Next Article in Journal
Multi-SNP Haplotypes in Circadian PER3 Gene Are Associated with Mood and Sleep Disorders in University Students
Previous Article in Journal
Teashirt and C-Terminal Binding Protein Interact to Regulate Drosophila Eye Development
Previous Article in Special Issue
Rainbow Trout (Oncorhynchus mykiss) Spleen-Derived Bioactive Compounds Suppress Pro-Inflammatory Gene Networks via NF-κB Pathway Modulation
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 16
Issue 9
10.3390/genes16091046
genes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Identification and Functional Speculation of Genes Related to Sex Pheromone Synthesis Expressed in the Gonads of Female Gynaephora qinghaiensis (Lepidoptera: Lymantriidae)

by
Zhanling Liu
1,
Shujing Gao
2,*,
Haibin Han
3,
Xiaorui Wang
1,
Guixiang Kou
1,
Haishun Wang
1 and
Yuantao Zhou
1,*
1
College of Agriculture and Animal Husbandry, Qinghai University, Xining 810016, China
2
Institute of Grassland, Chinese Academy of Agricultural Sciences, Hohhot 010010, China
3
College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot 010018, China
*
Authors to whom correspondence should be addressed.
Genes 2025, 16(9), 1046; https://doi.org/10.3390/genes16091046
Submission received: 11 July 2025 / Revised: 30 August 2025 / Accepted: 3 September 2025 / Published: 5 September 2025
(This article belongs to the Special Issue 15th Anniversary of Genes: Feature Papers in the “Molecular Genetics and Genomics” Section)
Browse Figures
Versions Notes

Abstract

Background: Grassland desertification has garnered significant attention as a pressing issue. Among the key pests affecting plateau meadows, the Gynaephora qinghaiensis (Lepidoptera: Lymantriidae) poses a substantial threat in the Qinghai-Tibet Plateau region, highlighting the urgent need for effective, environmentally friendly control strategies. Insect sex pheromones are increasingly employed in pest monitoring and management. Methods: This study aims to identify and analyze genes associated with sex pheromone synthesis in grassland caterpillars through transcriptome sequencing and tissue-specific expression analysis. Results: A total of 139,599 transcripts and 56,403 Unigenes were obtained from the sex pheromone glands transcriptome database. A total of 31 genes related to sex pheromone synthesis were identified, including 1 ACC, 8 DES, 6 AR, 7 FAR, 5 FAS, and 4 ACT genes. The expression levels of these genes varied significantly across different tissues in both male and female caterpillars (p < 0.05). GqinACC1, GqinDES1, GqinDES4, GqinDES8, GqinAR3, GqinFAR6, GqinACT2, and GqinACT3 exhibited significantly higher expression levels in the female gonads compared to other tissues (p < 0.01). Conclusions: We hypothesize that specific genes play specific roles in the pheromone synthesis pathways of pests, Key genes were identified based on expression patterns for subsequent functional studies. The results of this study offer valuable data support for subsequent investigations into the mechanisms underlying sex pheromone synthesis in G. qinghaiensis. Additionally, these findings may identify potential targets for future research on genes associated with pheromone biosynthesis, which could disrupt their chemical communication and contribute to grassland conservation efforts.

1. Introduction

In recent years, disruption of pheromone-mediated mating has proven effective for pest management in species such as Chilo suppressalis [1], Cnaphalocrocis medinalis [2], and Cossus insularis [3]. In many moths of the order Lepidoptera, reproductive isolation is largely dependent on sexually mature females producing and releasing species-specific sex pheromone components to attract males of the same species. Adult males utilize their antennae to detect these pheromone components, allowing them to locate females successfully and complete mating behaviors [4]. Insect sex pheromones, also referred to as sex pheromones, are trace chemical signaling substances secreted by specialized glands of sexually mature individuals of a specific sex within the same species. These pheromones are released into the environment and detected by the sensory organs of individuals of the opposite sex, facilitating successful courtship and mating. Most sex pheromones are produced by females [5]. In lepidopteran female moths, the glands that produce sex pheromones are typically located in the eighth and ninth abdominal segments [6,7,8,9]. However, in some species, these glands can be found on the dorsal surface of the thorax or on the wings [10]. In Lepidoptera, male moths primarily secrete sex pheromones from their abdomens and wings [11]. In 1959, Butenandt was the first to isolate sex pheromones from the Bombyx mori [12]. Since then, an increasing number of insect sex pheromone components have been identified, with lepidopteran sex pheromones being the most extensively studied. Most moths release mainly C10-C18 linear unsaturated fatty acids containing oxidative functional groups such as aldehyde, alcohol, or acetate [13,14,15,16,17,18,19,20]. Various enzymes are required for pheromone biosynthesis, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), desaturase (DES), fatty acyl reductase (FAR), acetyltransferase (ACT), and aldehyde reductase (AR), which collectively convert fatty acid precursors into species-specific pheromone components [21,22,23,24,25]. Transcriptomic studies have successfully identified pheromone biosynthesis genes in several lepidopteran species, revealing tissue-specific expression patterns in pheromone glands that correlate with functional roles in biosynthesis [6,26,27,28,29,30]. However, the quantities of sex pheromones secreted by insects are very small, necessitating reliance on artificial synthesis for pest control applications [31]. Therefore, studying the mechanisms of sex pheromone synthesis is crucial.
G. qinghaiensis belongs to the order Lepidoptera and the family Lymantriidae, and is a common herbivorous pest in alpine meadows. This insect mainly feeds on high-quality forage grasses of the families Poaceae and Cyperaceae. There is a significant morphological difference between male and female adults, with the thoracic legs, wings, and antennae of female adults being degenerate [32]. G. qinghaiensis is a holometabolous insect and has four developmental stages: eggs, larvae, pupae and adults. The larval stage is the nutritional stage for G. qinghaiensis to feed, and it is also the harmful stage of heavy grazing; When insect population density is high, typically ranging from 200 to 500 individuals per square meter [33], it can lead to a shortage of food for livestock. In cases of severe damage, these infestations can alter the structure of grassland plant communities, exacerbate grassland degradation, and contribute to the deterioration of the overall ecological environment [34,35,36]. It has been reported that G. qinghaiensis exhibits strong reproductive capabilities. The larvae possess poison glands on their backs, which can cause allergic reactions in livestock and humans. Additionally, they have adapted to extremely harsh environments, including strong ultraviolet radiation, low oxygen levels, and severe cold, making them difficult to prevent and control [37]. Despite extensive efforts toward their management since the 1960s, the population of G. qinghaiensis has not been fundamentally or effectively controlled [38].
In recent years, research on sex pheromones has highlighted their significant role in the monitoring and control of insects. Successful examples of using sex pheromones for the management of lepidopteran pests, such as Cydia nigriana [39] and Adoxophyes honmai [40], provide a new approach for monitoring and controlling caterpillar populations in the grasslands of Qinghai. Currently, research on the sex pheromones of G. qinghaiensis mainly focuses on the crude extracts of female moth sex pheromones and the attraction of male moths to synthetic sex pheromones [41,42]. However, the active components of these sex pheromones and the mechanisms underlying their synthesis remain unclear.
As a major pest of alpine meadows, further investigation into the genes involved in sex pheromone biosynthesis may provide potential targets for disrupting chemical communication and aiding in grassland conservation. In this study, we utilized the gonadal transcriptome database of G. qinghaiensis to screen and identify genes associated with sex pheromone synthesis. We analyzed the physicochemical properties, phylogenetic relationships, and tissue expression profiles of these genes using bioinformatics methods and RT-qPCR technology. This research aims to establish a foundation for a more in-depth understanding of the mechanisms underlying sex pheromone synthesis in G. qinghaiensis, ultimately offering new strategies for pest control.

