXP_035437791.1Spodoptera frugiperda0.0100
SfruFAS4LOC1182728991286NC_049726.113,387,36813,397,181fatty acid synthaseAGR49310.1Agrotis ipsilon0.085.25
SfruFAS5LOC1182749702384NC_049729.188,602113,004fatty acid synthase-likeXP_035448683.1Spodoptera frugiperda0.0100
SfruFAS6LOC1182750421580NC_049729.114,669,97714,692,392fatty acid synthase-likeXP_035448779.1Spodoptera frugiperda0.0100
SfruFAS7LOC1182746342422NC_049729.13,425,9203,481,831fatty acid synthase-likeXP_035448162.1Spodoptera frugiperda0.0100
SfruFAS8LOC1182747782348NC_049729.13,486,2273,504,960fatty acid synthase-likeXP_035448370.1Spodoptera frugiperda0.0100
SfruFAS9LOC1182749992276NC_049729.15,659,1085,676,788fatty acid synthase-likeXP_035448720.1Spodoptera frugiperda0.0100
SfruFAS10LOC1182746012321NC_049729.1117,349133,649fatty acid synthase-likeXP_047033130.1Helicoverpa zea0.068.91
SfruFAS11LOC118274969294NC_049729.184,89887,981fatty acid synthase-likeXP_035448682.1Spodoptera frugiperda0.0100
Fatty acid transport protein (FATP)
SfruFATP1LOC118273519700NC_049727.16,041,9986,085,508fatty acid transport proteinARD71229.1Spodoptera exigua0.096.43
SfruFATP2LOC118273025651NC_049727.16,086,0866,101,822fatty acid transport proteinARD71230.1Spodoptera exigua0.098.00
SfruFATP3LOC118277430661NC_049733.15,268,2825,285,638fatty acid transport proteinARD71231.1Spodoptera exigua0.096.37
SfruFATP4LOC118273026643NC_049727.16,103,5626,131,706fatty acid transport proteinARD71232.1Spodoptera exigua0.098.29
Acyl-CoA dehydrogenase (ACD) (β-oxidation enzyme)
SfruACD1LOC118274515408NC_049729.114,890,75914,893,193short-chain specific acyl-CoA dehydrogenase, mitochondrialXP_022831790.1Spodoptera litura0.099.02
SfruACD2LOC118264809410NC_049715.114,866,40814,875,265acyl-CoA dehydrogenaseQZC92122.1Dioryctria abietella0.072.68
SfruACD3LOC118276297418NC_049731.113,012,36313,027,495short/branched-chain specific acyl-CoA dehydrogenase, mitochondrialXP_022827910.1Spodoptera litura0.099.28
SfruACD4LOC118265626422NC_049710.113,581,97613,599,130putative medium-chain specific acyl-CoA dehydrogenaseAID66670.1Agrotis segetum0.096.45
SfruACD5LOC118278275626NC_049711.115,605,65515,609,993very long-chain specific acyl-CoA dehydrogenase, mitochondrialXP_047020249.1Helicoverpa zea0.089.97
Enoyl-CoA hydratase (ECH) (β-oxidation enzyme)
SfruECH1LOC118271967278NC_049725.18,995,0738,998,854enoyl-CoA hydratase domain-containing protein 3, mitochondrial-likeXP_035444142.1Spodoptera frugiperda0.0100
SfruECH2LOC118277211343NC_049733.110,231,63310,237,455putative enoyl-CoA hydrataseAID66686.1Agrotis segetum0.077.42
SfruECH3LOC118264501298NC_049715.12,819,3432,822,884enoyl-CoA hydratase domain-containing protein 2, mitochondrial-likeXP_047024252.1Helicoverpa zea5 × 10−18089.23
Hydroxyacyl-coenzyme A (HCD) (β-oxidation enzyme)
SfruHCD1LOC118272082313NC_049725.11,232,0631,234,251hydroxyacyl-coenzyme A dehydrogenase, mitochondrial-likeXP_035451823.1Spodoptera frugiperda0.0100
SfruHCD2LOC118272083307NC_049725.11,235,1371,239,192putative 3-hydroxyacyl-CoA dehydrogenaseAID66695.1Agrotis segetum0.094.14
SfruHCD3LOC11827208580NC_049725.11,234,6951,242,746hydroxyacyl-coenzyme A dehydrogenase, mitochondrial-likeXP_022823313.1Spodoptera litura3 × 10−49100
3-ketoacyl-CoA thiolase (KCT) (β-oxidation enzyme)
SfruKCT1LOC118266029396NC_049717.110,904,83910,911,2373-ketoacyl-CoA thiolase, mitochondrial-likeXP_035434809.1Spodoptera frugiperda0.0100
SfruKCT2LOC118266244400NC_049717.112,544,56512,548,6513-ketoacyl-CoA thiolase, mitochondrial-likeXP_035435538.1Spodoptera frugiperda0.0100
SfruKCT3LOC118266444398NC_049717.112,548,95412,551,9003-ketoacyl-CoA thiolase, mitochondrial-likeXP_035435800.1Spodoptera frugiperda0.0100
SfruKCT4LOC118266245394NC_049717.112,538,51212,544,4343-ketoacyl-CoA thiolase, mitochondrial-likeXP_035435539.1Spodoptera frugiperda0.0100
SfruKCT5LOC118266304396NC_049717.112,535,79612,538,1033-ketoacyl-CoA thiolase, mitochondrial-likeXP_035435600.1Spodoptera frugiperda0.0100
SfruKCT6LOC118265763396NC_049717.112,527,72912,534,1643-ketoacyl-CoA thiolase, mitochondrial-likeXP_035434809.1Spodoptera frugiperda0.0100
