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Transcriptome sequencing of Antheraea pernyi antennae for identification of olfactory-related genes

BMC Genomics volume 26, Article number: 499 (2025) Cite this article

Abstract

Background

In insects, the olfactory system governs physiological and behavioral processes by detecting various odorous molecules. Despite its economic importance and adaptability, the olfactory mechanism of Antheraea pernyi remains insufficiently understood, limiting its potential for pest management and as a model organism. Hence, we aimed to conduct transcriptome sequencing to explore olfactory-related genes in the antennae, serving as the most important olfactory organ in adult A. pernyi.

Results

Based on the datasets, 1184 differently expressed genes (DEGs), including 484 upregulated and 700 downregulated genes, were identified by comparing the transcriptome profiles of the male and female antennae of A. pernyi. Moreover, 20, 7, 30, 11, and 2 candidate genes encoding odorant‐binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs), respectively, involved in pheromone perception, odor binding, pesticide resistance, and growth and development regulation were screened, and most of which were expressed in both male and female antennae while the expression levels of these candidate genes varied significantly between males and females. Multiple sequence alignment indicated that the six OBPs exhibited typical characteristics, containing six conserved Cys residues with the sequence of C1-X26-30-C2-X3-C3-X41-42-C4-X8-10-C5-X8-C6. All CSPs followed a highly conserved pattern with four Cys residues arranged with an exact spacing of C1-X6-C2-X18-19-C3-X2-C4. Different numbers of transmembrane domains were found in ORs, IRs, and SNMPs. In addition, several DEGs involve signal transduction underlying chemoreception were also identified from the transcriptome data, including guanine nucleotide-binding protein (G protein), cGMP-dependent protein kinase (PKA), calmodulin-A (CaM-A), mitogen-activated protein kinase 1 (MAPK1), and phospholipase D2 (PLD2).

Conclusion

This study enriches the olfactory gene database of A. pernyi, providing insights into olfactory mechanisms crucial for mating and pest control, with implications for enhancing breeding strategies and ensuring the sustainability of the silk industry. These findings may serve as a theoretical foundation for a better understanding of the olfactory mechanisms of A. pernyi.

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Introduction

The olfactory system in insect antennae is essential for regulating physiological and behavioral actions by recognizing chemical odor molecules in the environment that are related to multiple olfactory proteins, including odor-binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs) [1].

Lepidopteran OBPs can be divided into three categories: pheromone-binding proteins (PBPs), general odorant-binding proteins (GOBPs), and antennal-binding proteins (ABPs). Based on the number of conserved Cys residues, OBPs are divided into the following categories: classical subgroups possessing six highly conserved Cys residues with a three-residue gap between the second and third Cys residues and an eight-residue gap between the fifth and sixth Cys residues; and non-classic subgroups with either more or fewer than six Cys sites [2]. Both OBPs and CSPs are soluble proteins with the N-terminal signal peptide removed during processing [3] and play similar roles in binding and transferring chemical pheromones [4]. CSPs contain four conserved Cys residues that form two disulfide bonds to maintain their structural stability [5]. ORs, GRs, and IRs belong to the chemoreceptor family and have different numbers of transmembrane domains in insects. ORs and GRs exhibit a similar membrane topology to the intracellular N-termini and extracellular C-termini [6], while a contrasting structure exists in IRs [7]. SNMPs are highly homologous to the human proteinCD36 which exhibits a large extracellular loop containing several Cys residues [8].

With the advancements in high-throughput sequencing technology, RNA-seq has been widely used to mine olfactory-related genes in the antennae of multiple lepidopteran species, with 71, 42, 171, 157, 85, and 88 genes identified in Spodoptera frugiperda [9], Histia Rhodope [10], Lobesia botrana [11], Spodoptera exigua [12], Dendrolimus houi [13], and Dendrolimus kikuchii [13], respectively. Antheraea pernyi is a typical lepidopteran insect that is important for the silk industry and accounts for a high proportion of agricultural economic output. Unlike the domesticated silkworm, Bombyx mori, A. pernyi lives in the wild and can adapt to harsh environments, resulting in a relatively sensitive olfactory system for feeding, mating, oviposition, and detecting harmful odor molecules. As an emerging model organism [14], the olfactory system of A. pernyi can be used as a reference for the prevention and control of agricultural and forestry lepidopteran pests. Hence, a comprehensive understanding of the olfactory mechanism would be beneficial in elucidating the biological characteristics of A. pernyi as an economically viable insect.