2. Materials and Methods

2.1. Insects

In June 2023, G. qinghaiensis larvae were collected in Haiyan County, Haibei Prefecture, Qinghai Province, and the larvae were reared in the laboratory until adult emergence. The heads, thorax, abdomens, male antennae (antennae of female adults were degenerated), ovary and sex pheromone glands of female adult worms were dissected and collected. A total of 9 samples were collected, with 3 biological replicates of each sample, each containing 10 adult tissues. The samples were labeled and immediately frozen in liquid nitrogen (Chengsheng calcium carbide supply station, Xining, China) and stored at −80 °C for later use. Note that in order to prevent decomposition and contamination of the glands during dissection, the glands were dissected on ice under a microscope (Osmicro Optical Instruments Co., Ltd., Shenzhen, China); after the head was removed with a scalpel, three or four sections of the abdomen were gently compressed to force the glands to protrude from the end of the abdomen, and the glands were cut with surgical scissors and quickly stored in liquid nitrogen [43].

2.2. Total RNA Extraction

Total RNA was extracted from each of the nine tissue samples using the TRIzol method. The integrity and DNA contamination of the RNA samples were evaluated via 1% agarose gel electrophoresis, whereas RNA concentration was determined using a fluorescence spectrophotometer. Additionally, the purity of RNA in each tissue sample was assessed with the same spectrophotometer(Olympic Scientific Instruments Co., Ltd., Shanghai, China). Samples with RNA purity within the acceptable range (OD260 nm/OD280 nm ratio of 1.8–2.2) were subjected to reverse transcription using the M-MLV Reverse Transcription Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The synthesized cDNA was stored at −20 °C for subsequent RT-qPCR analysis. For further validation, the integrity of RNA isolated from sex pheromone glands was accurately determined using an Agilent 2100 Bioanalyzer (Agilent Technologies Ltd., Beijing, China). The quality criteria for the extracted RNA were defined as follows: OD260 nm/OD280 nm ratio of 1.8–2.2, 28S/18S rRNA ratio > 2, OD260 nm/OD230 nm ratio > 1.8, and RNA Integrity Number (RIN) > 8.5. All these parameters confirmed that the extracted RNA was of high quality.

2.3. Library Construction and Quality Inspection

Once the RNA samples were deemed qualified, library construction commenced. Eukaryotic mRNA was enriched using magnetic beads with Oligo (dT). A fragmentation buffer was added to randomly fragment the mRNA. The first strand of cDNA was synthesized using the mRNA as a template, followed by the addition of the buffer, dNTPs, RNase H, and DNA polymerase I. The resulting cDNA was then purified using AMPure XP beads. The purified double-stranded cDNA underwent end repair, A-tailing, and ligation with sequencing adapters. Fragment size selection was performed using AMPure XP beads to ensure the desired insert size. Finally, the cDNA library was obtained through PCR enrichment. After library construction, quality checks were conducted. Initial quantification was performed using a Qubit 2.0, and the insert size of the library was assessed with an Agilent 2100. The insert size was confirmed to be as expected before proceeding to the next steps. The Q-PCR method was employed to accurately quantify the effective concentration of the library, ensuring it exceeded 2 nM. Once the library passed quality inspection, sequencing was performed on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA), utilizing a read length of PE150. Library construction and quality inspection were assisted by Beijing Novo Zhiyuan Technology Co., Ltd. (Beijing, China), DNA fragments are fragmented to approximately 350 bp, followed by end repair, A-tailing, adapter ligation, purification, and PCR amplification. (library quality control: concentration, ≥2 nmol/L (or 2 ng/μL); volume, ≥15–100 μL depending on data volume; insert size, approximately 350 bp; total passing filter rate, ≥55%; Q30 of Read1/Read2, ≥80%).

2.4. Transcriptome Data Assembly and Gene Annotation

The raw reads obtained from RNA-seq data underwent quality filtering to produce clean data. Trinity software (v2.11.0) was utilized for transcriptome assembly. Initially, the sequencing reads were fragmented into shorter segments (K-mers), which were then extended into longer contiguous sequences (Contigs). By analyzing overlaps between these fragments, a set of components was generated. The De Bruijn graph method, combined with sequencing read information, was used to identify transcript sequences within each component set. To annotate the Unigene sequences, we performed comparisons against several databases, including NR, Swiss-Prot, GO, COG, KOG, and KEGG, using BLAST software (2.14.0). Additionally, the amino acid sequences of the Unigenes were predicted and subsequently compared with the Pfam database using HMMER3.2.1 (default threshold) software to obtain comprehensive Unigene annotation information.

2.5. Bioinformatics Analysis

In the transcriptome database of the G. qinghaiensis gonads, genes related to sex pheromone synthesis were screened or identified as “ACC” (Acetyl-CoA carboxylase), “ACT” (Acetyltransferase), “FAR” (fatty acyl reductase), “DES” (Desaturase), “FAS” (fatty acid synthase), and “AR” (Aldehyde reductase) [7]. Relevant gene sequences were screened and subjected to BLASTn comparisons on the NCBI website, with a similarity threshold set to greater than 60%. The open reading frames (ORFs) were predicted using the online tool ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/ (accessed on 26 December 2023)). Additionally, the online tool ProtParam (https://web.expasy.org/protparam/ (accessed on 10 July 2025)) was employed to perform bioinformatics analyses on the identified genes. Using Signalp 4.1 Server (http://cbs.dtu.dk/services/Signalp/ (accessed on 28 December 2023)) to predict its signal peptide, MEGA7.0 was used to construct the phylogenetic tree. The amino acid sequences were aligned with ClustalW (1000 iterations of super fast self-expansion); maximum-likelihood trees of Des and FAR were constructed by the IQTree program using the LG + I + G4 and LG + F + R6 models, respectively, implemented with the default settings and 1000 ultrafast bootstrap approximation.

2.6. RT-qPCR Detection of Target Genes

The relative expression levels of each gene were based on the internal reference gene (RPS15). Using synthetic cDNA samples (head, thorax, abdomen, antennae, ovary, and sex pheromone glands of female adults) as templates. Amplification system (20 μL): positive and negative primers, 0.8 μL; Master Mix, 10 μL; ddH2O, 7.4 μL; and cDNA template, 1 μL. The reaction was performed using a real-time fluorescence quantitative PCR instrument (ABI7500 type). The PCR amplification conditions were as follows: initial denaturation at 95 °C for 1 min; denaturation at 95 °C for 15 s; and annealing at 60 °C for 15 s and, 72 °C for 45 s for, 40 cycles. The expression levels in the antennae of male adult worms were compared. Three biological replicates and negative control groups (no template)were set up for each sample. To calculate relative levels of expression, we followed the comparative 2−ΔΔCt method (amplification efficiency of all genes was recorded as almost to 100%).

2.7. Primer Design and Synthesis

Thirty-one gene sequences (1 ACC, 8 DES, 6 AR, 7 FAR, 5 FAS, and 4 ACT) were selected and identified from the sex pheromone glands transcriptomes database of female adult G. qinghaiensis. Primers were designed using Oligo7.60 software and synthesized by Beijing Ruboxingke Biotechnology Co., LTD. (Beijing, China). The reference gene (Ribosomal protein S15) primer was derived from the Insect Laboratory of Lanzhou University [44] (Table S1).

2.8. Data Analysis

The relative expression levels of genes related to sex pheromone synthesis in different tissues of G. qinghaiensis were calculated using the 2−ΔΔCt value method, compared with the expression levels in male antennae. The data were analyzed by one-way analysis of variance (ANOVA) and significance difference test (LDS) using SPSS 21.0 software [6]. At the same time, the software Graphpad Prism 10 was used for the t-test (p < 0.05), and the tissue expression profile was drawn. The expression difference between males and females was compared by Duncan’s new complex range method.