Desaturase (DES)
SfruDES1LOC118269923379NC_049722.14,551,2524,554,527acyl-CoA desaturase 1-likeXP_035441201.1Spodoptera frugiperda0.0100
SfruDES2LOC118264925375NC_049716.112,167,23112,217,589desaturaseAAQ74260.1Spodoptera littoralis0.096.54
SfruDES3LOC118268438444NC_049720.113,813,81313,831,130desaturaseARD71179.1Spodoptera exigua0.091.22
SfruDES4LOC118274250336NC_049711.111,647,11711,651,688desaturaseARD71180.1Spodoptera exigua0.098.21
SfruDES5LOC118276125339NC_049731.18,780,6098,784,814delta 11 desaturaseAGH12217.1Spodoptera litura0.091.15
SfruDES6LOC118268131322NC_049720.112,847,46412,851,906acyl-CoA Delta(11) desaturase-likeXP_035438325.1Spodoptera frugiperda0.0100
SfruDES7LOC118264926371NC_049716.112,252,07812,286,714stearoyl-CoA desaturase 5-likeXP_035433487.1Spodoptera frugiperda0.0100
SfruDES8LOC118280447321NC_049738.19,365,1259,401,887putative desaturase des8ALJ30231.1Spodoptera litura0.097.51
SfruDES9LOC118264929354NC_049716.112,362,28612,378,777desaturaseARD71183.1Spodoptera exigua0.098.02
SfruDES10LOC118268107390NC_049720.114,173,73314,179,282desaturaseARD71184.1Spodoptera exigua2 × 10−15195.43
SfruDES11LOC118264952377NC_049716.110,539,21210,545,018acyl-CoA Delta(11) desaturase-likeXP_035433515.1Spodoptera frugiperda0.0100
SfruDES12LOC118264931358NC_049716.112,104,70912,122,940desaturaseARD71185.1Spodoptera exigua0.092.12
SfruDES13LOC118264927369NC_049716.112,146,55812,164,738acyl-CoA Delta(11) desaturase-likeXP_035433488.1Spodoptera frugiperda0.0100
SfruDES14LOC118264933340NC_049716.112,346,23112,349,171acyl-CoA Delta(11) desaturase-likeXP_035433497.1Spodoptera frugiperda0.0100
SfruDES15LOC118268104453NC_049720.114,186,78114,189,958DesaturaseKOB71313.1Operophtera brumata0.064.52
SfruDES16LOC118269215360NC_049721.13,116,2483,129,873acyl-CoA Delta(11) desaturase-likeXP_035440106.1Spodoptera frugiperda0.0100
SfruDES17LOC118269922396NC_049722.14,554,9714,557,910acyl-CoA Delta(11) desaturase-likeXP_035441200.1Spodoptera frugiperda0.0100
Fatty acyl reductase (FAR)
SfruFAR1LOC118280370535NC_049738.17,174,2087,214,859fatty acyl reductaseARD71186.1Spodoptera exigua0.096.07
SfruFAR2LOC118265382462NC_049716.15,347,0905,355,248fatty acyl reductaseARD71187.1Spodoptera exigua0.082.93
SfruFAR3LOC118265592454NC_049716.11,589,1151,598,988putative fatty acyl reductase FAR3ALJ30237.1Spodoptera litura0.093.83
SfruFAR4LOC118280550498NC_049738.17,051,9367,079,859fatty acyl-CoA reductase 13AKD01774.1Helicoverpa armigera0.088.76
SfruFAR5LOC118280458525NC_049738.17,234,1487,242,711putative fatty acyl reductase FAR5ALJ30239.1Spodoptera litura0.094.26
SfruFAR6LOC118280491520NC_049738.11,004,9551,024,481fatty acyl reductaseARD71191.1Spodoptera exigua0.085.38
SfruFAR7LOC118263342520NC_049710.114,027,02414,057,897fatty acyl reductaseARD71192.1Spodoptera exigua0.089.62
SfruFAR8LOC118280282526NC_049738.17,122,3617,154,556fatty acyl-CoA reductase 1-likeXP_035456141.1Spodoptera frugiperda0.0100
SfruFAR9LOC118265401490NC_049716.11,634,2171,641,393fatty acyl-CoA reductase 1-likeXP_035434130.1Spodoptera frugiperda0.0100
SfruFAR10LOC118280227624NC_049738.110,266,63510,318,251putative fatty acyl reductase FAR10ALJ30244.1Spodoptera litura0.095.83
SfruFAR11LOC118280439510NC_049738.18,122,3628,131,120fatty acyl-CoA reductase 2AKD01763.1Helicoverpa armigera0.056.24
SfruFAR12LOC118280219523NC_049738.15,814,5485,844,700fatty acyl-CoA reductase 1-likeXP_047038603.1Helicoverpa zea0.068.84
SfruFAR13LOC118280549520NC_049738.17,017,6867,037,406putative fatty acyl-CoA reductase CG5065XP_035456531.1Spodoptera frugiperda0.0100
SfruFAR14LOC118280222528NC_049738.15,956,0016,001,728fatty acyl-CoA reductase 8QLI61998.1Streltzoviella insularis0.086.39
SfruFAR15LOC118280297512NC_049738.15,092,0435,105,984fatty acyl reductase 15ATJ44470.1Helicoverpa armigera0.078.52
SfruFAR16LOC118280293531NC_049738.17,103,7077,121,330fatty acyl reductase 16ATJ44527.1Helicoverpa assulta0.089.29
SfruFAR17LOC118265535450NC_049716.18,947,0088,959,114putative fatty acyl-CoA reductase CG5065XP_035434352.1Spodoptera frugiperda0.0100