In this study, transcriptome sequencing was performed for A. pernyi antennae to identify the olfactory-related genes encoding OBPs, CSPs, SNMPs, ORs, GRs, and IRs, which differential expression analyses were carried out between the male and female antennae. Then bioinformatics analysis was conducted for further characterizing these genes. Overall, the results will enrich the gene datasets related to A. pernyi olfaction and provide a theoretical foundation for further research into the mechanisms underlying chemoreception in A. pernyi.

Materials and methods

Collection of antennae samples

Adult A. pernyi (Xuanda strain) used for sample preparation was obtained from the Shandong Sericulture Research Institute (Yantai, China). The antennae samples were collected at night after the moths emerging from their cocoons. Newly emerged male and female moths were separated into two groups (referred to as M and F, respectively; Fig. 1A). Each group consisted of 150 moths and was set up with three biological replicates (referred to as M1, M2, M3 and F1, F2, F3, respectively); thus, 50 individuals were included in each replicate. The antennae samples were collected separately from each replicate and then stored at −80 °C for further use.

Fig. 1
figure 1

Statistical analysis of the transcriptomic data of A. pernyi antennae. A Male and female antennae of A. pernyi moth. B Number of identified genes from each sample in the M and F group. C PCA of the samples from the M and F group. D Correlation test between the samples in the M and F group. E Number of identified genes separately annotated in Swiss-Prot, Nr, GO, and KEGG databases. M and F in (B), (C), and (D) represent the male and female groups, respectively

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RNA extraction and library construction

Total RNA was extracted from the antennae samples using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. RNA purity and quantity were analyzed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Sequencing library was constructed separately for each replicate using the VAHTS Universal V5 RNA-seq Library Prep Kit following the manufacturer’s protocol.

RNA-Seq and data analysis

Illumina sequencing was also performed separately for each replicate by OE Biotech Co., Ltd. (Shanghai, China) with 150-bp paired-end reads generated on an Illumina Novaseq 6000 platform. The clean reads were obtained by filtering the low-quality reads by processing the raw reads in FASTQ format using a FASTQ preprocessor [15] and then mapping to the reference genome database of A. pernyi (https://bigd.big.ac.cn/gwh/, accession number: GWHABGR00000000) [16] using HISAT2 [17]. The FPKM measure [18] was used to calculate the expression of each gene, for which read counts were determined by HTSeq-count [19]. The biological duplication of samples was evaluated via principal component analysis (PCA) using R (v 3.2.0) (https://cloud.r-project.org/). Gene annotation was performed by aligning the gene sequence with the Swiss-Prot database and the NCBI non-redundant (NR) database to obtain the gene symbol and description, respectively, with a threshold of < 1e-5. GO classification was performed using the mapping relation between Swiss-Prot and GO term. The genes were mapped to the KEGG database to annotate potential pathways.

Analysis of DEGs

DESeq2 software was used for differential expression analysis [20]. Genes were considered significantly differentially expressed when foldchange ≥ 2 or ≤ 0.5 with a Q value < 0.05 was noted upon comparison of the two groups. The expression patterns of DEGs in different groups and samples were determined by hierarchical cluster analysis using R (v 3.2.0) (https://cloud.r-project.org/). GO function and KEGG pathway enrichment analyses of DEGs were conducted to screen for significantly enriched terms.

Identification and bioinformatics analysis of olfactory-related genes

Olfactory-related genes encoding OBPs, PBPs, ABPs, GOBPs, CSPs, OLRs, ORs, IRs, GRs, and SNMPs were screened from the transcriptome data of A. pernyi antennae. SignaIP 5.1, an online software package (http://www.cbs.dtu.dk/services/SignalP/), was used to predict the signal peptides. Sequence alignment was performed using ClustalX2 software (http://www.clustal.org/clustal2/), and the alignment results were analyzed using GeneDoc software (https://genedoc.software.informer. com/). Transmembrane (TM) domain prediction was performed using the online Deep TMHMM tool (https://services.healthtech.dtu.dk/service.php?DeepTMHMM-1.0) with default parameters. Phylogenetic analysis was performed in MEGA 7.0 software and the tree were constructed using neighbor-joining method with n = 1000 bootstrap replicates [21].