3. Results

3.1. Transcriptome Sequence Assembly

Transcriptome sequencing of three sex pheromone gland samples (three biological replicates) from the G. qinghaiensis was conducted using the Illumina HiSeq™ 2000 high-throughput sequencing platform. A total of 56,403 Unigenes were annotated, yielding an aggregate base count of 7.6 Gb. The quality metrics indicated robust sequencing data: Q20 was 98.60%, while the Q30 value (the percentage of bases with a quality score ≥ 30) was 96.26%. Additionally, the GC content was determined to be 39.51% (all the data above are averages) (Table 1). These results demonstrate that the sequencing data quality is high, providing a reliable foundation for subsequent data analysis.
A total of 139,599 transcripts were generated following the assembly process, resulting in the identification of 56,403 Unigenes. Among these, 22,694 unigenes fell within the length range of 300–500 bp, 16,624 genes were categorized in the 500–1000 bp range, 9273 genes were between 1000 and 2000 bp, and 7812 genes exceeded 2000 bp. This indicates a high level of assembly integrity (Table 2), confirming the data’s suitability for subsequent biological analyses.

3.2. Unigene Function Annotation

The gonadal transcriptomes of G. qinghaiensis were annotated across seven databases: COG, GO, KEGG, KOG, Pfam, Swiss-Prot, and NR. The analysis revealed that the KEGG database contained the highest number of annotated genes, with 20,576 entries (36.48%), followed by NR with 13,781 entries (24.43%). Other annotations included Pfam (11,603 genes, 20.57%), Swiss-Prot (11,001 genes, 19.50%), KOG (10,721 genes, 19.01%), GO (10,228 genes, 18.13%), and the COG database, which had the fewest annotations with 4865 genes (8.6%) (Table 3).

3.3. Bioinformatics Analysis

In this study, a total of 31 genes related to sex pheromone synthesis were identified in the gonadal transcriptome database of G. qinghaiensis. (Table 2). ORFfinder and NCBI BLASTp analysis revealed that the nucleotide length of the target gene was 381–1602 bp. TMHMM2.0 was used to predict its transmembrane domain. The analysis of the sex pheromone synthesis genes revealed that out of 31 genes examined, 13 exhibited between 1 to 5 transmembrane domains, indicating potential roles in membrane-associated processes. Notably, GqinFAR2 showed a high sequence similarity of 96.88% with the gene from Streltzoviella insularis (GenBank entry QLI61998.1), suggesting a close evolutionary relationship. Similarly, GqinFAS4 displayed a similarity of 87.23% with the Spodoptera litura gene (GenBank entry XP_022831505.1), while GqinDES2 showed an 86.60% similarity with the Spodoptera exigua gene (GenBank entry ARD71182.1). Additionally, the similarity between the other target genes and genes from various lepidopteran species was consistently above 50%, indicating a significant level of conservation among these genes across different species. This information may provide insights into the functional roles of these genes in pheromone synthesis and their evolutionary significance. These results indicate that the sex pheromone-related genes of the G. qinghaiensis may be homologous to those in other Lepidoptera (Table 4).

3.4. Physicochemical Properties of Genes

The prediction of physical and chemical properties of the genes related to sex pheromone synthesis of the G. qinghaiensis showed that the amino acid length was between 127 and 533 aa and the relative molecular weight was between 14.03 and 60.50 kDa. The aliphatic index is high, between 63.78 and 107.52, which belongs to the heat -stable protein. The isoelectric points of GqinACC1, GqinAR2, GqinAR4, GqinFAR2, GqinFAS1 and GqinACT1-2 were between 4 and 6 (acidic protein). The isoelectric points of GqinDES2-6, GqinDES8, GqinAR3, GqinAR5-6, GqinFAR4-7, GqinFAS4 and GqinACT4 were between 8 and 10 (partial basic protein). The isoelectric points of GqinDES1, GqinDES7, GqinAR1, GqinFAR3, GqinFAS2, GqinFAS5, and GqinACT3 were between 6 and 7 (weakly acidic protein). The positive and negative residues of the target gene ranged from 11 to 64 and 16 to 63 respectively. The instability index of GqinACC1, GqinDES1, GqinDES6, GqinAR6, GqinFAR5-6, GqinFAS1-2, and GqinACT2 was greater than 40 (unstable protein properties). The average hydropathicity of GqinACC1 and GqinACT1 was < −0.5 (hydrophilic protein). The average hydropathicity of the remaining genes was between −0.5 and 0.5 (amphoteric proteins) (Table 5).

3.5. Phylogenetic Analysis

Phylogenetic analysis was conducted to explore the evolutionary relationships between the identified GqinDES sequences and other desaturase protein sequences from lepidopteran species. A phylogenetic tree was constructed using appropriate methods, such as neighbor-joining, based on the amino acid sequences of the desaturases. The analysis revealed that the eight GqinDES sequences were distributed across various desaturase branches in insects. Specifically, GqinDES1 and GqinDES6 were found to cluster together, indicating a close evolutionary relationship. Meanwhile, GqinDES3, GqinDES5, and GqinDES7 were grouped within the ∆11 desaturase branch, suggesting a shared functional role in pheromone biosynthesis. Additionally, GqinDES4 was classified into the ∆9 (16C > 18C) desaturase homologous clade, and GqinDES8 was positioned within the ∆9 (18C > 16C) desaturase branch. In contrast, GqinDES2 exhibited a broader clustering pattern with other desaturases, indicating potential functional diversity (Figure 1). The GqinFARs phylogenetic analysis revealed that GqinFAR5 and GqinFAR7 were retained in the same clade. In contrast, GqinFAR3 and GqinFAR6 clustered together with other homologous species from lepidopterans. Additionally, GqinFAR2 and GqinFAR4 formed a separate group, clustering alongside the Achroia grisella, O. furnacalis, and Plodia interpunctella. It is noteworthy that among the seven GqinFARs, only GqinFAR6 was classified within a clade of lepidopteran pgFARs. The remaining GqinFARs were found to be closely related to these insect FARs, suggesting similar functional roles (Figure 2).
These findings contribute to our understanding of the evolutionary dynamics of desaturase genes and fatty acyl-CoA reductase in lepidopteran species and may provide insights into their roles in sex pheromone synthesis.

3.6. Analysis of Tissue Expression Profiles

In this study, RT-qPCR was used to study the expression of genes related to sex pheromone synthesis in different tissues (head, thorax, abdomen, antennae, ovary, and sex pheromone glands) of G. qinghaiensis by using the expression levels of each gene in male and female antennae as a control. The results showed that the expression levels of target genes in different tissues of male and female caterpillars were different. A total of 31 genes associated with sex pheromone synthesis were expressed in both PG and male antennae. Notably, GqinACC1, GqinDES1, GqinDES4, GqinDES8, GqinAR3, GqinFAR6, GqinACT2 and GqinACT3 were significantly expressed in PG. Among these, GqinDES1 and GqinFAS5 displayed the highest expression levels in the PG (p < 0.01), with GqinDES1 being approximately 100 times more highly expressed than other genes, and GqinFAS5 showing around 1000 times higher expression compared to the rest.
Additionally, GqinDES6, GqinDES7, GqinAR4-6, GqinFAR3, and GqinFAR5 genes were significantly expressed in male adult antennae. Except for GqinDES4, GqinAR2, GqinFAR2, GqinFAR3, GqinFAR7, GqinFAS1-5, and GqinACT1, the expression of other genes related to sex pheromone synthesis was relatively high in female ovaries. The expression of other genes was higher in the head, thorax or abdomen, and the expression was different between male and female. (p < 0.05) (Figure 3).