SfruFAR18LOC118280263511NC_049738.13,127,0283,152,947fatty acyl reductase 12ATJ44526.1Helicoverpa assulta0.075.15
SfruFAR19LOC118280403513NC_049738.15,124,4445,137,543fatty acyl-CoA reductase wat-likeXP_035456297.1Spodoptera frugiperda0.0100
SfruFAR20LOC118280192523NC_049738.17,287,5907,295,954fatty acyl-CoA reductase wat-likeXP_035455970.1Spodoptera frugiperda0.0100
SfruFAR21LOC118280547542NC_049738.16,979,0956,990,649fatty acyl reductase 13ATJ44468.1Helicoverpa armigera0.066.73
SfruFAR22LOC118280723537NC_049739.11,366,0621,372,472fatty acyl-CoA reductase wat-likeXP_035456945.1Spodoptera frugiperda0.0100
SfruFAR23LOC118267621535NC_049719.17,599,7507,608,971fatty acyl-CoA reductase wat-likeXP_035437588.1Spodoptera frugiperda0.0100
SfruFAR24LOC118267780538NC_049719.17,588,6807,597,048fatty acyl-CoA reductase 1-likeXP_035438040.1Spodoptera frugiperda0.069.60
SfruFAR25LOC118267895548NC_049719.17,574,8527,582,604fatty acyl-CoA reductase 1-likeXP_035438040.1Spodoptera frugiperda0.0100
SfruFAR26LOC118280456538NC_049738.17,251,5977,260,794fatty acyl-CoA reductase wat-likeXP_035456388.1Spodoptera frugiperda0.0100
SfruFAR27LOC118280298530NC_049738.17,271,6707,283,596fatty acyl-CoA reductase wat-likeXP_035456154.1Spodoptera frugiperda0.0100
SfruFAR28LOC118280321539NC_049738.13,362,7993,380,634fatty acyl reductase 5ATJ44463.1Helicoverpa armigera0.088.27
SfruFAR29LOC118280434538NC_049738.18,055,5088,085,920fatty acyl reductaseAID66647.1Agrotis segetum0.080.76
Acetyltransferase (ACT)
SfruACT1LOC118272936252NC_049726.17,310,2467,316,764N-alpha-acetyltransferase 60-likeXP_035445570.1Spodoptera frugiperda0.0100
SfruACT2LOC118267998176NC_049719.14,426,9764,431,010probable N-acetyltransferase sanXP_022826510.1Spodoptera litura1 × 10−127100
SfruACT3LOC118275415199NC_049730.11,741,1331,742,303N-alpha-acetyltransferase 80-likeXP_035449252.1Spodoptera frugiperda8 × 10−143100
SfruACT4LOC118281885107NW_023337168.195,71196,749N-alpha-acetyltransferase 38-BXP_022826548.1Spodoptera litura8 × 10−6691.35
SfruACT5LOC118272777710NC_049711.113,544,01813,554,256N-alpha-acetyltransferase 35, NatC auxiliary subunitXP_028155763.1Ostrinia furnacalis0.091.85
SfruACT6LOC118278521469NC_049735.18,174,3018,181,092putative acetyltransferase ACT9ALJ30256.1Spodoptera litura6 × 10−17899.25
SfruACT7LOC118281877294NW_023337168.141,78843,450N-alpha-acetyltransferase 30-likeXP_035458539.1Spodoptera frugiperda0.0100
SfruACT8LOC118267522173NC_049719.11,242,0301,243,082N-alpha-acetyltransferase 20XP_022826734.1Spodoptera litura9 × 10−127100
SfruACT9LOC118272523180NC_049726.16,776,0656,778,854N-alpha-acetyltransferase 10XP_022830428.1Spodoptera litura6 × 10−13299.44
SfruACT10LOC118276179396NC_049731.13,555,4743,572,627acetyltransferaseARD71213.1Spodoptera exigua0.098.49
SfruACT11LOC118276445512NC_049731.13,602,4423,605,363fatty alcohol acetyltransferaseAIN34682.1Agrotis segetum0.088.85
SfruACT12LOC118278776479NC_049735.14,751,8374,791,014acetyltransferaseARD71206.1Spodoptera exigua0.099.79
SfruACT13LOC118275943245NC_049730.15,635,2345,651,704N-alpha-acetyltransferase 40-likeXP_035449986.1Spodoptera frugiperda0.0100
SfruACT14LOC118267183480NC_049710.116,346,53916,357,150acetyltransferase 18ATJ44585.1Helicoverpa assulta0.077.90
SfruACT15LOC118264603505NC_049715.18,586,4078,600,294acetyltransferaseARD71205.1Spodoptera exigua0.090.69
SfruACT16LOC118262813195NC_049713.114,325,49314,326,654N-acetyltransferase 9-likeXP_035430320.1Spodoptera frugiperda1 × 10−194100
SfruACT17LOC118271269869NC_049724.14,989,0915,073,434N-alpha-acetyltransferase 15XP_035442996.1Spodoptera frugiperda0.099.88
Acyl-CoA-binding protein (ACBP)
SfruACBP1LOC11826720685NC_049718.14,519,3134,523,840putative acyl-CoA-binding proteinXP_021185372.1Helicoverpa armigera2 × 10−5294.12
SfruACBP2LOC118266031470NC_049717.110,902,55810,904,528acyl-CoA-binding domain-containing protein 6-likeXP_035434810.1Spodoptera frugiperda0.0100
SfruACBP3LOC118279722265NC_049737.16,551,4746,584,040acyl-CoA-binding domain-containing protein 5-like isoform X4XP_035455327.1Spodoptera frugiperda0.0100
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MDPI and ACS Style