Verification of RNA-Seq data using qRT-PCR

Total RNA used for RNA-Seq was applied for cDNA synthesis, and subsequent qRT-PCR validation was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, USA) with a 20-μL reaction system using TB Green® Premix Ex Taq™ II (Takara, China) following the manufacturer’s protocol. The reaction procedure was as follows: pre-denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, and annealing/extension at 60 °C for 30 s. All reactions were performed in triplicate. The relative transcript level of each gene was quantified using the Ct value obtained from the reaction. The housekeeping gene Apactin1 of A. pernyi was used as a normalized internal control. Primers (Table S1) for the selected genes were designed based on their coding sequences reported in the online genome database of A. pernyi [16].

Results

Statistical analysis of A. pernyi antennae transcriptome data

The transcriptome data of A. pernyi antennae from the M and F groups generated 279.29 M clean reads after filtering for quality from 279.58 M raw reads, with the Q30 base ranging from 96.98% to 97.33% and the average GC content of 37.89% (Table 1). The match ratio ranged from 64.37% to 77.50%, as observed upon aligning the clean reads of each sample to the reference genome (Table 1). After assembling the short-read sequences, 13,869, 13,838, 13,855 and 13,556, 13,651, 13,513 genes were produced in each sample of the M and F groups, respectively, and 16,458 genes were obtained after removing redundant entries from all samples (Fig. 1B, Table S2). High intragroup repeatability and significant intergroup differences were observed using PCA and correlation tests (Fig. 1C & D). A total of 9874, 14,934, 9666, and 4503 genes were annotated separately in the Swiss-Prot, Nr, GO, and KEGG databases, respectively (Table 1, Fig. 1E).

Table 1 Statistical analysis of the transcriptome data of A. pernyi antennae
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Differential expression analysis

Based on the screening criteria for significant differential expression, 1184 DEGs, including 484 upregulated (Table S3) and 700 downregulated genes (Table S4), were identified between the male and female antennae of A. pernyi using the M group as a control (Fig. 2A). The expression patterns of DEGs between each antennae sample in the M and F groups were determined using hierarchical cluster analysis (Fig. 2B). The results of GO enrichment analysis indicated that 715 DEGs were enriched in 1746 GO terms, which primarily involved integral component of membrane, plasma membrane, extracellular region, cytoplasm, multicellular organism development, proteolysis, metal ion binding, structural constituent of cuticle, and ATP binding (Fig. 2C, Table S5). KEGG enrichment analysis showed that 268 DEGs were enriched in 281 pathways, of which protein processing in endoplasmic reticulum, longevity-regulating pathway, peroxisome, glutathione metabolism, and metabolism of xenobiotics by cytochrome P450 were the most enriched (Fig. 2D, Table S6).

Fig. 2
figure 2

Differential expression analysis between male and female antennae of A. pernyi. A DEGs are displayed in the volcano plot. The X- and Y-axes separately represent the fold changes in gene expression and the statistical significance of changes in gene expression. Different colored dots represent genes with different expression patterns. B Hierarchical cluster analysis of the DEGs. Different colors represent different levels of gene expression in each sample. The color gradient from red to blue represents the levels of expression of genes from high to low. C GO enrichment analysis of the DEGs. The top 15 GO terms with significant enrichment in cellular component, molecular function, and biological process are presented. D KEGG enrichment analysis of the DEGs. The top 45 pathways with significant enrichment are provided

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Screening and analysis of the expression patterns of olfactory-related genes

A total of 70 olfactory-related genes were screened based on gene annotations from the transcriptome data (Table 2). Among the 20 OBPs, 2 PBPs, 1 ABP, 8 OBPs, and 9 GOBPs were classified (Table 2). A total of 7, 30, 11, and 2 genes encoding CSPs, ORs, IRs, and SNMPs, respectively, were identified (Table 2). Hierarchical clustering analysis showed that most olfactory-related genes were expressed in both male and female antennae and that the expression levels of all these genes varied significantly between the two groups (Fig. 3). Several genes encoding ORs, including ApOR (GWHGABGR010755, GWHGABGR005934, GWHGABGR013978) and ApOR23a (GWHGABGR021059), were highly expressed in male antennae compared to those in females, whereas ApOR20 (GWHGABGR011421), ApOR (GWHGABGR021062), and ApOR30a (GWHGABGR011390) displayed high expression levels in female antennae (Fig. 3). In addition, a series of genes such as ApOBP31 (GWHGABGR003019), ApOBP32 (GWHGABGR003119), ApOBP38 (GWHGABGR019142), ApOR20 (GWHGABGR011405), ApIR75a (GWHGABGR012255), and ApIR3 (GWHGABGR014182) exhibited relatively low expression levels in the two groups (Fig. 3).