4. Discussion

For non-model species, transcript sequences can be obtained through de novo assembly by assembling cDNA fragments derived from sequencing. This approach establishes a foundation for various transcriptome studies and other research involving non-model organisms. In this study, a series of bioinformatics analyses were performed, including gene structure annotation, gene expression analysis, and gene function annotation, providing a molecular basis for biological research projects. Transcriptome sequencing was conducted on three samples, resulting in a total of 22.82 Gb of clean data. Each sample yielded clean data of 7.40 Gb or more, with a Q30 base percentage of 96.14% or higher. The analysis produced a total of 139,599 transcripts and 56,403 unigenes, with N50 values of 2135 for transcripts and 1757 for unigenes. These results indicate high assembly integrity and ensure the accuracy of the transcriptome analysis [45].
The sex pheromones released by female moths consist of a mixture of components in specific proportions, exhibiting high species specificity. The synthesis of these specific sex pheromone mixtures necessitates the coordinated action of multiple enzymes. Utilizing the gonadal transcriptome data of caterpillars from the G. qinghaiensis, this study identified a total of 31 enzyme genes associated with sex pheromone synthesis. Phylogenetic analysis and tissue expression profiling primarily highlighted three genes that are particularly relevant to sex pheromone biosynthesis. These genes represent promising candidates for further functional analysis and may also serve as potential targets for pest control strategies.
In moths, saturated long-chain fatty acids serve as precursors to sex pheromones, with their biosynthesis initiated by acetyl-CoA carboxylase (ACC), which catalyzes the conversion of acetyl-CoA to malonyl-CoA, as the first step in sex pheromone biosynthesis [46]. In the gland of the G. qinghaiensis, we identified a transcript encoding ACC. Notably, GqinACC1, a 381 bp coding sequence, exhibited 61.07% amino acid identity with the ACC of H. assulta (Protein ID: AKD01721.1). Furthermore, GqinACC1 was significantly expressed in the female gonads, indicating its crucial role in the acetylation reaction involved in the biosynthesis of sex pheromone fatty acids in G. qinghaiensis.
Fatty acid synthase (FAS) catalyzes the conversion of malonyl-CoA and NADPH to produce saturated fatty acids. In the gonadal transcriptome analysis [47,48], we identified five FAS proteins, with lengths ranging from 495 to 783 bp. Notably, the 564 bp coding sequence of FAS4 exhibited 87.23% amino acid identity with the FAS from S. litura (Protein ID: XP_022831505.1). Expression profiling revealed that GqinFAS5 had an extremely high expression level in the gonads, suggesting that this gene may play a significant role in the synthesis of saturated fatty acids in conjunction with malonyl-CoA and NADPH [49].
Specific double bonds are introduced into fatty acid precursors to form fatty acyl-CoA precursors, which are subsequently shortened by β-oxidation to generate various short-chain fatty acyl-CoA precursors. These precursors are further reduced by fatty acyl reductase (FAR) to produce the corresponding alcohols [21,50]. In our analysis of the gonadal transcriptome data, we identified seven FARs homologous to the putative FAR gene. Among these, the 384 bp coding sequence of FAR2 exhibited a high amino acid identity of 96.88% with the FAR from S. insularis (Protein ID: QLI61998.1), while the remaining six protein-coding transcripts showed amino acid identities ranging from 64.77% to 81.19% with H. armigera and other species. Phylogenetic analysis revealed that GqinFAR6 clustered with pgFARs, and the FAR in this clade, including pgFAR from B. mori [50] and SlitFAR3 from S. litura [8], exhibited significant expression in pheromone glands. This suggests that GqinFAR6 may play a crucial role in the synthesis of sex pheromones.
Acyltransferase (ACT) is a key enzyme in the biosynthesis pathway of moth pheromones, facilitating the conversion of fatty acid alcohols into their corresponding esters [51,52]. In this study, we identified a total of four ACT transcripts from related species, including O. furnacalis, D. plexippus, T. ni, and Spodoptera littoralis, with amino acid identities ranging from 60% to 75%. The gonad-specific expression of ACT in the Atrijuglans hetaohei suggests its involvement in the synthesis of corresponding acetates [53]. Notably, GqinACT3 and GqinACT4 were specifically expressed in the gonads of female G. qinghaiensis, leading to speculation that these two genes also play a role in the synthesis of the corresponding acetates.
Desaturase (DES) enzymes are responsible for introducing double bonds into fatty acyl precursors, a critical step in the biosynthesis of sex pheromones [54]. Previous studies in lepidopteran species have demonstrated the role of Δ11-desaturase in modifying palmitic acid to produce components of sex pheromones [49]. Additionally, Δ9-desaturase converts stearic acid (18:0) to oleic acid and may also convert palmitic acid to palmitoleic acid, although the latter is not involved in the biosynthesis of sex pheromones in Agrotis ipsilon [9]. In our analysis, we identified eight DES proteins in the gonads, which exhibited high homology with DES sequences from other lepidopteran insects, ranging from 67.45% to 86.60%. Phylogenetic analysis indicated that GqinDES1, GqinDES6, GqinDES3, GqinDES5, and GqinDES7 clustered within the Δ11-desaturase branch. GqinDES4 was positioned in the Δ9 (16C > 18C) desaturase branch, while GqinDES8 fell into the Δ9 (18C > 16C) desaturase clade. These findings suggest that the sex pheromone biosynthesis pathway in G. qinghaiensis may involve desaturation steps mediated by both Δ9 and Δ11 desaturases. Additionally, the expression levels of GqinDES1, GqinDES4, and GqinDES8 in the gonads were significantly higher than those in other tissues, suggesting that these three genes may play a crucial role in producing fatty acyl precursors for sex pheromone biosynthesis. They are likely involved in the desaturation step that converts saturated fatty acids (16C) to unsaturated fatty acids by introducing double bonds at the 9th or 11th positions of the carbon chain. In contrast, other DES genes did not exhibit gonad-specific expression and may not be involved in sex pheromone desaturation, though they could serve other physiological functions. Further detailed studies are needed to validate this hypothesis [7].
Aldehyde reductase (AR) catalyzes the reduction of aldehyde substrates to their corresponding alcohols in the synthesis of sex pheromones [55]. In our analysis, we identified six transcripts in the gonads of steppe caterpillars that were homologous to the fatty acyl reductase (FAR) of H. armigera and H. assulta. The proteins encoded by these transcripts exhibited amino acid identities ranging from 58.40% to 80.65% with the homologous FARs (ProteinID: ATJ44537.1, ATJ44541.1, ATJ44498.1, ATJ44502.1, ATJ44539.1, ATJ44505.1). Notably, GqinAR3 was specifically expressed in the gonads, suggesting its primary role as a reductase in converting sex pheromone components to their corresponding alcohols. In contrast, GqinAR4-6 were predominantly expressed in the antennae of male adults, indicating their potential involvement in olfactory functions. Additionally, ARs that are highly expressed in other tissues may have roles beyond olfaction [7].
The β-oxidation and fatty acid synthesis pathways involved in moth sex pheromone production are generally similar to those found in the normal metabolism of organisms. Therefore, we would expect no significant differences in the expression of acetyl-CoA carboxylase, fatty acid synthase, and β-oxidation enzyme systems between males and females. However, our experiment revealed notable differences in the expression levels of certain genes between the sexes. This observation leads us to speculate that the sex pheromone synthesis pathway in G. qinghaiensis may possess unique characteristics in its β-oxidation and fatty acid synthesis processes. We propose that key genes involved in sex pheromone production—specifically GqinDES1, GqinDES8, and GqinFAR6, which exhibit significant expression in the gonads—should be prioritized for further functional analysis. Additionally, genes that are not specifically expressed in the gonads may be investigated using alternative methodologies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes16091046/s1, Table S1: Primers used in the study.

Author Contributions

Conceptualization, Z.L. and Y.Z.; methodology, S.G. and X.W.; software, H.H., H.W. and X.W.; validation, Z.L. and G.K.; formal analysis, Z.L. and S.G.; investigation, Z.L., G.K. and H.H.; resources, Y.Z. and H.H.; writing—original draft preparation, Z.L.; writing—review and editing, Z.L., H.H. and Y.Z.; supervision, Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Key R&D Program of China, “Research on Monitoring and Control Technology of Grassland Caterpillars and Other Important Pests” (2022YFD1401102).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials; further inquiry can be directed to the corresponding authors.