Qu, C.; Kang, Z.; Zhang, B.; Fang, Y.; Wang, R.; Li, F.; Zhao, H.; Luo, C. Genome-Wide Identification and Expression Profiling of Candidate Sex Pheromone Biosynthesis Genes in the Fall Armyworm (Spodoptera frugiperda). Insects 2022, 13, 1078. https://doi.org/10.3390/insects13121078

AMA Style

Qu C, Kang Z, Zhang B, Fang Y, Wang R, Li F, Zhao H, Luo C. Genome-Wide Identification and Expression Profiling of Candidate Sex Pheromone Biosynthesis Genes in the Fall Armyworm (Spodoptera frugiperda). Insects. 2022; 13(12):1078. https://doi.org/10.3390/insects13121078

Chicago/Turabian Style

Qu, Cheng, Zhiwei Kang, Biyun Zhang, Yong Fang, Ran Wang, Fengqi Li, Haipeng Zhao, and Chen Luo. 2022. "Genome-Wide Identification and Expression Profiling of Candidate Sex Pheromone Biosynthesis Genes in the Fall Armyworm (Spodoptera frugiperda)" Insects 13, no. 12: 1078. https://doi.org/10.3390/insects13121078

APA Style

Qu, C., Kang, Z., Zhang, B., Fang, Y., Wang, R., Li, F., Zhao, H., & Luo, C. (2022). Genome-Wide Identification and Expression Profiling of Candidate Sex Pheromone Biosynthesis Genes in the Fall Armyworm (Spodoptera frugiperda). Insects, 13(12), 1078. https://doi.org/10.3390/insects13121078

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