Table 2 Olfaction related genes identified in the adult antennal transcriptome of A. pernyi
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Fig. 3
figure 3

Hierarchical clustering analysis of olfactory-related gene expression between A. pernyi male and female antennae. The color gradient from red to blue represents the levels of expression of genes from high to low

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Sequence characteristics of the olfactory-related genes

Sequence analysis showed that the full length of the olfactory related genes ranged from 51 to 834 aa with 62 complete open reading frames and 16 out of the OBPs and CSPs with signal peptide cleavage site between 16 and 25 aa (Table 2). The results of amino acid multiple sequence alignment indicated low homology among the OBPs, several of which, including ApPBP1 (GWHGABGR012197), ApPBP3 (GWHGABGR012195), ApABP (GWHGABGR018897), ApGOBP1 (GWHGABGR012186), ApOBP38 (GWHGABGR019142), and ApGOBP2 (GWHGABGR012194), exhibited typical characteristics comprising six conserved Cys residues, with the sequence of C1-X26–30-C2-X3-C3-X41–42-C4-X8–10-C5-X8-C6, while ApOBP6 (GWHGABGR001129) and ApGOBP70 (GWHGABGR004634) missing C1 (Fig. 4A). In addition, all CSPs followed a highly conserved pattern with four Cys residues arranged with an exact spacing of C1-X6-C2-X18–19-C3-X2-C4 (Fig. 4B). TM domain prediction showed that eight and seven TM domains were found in ApOR20 (GWHGABGR011405) and ApOR (GWHGABGR021060), respectively, whereas 0–6 domains were detected in other ORs. Among the IRs, three TM domains were observed in ApIR25a (GWHGABGR003112), ApIR93a (GWHGABGR005160), and ApIR4 (GWHGABGR005485), and four were identified in ApIR3 (GWHGABGR014182 and GWHGABGR014192). Besides, ApSNMP2 (GWHGABGR005182) was displayed with 2 TM domains.

Fig. 4
figure 4

Sequence alignment of the identified ApOBPs (A) and ApCSPs (B). The conserved Cys residues are highlighted by red wireframes

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Validation of reliability of RNA-Seq data using qRT-PCR

To validate the accuracy of RNA-Seq data, 14 candidates with seven upregulated and seven downregulated genes were selected from the DEGs for qRT-PCR analysis. The results showed expression patterns at the transcriptional level similar to those in the transcriptome of the selected genes (Fig. 5), further confirming the validity of the transcriptome data.

Fig. 5
figure 5

Validation of transcriptome data by qRT-PCR. The X- and Y-axes separately represent the selected genes and the relative expression. The selected genes include pheromone binding protein 1 (GWHGABGR012197), pheromone binding protein 3 (GWHGABGR012195), odorant-binding protein 6 (GWHGABGR001129), odorant-binding protein 14 (GWHGABGR014086), general odorant-binding protein 2 (GWHGABGR012194), general odorant-binding protein 56a (GWHGABGR017175), chemosensory protein (GWHGABGR012364, GWHGABGR001096), olfactory receptor 23 (GWHGABGR008832), olfactory receptor 20 (GWHGABGR011405), odorant receptor 44 (GWHGABGR012233), odorant receptor 53 (GWHGABGR012613), ionotropic receptor 93a (GWHGABGR005160), sensory neuron membrane protein 2 (GWHGABGR000255)

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Discussion

During olfactory recognition in insects, odorant molecules enter the olfactory sensilla in the antennae via micropores and interact with ORs or IRs. The molecules are transported via the sensilla lymph to the dendrite membrane upon binding with OBPs or CSPs, thus activating receptor neurons and converting chemical signals into electrical signals [3, 22]. Based on the antennal transcriptome data, 70 olfactory-related genes were identified in this study except the GRs involved in detecting sugars, bitter compounds, and carbon dioxide [23, 24], which may be attributed to that GRs are mainly expressed in the maxillary palps instead of antennae [25] and the moths no longer feeds during the adult stage of A. pernyi which primary physiological process is reproduction.