Acknowledgments

We would like to thank Yuantao Zhou for their suggestions in performing this research.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Lucchi, A.; Sambado, P.; Juan Royo, A.B.; Bagnoli, B.; Conte, G.; Benelli, G. Disrupting mating of Lobesia botrana using sex pheromone aerosol devices. Environ. Sci. Pollut. Res. 2018, 25, 22196–22204. [Google Scholar] [CrossRef]
  2. Liang, Y.Y.; Luo, M.; Fu, X.G.; Zheng, L.X.; Wei, H.Y. Mating disruption of Chilo suppressalis from sex pheromone of another pyralid rice pest Cnaphalocrocis medinalis (Lepidoptera: Pyralidae). J. Insect Sci. 2020, 20, 19. [Google Scholar] [CrossRef]
  3. Hoshi, H.; Takabe, M.; Nakamuta, K. Mating disruption of a carpenter moth, Cossus insularis (Lepidoptera: Cossidae) in apple orchards with synthetic sex pheromone, and registration of the pheromone as an agrochemical. J. Chem. Ecol. 2016, 42, 606–611. [Google Scholar] [CrossRef]
  4. Wyatt, T.D. Fifty years of pheromones. Nature 2009, 457, 262–263. [Google Scholar] [CrossRef] [PubMed]
  5. Raina, A.K. Neuroendocrine control of sex pheromone biosynthesis in Lepidoptera. Annu. Rev. Entomol. 1993, 38, 329–349. [Google Scholar] [CrossRef] [PubMed]
  6. Zhu, S.Y.; Shaheen, T.Y.; Li, R.; Pei, P.; Zhao, X.; Li, Y.; Li, F.F.; Tang, G.G. Transcriptome analysis and identification of sex pheromone biosynthesis and transport related genes in Atrijuglans hetaohei (Lepidoptera: Gelechioidea). Insect Mol. Biol. 2021, 31, 273–285. [Google Scholar] [CrossRef] [PubMed]
  7. He, P.; Zhang, Y.F.; Hong, D.Y.; Wang, J.; Wang, X.L.; Zuo, L.H.; Tang, X.F.; Xu, W.M.; He, M. A reference gene set for sex pheromone biosynthesis and degradation genes from the diamondback moth, Plutella xylostella, based on genome and transcriptome digital gene expression analyses. BMC Genom. 2017, 18, 219. [Google Scholar] [CrossRef]
  8. Zhang, Y.N.; Zhu, X.Y.; Fang, L.P.; He, P.; Wang, Z.Q.; Chen, G.; Sun, L.; Ye, Z.F.; Deng, D.G.; Li, J.B. Identification and expression profiles of sex pheromone biosynthesis and transport related genes in Spodoptera litura. PLoS ONE 2015, 10, e0140019. [Google Scholar] [CrossRef]
  9. Gu, S.H.; Wu, K.M.; Guo, Y.Y.; Pickett, J.A.; Field, L.M.; Zhou, J.J.; Zhang, Y.J. Identification of genes expressed in the sex pheromone gland of the black cutworm Agrotis ipsilon with putative roles in sex pheromone biosynthesis and transport. BMC Genom. 2013, 14, 636. [Google Scholar] [CrossRef]
  10. Li, D.M.; Li, J.; Li, L.C. Study of Sex Pheromones in Illiberis pruni III: Observation of the morphological structure of sex pheromone secretion glands. Acta Agric. Boreali-Sin. 1997, 113–117. [Google Scholar]
  11. Yang, M.W.; Dong, S.L. A review of the male moth sex pheromone and its roles in the sex communication. J. Biosaf. 2006, 3, 179–186. [Google Scholar]
  12. Butenandt, V. Uber den sexsual-lockstoff des seidenspinners Bombyx mori. Reindarstellung und konstitution. Z. Naturforschung B 1959, 14, 283. [Google Scholar] [CrossRef]
  13. Matsumoto, S.; Ozawa, R.; Uchiumi, K.; Kurihara, M.; Mitsui, T. Intracellular signal transduction of PBAN action in the common cutworm, Spodoptera litura: Effects of pharmacological agents on sex pheromone production in vitro. Insect Biochem. Mol. Biol. 1995, 25, 1055–1059. [Google Scholar] [CrossRef]
  14. Luo, J.; Liu, X.Y.; Liu, L.; Zhang, P.Y.; Chen, L.J.; Gao, Q.; Ma, W.H.; Chen, L.Z.; Lei, C.L. De novo analysis of the Adelphocoris suturalis Jakovlev metathoracic scent glands transcriptome and expression patterns of pheromone biosynthesis-related genes. Gene 2014, 551, 271–278. [Google Scholar] [CrossRef]
  15. Tamaki, Y.Z. Z-11-hexadecenal and Z-11-hexadecenyl acteact: Sex pheromone components of the diamondback moth (Lepideptera: Plutellidae). Appl. Entomol. Zool. 1977, 12, 208–210. [Google Scholar] [CrossRef]
  16. Môttus, E.; Nômm, V.; Williams, I.H.; Liblikas, I. Optimization of Pheromone Dispensers for Diamondback Moth Plutella xylostella. J. Chem. Ecol. 1997, 23, 2145–2159. [Google Scholar] [CrossRef]
  17. Chisholm, M.D.; Steck, W.F.; Underhill, E.W.; Palaniswamy, P. Field trapping of diamondback moth Plutella xylostella using an improved four-component sex attractant blend. J. Chem. Ecol. 1983, 9, 113–118. [Google Scholar] [CrossRef]
  18. Koshihara, T.; Yamada, H. Attractant activity of the female Sex pheromone of diamondback moth, Plutella xylostella (L.), and analogue. Jpn. J. Appl. Entomol. Zool. 1980, 24, 6–12. [Google Scholar] [CrossRef]
  19. Ando, T.; Koshihara, T.; Yamada, H.; Vu, M.H.; Takahashi, N.; Tamaki, Y. Electroantennogram activities of Sex pheromone analogues and their synergistic effect on field attraction in the diamondback moth. Appl. Entomol. Zool. 1979, 14, 362–364. [Google Scholar] [CrossRef]
  20. Hao, X.Y.; Zhang, J.T.; Wang, R.; Cao, T.W.; Li, Y.L. Component Analysis of the Sex Pheromone Gland Secretion for Atrijuglans hetaohei Yang. J. Shanxi Agric. Univ. (Nat. Sci. Ed.) 2007, 27, 409–411. [Google Scholar]
  21. Jurenka, R. Insect pheromone biosynthesis. Top. Curr. Chem. 2004, 239, 97–132. [Google Scholar] [PubMed]
  22. Jurenka, R.A.; Subchev, M.; Abad, J.L.; Choi, M.Y.; Fabrias, G. Sex pheromone biosynthetic pathway for disparlure in the gypsy moth, Lymantria dispar. Proc. Natl. Acad. Sci. USA 2003, 100, 809–814. [Google Scholar] [CrossRef] [PubMed]
  23. Hagström, Å.K.; Albre, J.; Tooman, L.K.; Thirmawithana, A.H.; Corcoran, J.; Löfstedt, C.; Newcomb, R.D. A novel fatty acyl desaturase from the pheromone glands of Ctenopseustis obliquana and C. herana with specific Z5-desaturase activity on myristic acid. J. Chem. Ecol. 2014, 40, 63–70. [Google Scholar] [CrossRef] [PubMed]
  24. Matsumoto, S.; Ohnishi, A.; Lee, J.M.; Hull, J.J. Unraveling the pheromone biosynthesis activating neu ropeptide (PBAN) signal transduction cascade that regulates sex pheromone production in moths. Vitam. Horm. 2010, 83, 425–445. [Google Scholar]
  25. Roelofs, W.L.; Rooney, A.P. Molecular genetics and evolution of pheromone biosynthesis in Lepidoptera. Proc. Natl. Acad. Sci. USA 2003, 100, 9179–9184. [Google Scholar] [CrossRef]
