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

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-X_26-30-C2-X₃-C3-X_41-42-C4-X_8-10-C5-X₈-C6. All CSPs followed a highly conserved pattern with four Cys residues arranged with an exact spacing of C1-X₆-C2-X_18-19-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.

Keywords: Antheraea pernyi; Antennae; Bioinformatics analysis; Olfactory-related genes; Transcriptome.

Fig. 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

Fig. 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

Fig. 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

Fig. 4

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

Fig. 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)

Fig. 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

Fig. 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