OBPs are key factors involved in odor reception in the olfactory system of insects and are mainly expressed in olfactory organs such as the antennae and labia [26]. As the first medium for insects to communicate with the external environment, OBPs carry hydrophobic odor molecules through the aqueous lymph to odor receptors, thereby guiding the biochemical processes of insects to odors. PBPs are a subfamily of OBPs that selectively bind to sex pheromones in water-soluble hemolymph while filtering out other odor molecules [3]. In this study, the expression level of ApPBP1 (GWHGABGR012197) was higher in male antennae than that in females, whereas a contrasting trend was observed in ApPBP3 (GWHGABGR012195) expression (Figs. 3 & 5). This expression pattern corresponds to that of PBPs in males and females of Spodoptera litura [27]. In S. litura, the PBPs also exhibited a very different sex-biased expression in adult antenna with male-biased for SlitPBP1 which was supposed to play critical roles in the perception of female sex pheromones, while female-biased for SlitPBP3 with very weak binding affinities to sex pheromones [27]. Meanwhile, in Agrotis ipsilon, AipsPBP1 showed high binding affinities with the major female sex pheromone components Z7-12: Ac and Z9-14: Ac, while AipsPBP3 displayed low binding affinities with them [28]. We speculated that the identified PBPs from the transcriptome data may display sexual dimorphism in A. pernyi, and ApPBP1 with high expressions in male antennae may play essential roles in perceiving female sex pheromones, while ApPBP3 expressed highly in female antennae may have other functions. GOBPs predominantly perceive general odorants, such as plant volatiles or pheromones [29]. Among the identified GOBPs, ApGOBP19 d (GWHGABGR014092) and ApGOBP56a (GWHGABGR017175) were preferentially expressed in the female antennae, indicating that they may participate in recognizing and binding host plant odorants, thereby helping females select oviposition sites [30]. ABPs are specifically expressed in the antennae, yet they remain to be further functionally characterized [3]. Phylogenetic analysis showed that ApABPX (GWHGABGR018897) clustered with the ABPXs from M. sexta and B. mori (Fig. 6A), indicating a relatively close genetic relationship between these ABPs.

Fig. 6
figure 6

Construction of phylogenetic trees for ApABPX and ApCSPs. A Phylogenetic analysis of ApABPX and selected ABPs from several lepidoptera insects including Chilo suppressalis (CsABPX), Manduca sexta (MsABPX), Peridroma saucia (PsABP), Heliothis virescens (HvABPX), Agrotis ipsilon (AiABPX1), Argyresthia conjugella (AcABPX), Amyelois transitella (AtABPX), Spodoptera exigua (SeABP), Danaus Plexippus (DpABPX), Bombyx mori (BmABPX), Plutella xylostella (PxABP). B Phylogenetic analysis of ApCSPs and selected lepidoptera insect CSPs including Bombyx mori (BmCSP1/2/10/15), Clostera restitura (CrCSP1/2/3/4/5/6/7), Plutella xylostella (PxCSP1/2/3/4/5/11), Helicoverpa armigera (HaCSP8/9/10/11/12), Pieris rapae (PrCSP7/16/18/20), Spodoptera litura (SlCSP3). Neighbor-joining tree is constructed using MEGA 7 software with 1000-fold bootstrap resampling. The numbers at the nodes of the branches represent the level of bootstrap support for each branch. The accession numbers of the ABPs and CSPs were marked in the brackets

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Although share certain similarities with OBPs in their roles, CSPs participate in the olfactory recognition process of insects by binding to non-volatile chemical pheromones [31]. Unlike the expression patterns of OBPs, CSPs are widely expressed in olfactory and non-olfactory sensory organs with multiple physiological functions involved in regulating reproduction, growth and development, and physiological rhythms [32]. In Clostera restitura, CrCSP1/2/3/4/5/6/7/8 regulate growth, development, reproduction, and the perception of chemical molecules such as plant volatile substances and sex pheromones [33]. In B. mori, BmCSP1 is associated with insecticide resistance [34]. In this study, the close phylogenetic relationship between ApCSP (GWHGABGR001096) and CrCSP7, as well as between ApCSP15 (GWHGABGR001108) and CrCSP5 (Fig. 6B), may imply their possible roles in chemo sensing, odor binding, pheromone perception, and regulation of the lifecycle of A. pernyi. ApCSP (GWHGABGR012364) and ApCSP9 (GWHGABGR001097) were hypothesized to be associated with pesticide resistance, given their homology with BmCSP1 (Fig. 6B).