  26. Knipple, D.C.; Rosenfield, C.L.; Nielsen, R.; You, K.M.; Jeong, S.E. Evolution of the integral membrane desaturase gene family in moths and flies. Genetics 2002, 162, 1737–1752. [Google Scholar] [CrossRef]
  27. Chen, D.S.; Dai, J.Q.; Han, S.C. Identification of the pheromone biosynthesis genes from the sex pheromone gland transcriptome of the diamondback moth, Plutella xylostella. Sci. Rep. 2017, 7, 16255. [Google Scholar] [CrossRef]
  28. Grapputo, A.; Thrimawithana, A.H.; Steinwender, B.; Newcomb, R.D. Differential gene expression in the evolution of sex pheromone communication in New Zealand’s endemic leafroller moths of the genera Ctenopseustis and Planotortrix. BMC Genom. 2018, 19, 94. [Google Scholar] [CrossRef]
  29. Yang, Y.; Tao, J.; Zong, S. Identification of putative Type-I sex pheromone biosynthesis-related genes expressed in the female pheromone gland of Streltzoviella insularis. PLoS ONE 2020, 15, e0227666. [Google Scholar] [CrossRef]
  30. Yan, Q.; Yang, T.T.; Dong, S.L. Review of research on the hybrid pheromone systemin lepidopteran insects. Chin. J. Appl. Entomol. 2023, 60, 323–334. [Google Scholar]
  31. Yu, R.; Yang, S.; Zhao, Y.H.; Ji, L.L. Overview Of Pheromone Pesticide Registration in China and Abroad and Policy Suggestions. Pestic. Sci. Adm. 2019, 40, 6–9+20. [Google Scholar]
  32. Nan, Y.B.; Liu, Z.L.; Kou, G.X.; Wang, K.X.; Li, R.R.; Zhou, Y.T. Observations on the Ultrastructure of adult Gynaephora qinghaiensis antennal sensilla and tissue expression analysis of GqinOBPs. Plant Prot. 2024, 50, 15–25+71. [Google Scholar]
  33. Yang, A.L. Grassland caterpillars in some areas of Qinghai in Tibet are seriously infested. Pratacult. Sci. 2002, 5, 73. [Google Scholar]
  34. Wang, H.Z.; Zhong, X.; Lin, H.F.; Li, S.S.; Yi, J.Q.; Zhang, G.R.; Liu, X.; Gu, L. Genetic Diversity and Population Structure of Gynaephora qinghaiensis in Yushu Prefecture, Qinghai Province Based on the Mitochondrial COI Gene. Biochem. Genet. 2021, 59, 1396–1412. [Google Scholar] [CrossRef]
  35. Cao, J.; Xu, H.; Pan, X.B.; Rong, Y.P. Study on the Status of Invasive Plants in Chinese Grassland. Acta Agrestia Sin. 2020, 1, 1–11. [Google Scholar]
  36. Wang, X.L.; Zhang, W.G. Feeding Habit and Spatial Pattern of Gynaephora alpherakii Larvae. Acta Agrestia Sin. 2006, 14, 84–88. [Google Scholar]
  37. Nan, Y.B.; Tang, D.J.; Yang, Y.C.; Kong, Y.S.; Zhou, Y.T. Identification and Expression Profiling of Chemoreceptor protein Genes in Gynaephora qinghaiensis (Lepidoptera: Lymantriidae). Acta Agrestia Sin. 2023, 31, 3299–3309. [Google Scholar]
  38. Zhang, Q.L.; Yuan, M.L. Current status and prospect of Gynaephora qinghaiensis research. Pratacult. Sci. 2013, 30, 638–646. [Google Scholar]
  39. Wall, C.; Garthwaite, D.G.; Smyth, J.A.; Sherwood, A.J. The efficacy of sex-attractant monitoring for the pea moth, Cydia nigricana, in England, 1980–1985. Ann. Appl. Biol. 2010, 110, 223–229. [Google Scholar] [CrossRef]
  40. Tamaki, Y.; Noguchi, H.; Sugie, H.; Sato, R.; Kariya, A. Minor component of the female sex-attractant pheromone of smaller tea tortrix moth (Lepidoptera: Tortricidae): Isolation and identification. Appl. Entomol. Zool. 1979, 14, 101–113. [Google Scholar] [CrossRef]
  41. Bao, G.S.; Wang, H.S. Effects of female’s sex pheromone of Gynaephora alpherakii on attracting male moth by extracting four organic solvents. Chin. Qinghai J. Anim. Vet. Sci. 2015, 45, 10–12. [Google Scholar]
  42. Shen, N.Y.; Zen, L.; Zhang, X.C.; Shan, Z.; Zhou, Z.R.; Yan, S.S.; Gao, X.C. A study of sex pheromones in Gynaephora qinghaiensis. Chin. J. Grassl. 1983, 1, 43–49. [Google Scholar]
  43. Batista-Pereira, L.G.; Santangelo, E.M.; Stein, K.; Unelius, C.R.; Eiras, A.E.; Corrêa, A.G. Electrophysiological studies and identification of possible sex pheromone components of Brazilian populations of the sugarcane borer, Diatraea saccharalis. Z. Naturforschung C 2002, 57, 753–758. [Google Scholar] [CrossRef] [PubMed]
  44. Zhang, L.; Zhang, Q.L.; Wang, X.T.; Yang, X.Z.; Li, X.P.; Yuan, M.L. Selection of reference genes for qRT-PCR and expression analysis of high-altitude-related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient. Ecol. Evol. 2017, 7, 9054–9065. [Google Scholar] [CrossRef]
  45. Yang, P.; Xie, S.A.; Gon, X.F.; Che, X.R.; Wang, Y.L.; Lv, S.J. Analysis of the transcriptome and chemoreception-related genes of Agrilus zanthoxylumi (Coleoptera: Buprestidae). Acta Entomol. Sin. 2019, 62, 547–560. [Google Scholar]
  46. Pape, M.E.; Lopez, C.F.; Kim, K.H. Physiological regulation of acetyl-CoA carboxylase gene expression: Effects of diet, diabetes, and lactation on acetyl-CoA carboxylase mRNA. Arch. Biochem. Biophys. 1988, 267, 104–109. [Google Scholar] [CrossRef]
  47. Volpe, J.J.; Vagelos, P.R. Saturated fatty acid biosynthesis and its regulation. Annu. Rev. Biochem. 1973, 42, 21–60. [Google Scholar] [CrossRef]
  48. Tang, J.D.; Charlton, R.E.; Jurenka, R.A.; Wolf, W.A.; Phelan, P.L.; Sreng, L.; Roelofs, W.L. Regulation of pheromone biosynthesis by a brain hormone in two moth species. Proc. Natl. Acad. Sci. USA 1989, 86, 1806–1810. [Google Scholar] [CrossRef]
  49. Bjostad, L.B.; Roelofs, W.L. Sex pheromone biosynthesis from radiolabeled fatty acids in the redbanded leafroller moth. J. Biol. Chem. 1981, 256, 7936–7940. [Google Scholar] [CrossRef]
  50. Moto, K.; Yoshiga, T.; Yamamoto, M.; Takahashi, S.; Okano, K.; Ando, T.; Nakata, T.; Matsumoto, S. Pheromone gland-specific fatty-acyl reductase of the silkmoth, Bombyx mori. Proc. Natl. Acad. Sci. USA 2003, 100, 9156–9161. [Google Scholar] [CrossRef]
  51. Beekwilder, J.; Alvarez-Huerta, M.; Neef, E.; Verstappen, F.W.A.; Bouwmeester, H.J.; Aharoni, A. Functional characterization of enzymes forming volatile esters from strawberry and banana. Plant Physiol. 2004, 135, 1865–1878. [Google Scholar] [CrossRef]
  52. Kalscheuer, R.; Stoveken, T.; Luftmann, H.; Malkus, U.; Reichelt, R.; Steinbuchel, A. Neutral lipid biosynthesis in engineered Escherichia coli: Jojoba oil-like wax esters and fatty acid butyl esters. Appl. Environ. Microbiol. 2006, 72, 1373–1379. [Google Scholar] [CrossRef]
  53. Zhu, S.Y. Analysis of Genes Related to Sex Pheromone Biosynthesis and Transport in Atrijuglans hetaohei Yang. Ph.D. Thesis, Northwest A&F University, Xianyang, China, 2021; pp. 27–53. [Google Scholar]