ORs are the main olfactory receptors distributed in the dendritic membrane of olfactory sensory neurons and play essential roles in detecting and recognizing a vast array of chemically diverse odorants in insects [35, 36]. In the current study, 30 ApORs were screened from the transcriptome. However, this number was lower than that in Helicoverpa armigera (81 ORs) [37], Tuta absoluta (58 ORs) [38], and Phthorimaea operculella (72 ORs) [39]. This discrepancy may be attributed to behavioral and ecological diversity among different lepidopteran species. Low sequence similarity was observed among ApORs (Fig. S1), suggesting their respective characteristics in recognizing different odors. Meanwhile, the identified ORs displayed different expression patterns between male and female A. pernyi (Fig. 3). In A. polyphemus, the OR (ApolOR1) is predominantly expressed in the pheromone‐sensitive sensilla trichodea of the male antennae, and is concluded to be involved in perception of the sex pheromone [40]. In H. armigera, HarmOR56 expressed specifically in sensilla trichodea on female antennae is demonstrated to mediate oviposition deterrence by detecting oviposition-deterring pheromones [41]. We speculated that the ApORs with high expression levels in male antennae may be involved in the regulation of the male moths of A. pernyi recognizing and binding the female pheromones, while the individuals highly expressed in females were supposed to facilitate the female A. pernyi to recognize the odor molecules in the environment thus select oviposition sites. IRs are the second class of olfactory receptors that are mainly expressed in sensory neurons without ORs and GRs [42]. A total of 11 ApIRs were identified in A. pernyi antennae including two ApIR25as (GWHGABGR003112 and GWHGABGR019950) conserved among lepidopteran IR25as (Fig. S2) possessing an extracellular amino-terminal domain that is contained in ionotropic glutamate receptors [43], which was thought to act as co-receptors since IR25as are typically co-expressed along with other IRs in insects [44]. In addition, two genes (GWHGABGR000255 and GWHGABGR005182) encoding ApSNMP2 were identified, which may be involved in the recognition and transportation of lipophilic odor molecules, especially pheromones [45].

Meanwhile, based on KEGG enrichment analysis, a series of DEGs related to regulating signal transduction in Ras, Rap1, Calcium, cGMP-PKG, and cAMP signaling pathways which were believed to be involved in olfactory sensory transduction in both vertebrates and insects [46] were identified from the transcriptome data (Table S6, Fig. 7A), such as guanine nucleotide-binding protein (G protein, GWHGABGR007673), cGMP-dependent protein kinase (PKA, GWHGABGR009163), calmodulin-A (CaM-A, GWHGABGR011096), mitogen-activated protein kinase 1 (MAPK1, GWHGABGR010342), and phospholipase D2 (PLD2, GWHGABGR021202), and interactions were observed between these proteins according to the protein–protein interaction network exhibition (Fig. 7B). G proteins are key components of the GPCR signaling pathway and act as molecular switches inside the cells, which involves transmitting a variety of stimuli from extracellular to intracellular [47]. MAPK1, PKG, and PLD are the critical regulators of cell signal transduction. In addition, the cGMP-PKG signaling pathway plays important roles in regulating multiple behaviors, such as feeding, learning, and memory, in the insect nervous system [48, 49] and is involved in modulating the activity of the extracellular stimuli-responsive kinase pathway [50]. It has been demonstrated that CaM participating in the calcium signaling pathway could regulate the olfactory responses, including sensitization of ORs and odor localization by targeting co-receptors in Drosophila melanogaster [51]. The higher expression levels of the G protein, PKG, MAPK1, and PLD2 in male antennae and CaM-A in females (Fig. 7A) may indicate different capabilities in processing the transduction of different external stimuli into cells between male and female A. pernyi.

Fig. 7
figure 7

Hierarchical clustering analysis (A) and the protein–protein interaction network (B) for the DEGs involved in olfactory transduction between male and female antennae of A. pernyi. The color gradient from red to blue in (A) represents the levels of expression of genes from high to low. The green and red dots in (B) separately represent the up-regulated and down-regulated genes

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In this study, the number of identified olfactory-related genes may not represent the complete set of gene families involved in the olfactory recognition of A. pernyi. Certain genes may have been missed during the RNA-seq process due to low expression levels or lack of expression in the tested transcriptome. A few olfactory genes with significant differences with respect to the known query gene sequences may not have been detected by transcriptome analysis using homology search methods [52]. In addition, A. pernyi live in wild and harsh condition, the environmental factors (such as temperature and humidity) or dietary factors (such as exposure to host plants) may also affect olfactory gene expression.

Recently, olfactory genes have been considered as important targets for pest control since their importance for insects in recognizing odors in the external environment [53]. In the future, further functional studies of the olfactory-related genes in the present study would provide references for investigating target genes for controlling the pests that cause damage to oaks, which are the primary source of food for A. pernyi. Meanwhile, the olfactory recognition by antennae between male and female moths is crucial for the courtship and mating behavior which promise the life circle completion thus ensure the silk industry continuity of A. pernyi. Overall, the findings of this study enrich the gene database related to the olfactory system, and also provide a foundation for better understanding the mechanisms underlying olfactory recognition, which could be further served as theoretical reference for well-breeding of A. pernyi.

Conclusions

By transcriptome sequencing for the adult A. pernyi antennae, we identified 70 olfactory-related genes including 20, 7, 30, 11, and 2 candidates encoding OBPs, CSPs, ORs, IRs, and SNMPs, respectively, which were suggested to involve pheromone perception, odor binding, pesticide resistance, and growth and development regulation. Six OBPs were categorized into classical subgroup containing six conserved Cys residues with the sequence of C1-X26–30-C2-X3-C3-X41–42-C4-X8–10-C5-X8-C6. All CSPs followed the sequence pattern of C1-X6-C2-X18–19-C3-X2-C4 with four highly conserved Cys residues. The number of transmembrane domains were varied among ORs, IRs, and SNMPs. Meanwhile, several DEGs including G protein, PKA, CaM-A, MAPK1, and PLD2 were supposed to be involved in signal transduction underlying chemoreception of A. pernyi by differential expression analysis for the transcriptome profiles between the male and female antennae. In general, the results of the present study would provide theoretical basis for further elucidating the olfactory mechanisms in A. pernyi.

Data availability

The raw datasets generated during the current study are available in the Gene Expression Omnibus repository under accession number GSE283343.

Abbreviations

OBPs:

Odor-binding proteins

CSPs:

Chemosensory proteins

ORs:

Odorant receptors

GRs:

Gustatory receptors

IRs:

Ionotropic receptors

SNMPs:

Sensory neuron membrane proteins

PBPs:

Pheromone-binding proteins

GOBPs:

General odorant-binding proteins

ABPs:

Antennal-binding proteins

DEGs:

Differentially expressed genes

GO:

Gene Ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

NR:

NCBI non-redundant

TM:

Transmembrane

PCA:

Principal component analysis

G protein:

Guanine nucleotide-binding protein

PKA:

CGMP-dependent protein kinase

CaM-A:

Calmodulin-A

MAPK1:

mitogen-activated protein kinase 1

PLD2:

Phospholipase D2

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Funding

This work was supported by Youth Innovation Team Project in Institutions of Higher Learning of Shandong Province (2022 KJ259), and Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences (CXGC2022B02).

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  1. College of Biology and Oceanography, Weifang University, No.5147 Dongfeng East Street, Weifang, 261061, China

    Xueting Liu, Shuwei Ma, Xinxue Zhang, Xue Li & Guobao Wang

  2. Shandong Sericulture Research Institute, Shandong Academy of Agricultural Sciences, Yantai, 264002, China

    Lei Nie

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Contributions

XTL: Formal analysis; Project administration; Writing‐original draft. SWM: Software; Data curation. XXZ: Investigation. XL: Supervision. LN: Resources; Visualization. GBW: Conceptualization; Funding acquisition; Methodology; Writing‐review & editing.

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Liu, X., Ma, S., Zhang, X. et al. Transcriptome sequencing of Antheraea pernyi antennae for identification of olfactory-related genes. BMC Genomics 26, 499 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11698-4

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BMC Genomics

ISSN: 1471-2164

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