  54. Cook, O.; Hildebrand, M. Enhancing LC-PUFA production in Thalassiosira pseudonana by overexpressing the endogenous fatty acid elongase genes. J. Appl. Phycol. 2016, 28, 897–905. [Google Scholar] [CrossRef]
  55. Morse, D.; Meighen, E.A. Pheromone biosynthesis: Enzymatic studies in lepidoptera. In Pheromone Biochemistry; Academic Press: Cambridge, MA, USA, 1987; pp. 121–158. [Google Scholar]
Genes 16 01046 g001
Figure 1. Phylogenetic tree of GqinDES and other identified Lepidopteran DESs. A. grisella (Agri), A. segetum (Aseg), Amyelois transitella (Atra), Bicyclus anynana (Bany), B. mori (Bmor), Cydia pomonella (Cpom), Dendrolimus punctatus (Dpun), Galleria mellonella (Gmel), H. assulta (Hass), Helicoverpa zea (Hzea), Heliothis virescens (Hvir), Manduca sexta (Msex), Melitaea cinxia (Mcin), Spodoptera frugiperda (Sfru), S. litura (Slit), T. ni (Tni), Lampronia capitella (Lcap), Ascotis selenaria (Asel), Planotortrix octo (Poct), E. postvittana (Epos), Planotortrix notophaea (Pnot), Planotortrix excessana (Pexc), O. furnacalis (Ofur), Ostrinia nubilalis (Onub), Antheraea pernyi (Aper), Choristoneura parallela (Cpar), Mamestra brassicae (Mbra), Argyrotaenia velutinana (Avel), Choristoneura rosaceana (Cros), Ctenopseustis obliquana (Cobl), Ctenopseustis herana (Cher), Thaumetopoea pityocampa (Tpit). G. qinghaiensis isrepresented by black circles.
Figure 1. Phylogenetic tree of GqinDES and other identified Lepidopteran DESs. A. grisella (Agri), A. segetum (Aseg), Amyelois transitella (Atra), Bicyclus anynana (Bany), B. mori (Bmor), Cydia pomonella (Cpom), Dendrolimus punctatus (Dpun), Galleria mellonella (Gmel), H. assulta (Hass), Helicoverpa zea (Hzea), Heliothis virescens (Hvir), Manduca sexta (Msex), Melitaea cinxia (Mcin), Spodoptera frugiperda (Sfru), S. litura (Slit), T. ni (Tni), Lampronia capitella (Lcap), Ascotis selenaria (Asel), Planotortrix octo (Poct), E. postvittana (Epos), Planotortrix notophaea (Pnot), Planotortrix excessana (Pexc), O. furnacalis (Ofur), Ostrinia nubilalis (Onub), Antheraea pernyi (Aper), Choristoneura parallela (Cpar), Mamestra brassicae (Mbra), Argyrotaenia velutinana (Avel), Choristoneura rosaceana (Cros), Ctenopseustis obliquana (Cobl), Ctenopseustis herana (Cher), Thaumetopoea pityocampa (Tpit). G. qinghaiensis isrepresented by black circles.
Genes 16 01046 g001
Genes 16 01046 g002
Figure 2. Phylogenetic tree of GqinFAR and other identified Lepidopteran FARs. A. grisella (Agri), Agrotis ipsilon (Aips), A. transitella (Atra), B. anynana (Bany), Bombyx mandarina (Bman), B. mori (Bmor), C. suppressalis (Csup), G. mellonella (Gmel), H. armigera (Harm), H. assulta (Hass), Helicoverpa zea (Hzea), M. sexta (Msex), Maniola hyperantus (Mhyp), Maniola jurtina (Mjur), M. cinxia (Mcin), Mythimna separata (Msep), Nymphalis io (Nio), O. furnacalis (Ofur), Papilio machaon (Pmac), Papilio polytes (Ppol), Papilio xuthus (Pxut), Pararge aegeria (Paeg), P. gossypiella (Pgos), P. interpunctella (Pint), Spodoptera frugiperda (Sfru), S. litura (Slit), S. insularis (Sins), T. ni (Tni), Vanessa cardui (Vcar), Vanessa tameamea (Vtam). G. qinghaiensis is represented by black circles.
Figure 2. Phylogenetic tree of GqinFAR and other identified Lepidopteran FARs. A. grisella (Agri), Agrotis ipsilon (Aips), A. transitella (Atra), B. anynana (Bany), Bombyx mandarina (Bman), B. mori (Bmor), C. suppressalis (Csup), G. mellonella (Gmel), H. armigera (Harm), H. assulta (Hass), Helicoverpa zea (Hzea), M. sexta (Msex), Maniola hyperantus (Mhyp), Maniola jurtina (Mjur), M. cinxia (Mcin), Mythimna separata (Msep), Nymphalis io (Nio), O. furnacalis (Ofur), Papilio machaon (Pmac), Papilio polytes (Ppol), Papilio xuthus (Pxut), Pararge aegeria (Paeg), P. gossypiella (Pgos), P. interpunctella (Pint), Spodoptera frugiperda (Sfru), S. litura (Slit), S. insularis (Sins), T. ni (Tni), Vanessa cardui (Vcar), Vanessa tameamea (Vtam). G. qinghaiensis is represented by black circles.
Genes 16 01046 g002
Genes 16 01046 g003aGenes 16 01046 g003b
Figure 3. Tissue expression profile analysis of sex pheromone biosynthesis related genes in G. qinghaiensis. Male antennae (An); head (with antennae removed) (Hd); thorax (Th); abdomen (Ab); ovary (OV); sex pheromone glands (PG). RPS15 was used as the internal reference gene to correct the expression level of each tissue, and the expression level of the target gene in the PG was used as a positive control. Error bars indicate the standard error of 3 independent experiments, different uppercase and lowercase letters indicate significant differences between male and female tissues, and single and double asterisks indicate significant differences in the relative expression levels of target genes in the same tissue, and between males and females (t-test, * p < 0.05; ** p < 0.01; ns: not significant). (A) ACC1, DES1~8; (B) AR1~6, FAR1~3; (C) FAR4~7, FAS1~5; (D) ACT1~4.
Figure 3. Tissue expression profile analysis of sex pheromone biosynthesis related genes in G. qinghaiensis. Male antennae (An); head (with antennae removed) (Hd); thorax (Th); abdomen (Ab); ovary (OV); sex pheromone glands (PG). RPS15 was used as the internal reference gene to correct the expression level of each tissue, and the expression level of the target gene in the PG was used as a positive control. Error bars indicate the standard error of 3 independent experiments, different uppercase and lowercase letters indicate significant differences between male and female tissues, and single and double asterisks indicate significant differences in the relative expression levels of target genes in the same tissue, and between males and females (t-test, * p < 0.05; ** p < 0.01; ns: not significant). (A) ACC1, DES1~8; (B) AR1~6, FAR1~3; (C) FAR4~7, FAS1~5; (D) ACT1~4.
Genes 16 01046 g003aGenes 16 01046 g003b
Table 1. Results of transcriptome data from sex pheromone glands of G. qinghaiensis.
Table 1. Results of transcriptome data from sex pheromone glands of G. qinghaiensis.
SampleRead SumBaseSum (Gb)GC (%)N (%)Q20 (%)CycleQ20 (%)Q30 (%)
X.125,560,5767.71939.60.0198.5610096.14
X.224,505,8237.40039.280.0198.6710096.42
X.325,499,0537.70039.640.0198.5710096.21
Average25,188,4847.60739.510.0198.6010096.26
Table 2. Results of the transcriptome assembly from sex pheromone glands of G. qinghaiensis.
Table 2. Results of the transcriptome assembly from sex pheromone glands of G. qinghaiensis.
Length RangeTranscriptsPercentage/%UnigenesPercentage/%
300–50039,92828.60%22,69440.24%
500–100041,16429.49%16,62429.47%
1000–200030,92222.15%927316.44%
2000+27,58519.76%781213.85%
Total139,599 56,403
Table 3. Statistics of gene functional annotation for transcriptomes from sex pheromone glands of G. qinghaiensis.
Table 3. Statistics of gene functional annotation for transcriptomes from sex pheromone glands of G. qinghaiensis.
Anno_DatabaseAnnotated_NumberPercentage/%300 ≤ Length < 1000Length ≥ 1000
COG_Annotation48658.69353930
GO_Annotation10,22818.1326917537
KEGG_Annotation20,57636.48882411,752
KOG_Annotation10,72119.0128267895
Pfam_Annotation11,60320.5727878816
Swissprot_Annotation11,00119.5029198082
NR_Annotation13,78124.4340009781
All_Annotated20,72936.75894111,788
Note: 300 ≤ Length < 1000-, indicates the number of Unigene notes to the database with a length between 300 and 1000 bases; Length ≥ 1000-, indicates the number of Unigene comments with a length of more than 1000 bases to the database.
Table 4. Bioinformatics analysis of sex pheromone biosynthesis related genes in G. qinghaiensis.
Table 4. Bioinformatics analysis of sex pheromone biosynthesis related genes in G. qinghaiensis.
GeneNucleotideTransmembrane DomainsCDSAmino AcidBlastp Test and Verify
Length/bpIntegrityLength/aaSpeciesGenBank AccessionE-ValueIdentity/%
ACC13811No126Helicoverpa assultaAKD01721.12 × 10−4161.07%
DES19905Yes329S. exiguaARD71185.1074.61%
DES29664Yes321S. exiguaARD71182.1086.60%
DES310985Yes365Grapholita molestaAUC64291.11 × 10−14767.45%
DES49934Yes330S. insularisQLI61972.13 × 10−16169.13%
DES513685Yes455Agrotis segetumAID66662.1076.77%
DES69392Yes312Danaus plexippus plexippusOWR48667.13 × 10−15270.67%
DES711164Yes371Spodoptera littoralisAAQ74260.1079.40%
DES810654Yes354Sesamia inferensAII21941.1082.77%
AR110230Yes340H. assultaATJ44537.1077.35%
AR210140Yes337H. assultaATJ44541.1071.77%
AR35040Yes167H. assultaATJ44498.12 × 10−8168.86%
AR48010Yes266Helicoverpa armigeraATJ44502.12 × 10−12959.63%
AR59270Yes308H. assultaATJ44539.1080.65%
AR611670Yes388H. armigeraATJ44505.13 × 10−16558.40%
FAR115572Yes518Pectinophora gossypiellaXP_049883830.1068.42%
FAR23840NO127S. insularisQLI61998.19 × 10−8396.88%
FAR316022Yes533H. armigeraAKD01766.1078.30%
FAR413950Yes464H. assultaAKD01789.1064.77%
FAR515661Yes521H. armigeraAKD01770.1076.06%
FAR67050Yes234H. armigeraAKD01771.12 × 10−12081.19%
FAR715782Yes525Helicoverpa zeaXP_047038839.1080.04%
FAS14950Yes164H. armigeraXP_049700707.13 × 10−8277.44%
FAS27440Yes247Dioryctria abietellaQZC92075.11 × 10−12369.35%
FAS37830Yes260Spodoptera frugiperdaXP_050550085.13 × 10−12068.58%
FAS45640Yes187S. lituraXP_022831505.12 × 10−10787.23%
FAS55190Yes172H. armigeraXP_049699195.12 × 10−7167.82%
ACT15310Yes176Ostrinia furnacalisXP_028157379.12 × 10−9174.12%
ACT27320Yes243D. p. plexippusOWR41901.11 × 10−9363.51%
ACT37830Yes260Trichoplusia niXP_026730350.13 × 10−13773.46%
ACT414100Yes469Spodoptera littoralisCAB3515140.1069.79%
Table 5. Physicochemical properties of sex pheromone biosynthesis related proteins in G. qinghaiensis.
Table 5. Physicochemical properties of sex pheromone biosynthesis related proteins in G. qinghaiensis.
ProteinAmino AcidsMolecular Weight/KDIsoelectricpoint/PINegatively Charged ResiduesPositively Charged ResiduesInstability IndexAliphatic IndexAverage Hydropathicity
ACC112714.054.96201355.1263.78−0.613
DES132937.566.89313040.27104.620.141
DES232137.288.93263139.1497.730.117
DES336541.779.3253737.384.88−0.095
DES433038.559.04273430.0289.27−0.111
DES545553.348.65424739.0989.82−0.164
DES631236.148.77283244.4181.92−0.241
DES737142.786.43403636.4994.07−0.091
DES835440.958.81333734.5287.34−0.149
AR134039.126.55444336.4388.24−0.389
AR233737.835.77403536.4296.26−0.166
AR316719.069.23182329.4692.75−0.249
AR426630.125.86312833.3497.48−0.161
AR530934.988.78354024.6389.58−0.286
AR638843.878.37434640.2797.99−0.147
FAR151859.157.87616335.18101.62−0.069
FAR212814.035.1161121.41101.170.022
FAR353360.506.24636027.8194.93−0.082
FAR446453.118.99516033.0190.32−0.138
FAR552159.308.82556240.43107.52−0.052
FAR623427.179.43223042.35100.73−0.024
FAR752559.448.53596436.5699.90.025
FAS116518.915.29251645.1786.79−0.339
FAS224827.396.05262144.06100.280.025
FAS326129.177.08313131.0390.04−0.324
FAS418820.508.52202231.73107.340.128
FAS517318.776.41171638.0595.950.042
ACT117619.935.81262438.9265.97−0.553
ACT224327.434.71372542.1777.45−0.322
ACT326029.416.61363520.3183.35−0.361
ACT446951.878.75505731.1484.22−0.359
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Liu, Z.; Gao, S.; Han, H.; Wang, X.; Kou, G.; Wang, H.; Zhou, Y. Identification and Functional Speculation of Genes Related to Sex Pheromone Synthesis Expressed in the Gonads of Female Gynaephora qinghaiensis (Lepidoptera: Lymantriidae). Genes 2025, 16, 1046. https://doi.org/10.3390/genes16091046

AMA Style

Liu Z, Gao S, Han H, Wang X, Kou G, Wang H, Zhou Y. Identification and Functional Speculation of Genes Related to Sex Pheromone Synthesis Expressed in the Gonads of Female Gynaephora qinghaiensis (Lepidoptera: Lymantriidae). Genes. 2025; 16(9):1046. https://doi.org/10.3390/genes16091046

Chicago/Turabian Style

Liu, Zhanling, Shujing Gao, Haibin Han, Xiaorui Wang, Guixiang Kou, Haishun Wang, and Yuantao Zhou. 2025. "Identification and Functional Speculation of Genes Related to Sex Pheromone Synthesis Expressed in the Gonads of Female Gynaephora qinghaiensis (Lepidoptera: Lymantriidae)" Genes 16, no. 9: 1046. https://doi.org/10.3390/genes16091046

APA Style

Liu, Z., Gao, S., Han, H., Wang, X., Kou, G., Wang, H., & Zhou, Y. (2025). Identification and Functional Speculation of Genes Related to Sex Pheromone Synthesis Expressed in the Gonads of Female Gynaephora qinghaiensis (Lepidoptera: Lymantriidae). Genes, 16(9), 1046. https://doi.org/10.3390/genes16091046

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop