Genome-Wide Analysis of Odorant-Binding Proteins and Chemosensory Proteins in the Bean bug Riptortus pedestris

Abstract

Insects have sensitive olfactory systems to interact with environment and respond to the change in host plant conditions. Key genes in the system can be potential targets for developing new and efficient pest behaviour control methods. Riptortus pedestris is an important soybean pest in East Asia and has caused serious damage to the soybean plants in Huang-Huai-Hai region of China. However, the current treatment of pests is dominated by chemical insecticides and lacks efficient sustainable prevention and control technologies. In this study, we identified 49 putative odorant-binding proteins (OBPs) (43 were new genes) and 25 chemosensory proteins (CSPs) (17 were new genes) in R. pedestris genome. These OBP and CSP genes are clustered in highly conserved groups from other hemipteran species in phylogenetic trees. Most RpedOBPs displayed antennal-biased expression. Among the 49 RpedOBPs, 33 were significantly highly expressed in the antennae, including three male-biased and nine female-biased. While many RpedCSPs were detected both in the antennae and in non-antennal tissues, only 11 RpedCSPs displayed antennal-biased expression, in which four RpedCSPs were male-biased and five RpedCSPs were female-biased. Some OBP and CSP genes showed sex-biased expression profiles. Our results not only provide a foundation for future exploration of the functions of RpedOBPs and RpedCSPs but also aid in developing environmentally friendly insecticides in the future.

Introduction

Insects have a sensitive olfactory system, which enhances their ability to adapt to the complex external environment to accurately complete behavioural reactions such as feeding, mating, and avoiding natural enemies (Leal, 2013; Robertson, 2019). A large amount of studies on the molecular mechanisms of insect olfactory systems have found that the accurate operation of the system is inseparable from the participation of olfactory genes such as odorant-binding proteins (OBPs), chemosensory proteins (CSPs), and olfactory receptors (ORs) (Zhang et al., 2013; Glaser et al., 2015; Li et al., 2015; Elfekih et al., 2016; Larter et al., 2016; Paula et al., 2016; Renou and Anton, 2020; Rihani et al., 2021).

OBPs and CSPs are located in the lymph of insect antennal sensilla, and can accurately bind to external odorants and transport them to the corresponding ORs, ionotropic receptors (IRs) or gustatory receptors (GRs) to initiate behavioural responses. Therefore, the action of OBPs and CSPs is the first step in activating insect olfactory perception (Zhou, 2010; Dani et al., 2011; Pelosi et al., 2018; Rihani et al., 2021), which can be used as potential target genes to develop new and efficient pest behaviour control agents. Since the discovery of the first OBP and CSP in Antheraea polyphemus (Vogt and Riddiford, 1981) and Drosophila melanogaster (McKenna et al., 1994), respectively, a large number of OBPs and CSPs have been confirmed and studied in different insects (Latorre-Estivalis et al., 2021; Li et al., 2021; He et al., 2022).

These two types of genes have been the subject of studies on evolution, molecular structure, tissue distribution, and functional analysis (Spinelli et al., 2012; Pelosi et al., 2018; Li et al., 2021). It is now clear that both OBPs and CSPs are soluble small-molecule proteins. Generally, OBPs use six positionally conserved cysteines to form three interlocking disulfide bridges that stabilise the three-dimensional structure of the proteins. OBPs can be divided into three distinct subfamilies: classic OBPs (six conserved cysteines), minus-C OBPs (four conserved cysteines), and plus-C OBPs (more than six conserved cysteines) (Zhou, 2010; Schultze et al., 2012; Spinelli et al., 2012; Li et al., 2013; He and He, 2014; Zhang et al., 2017b). Compared with OBPs, CSPs are smaller, display greater evolutionary conservation, and have only two disulfide bridges with four conserved cysteines (Maleszka and Stange, 1997; Bohbot et al., 1998; Pelosi et al., 2005; Zhang et al., 2014; Zhu J. et al., 2016; Yi et al., 2017; Pelosi et al., 2018). Additionally, OBPs are often specifically or highly expressed in the antennae and are mainly involved in odorant recognition (Krieger et al., 1996; Sengul and Tu, 2010; Missbach et al., 2015; Zhang et al., 2017a), whereas many CSPs are expressed in the antennal and other non-olfactory organs (Pelosi et al., 2005; Vogt, 2005; Zhang et al., 2013; Zhang et al., 2014; Zhang L.-W. et al., 2016; Chen G.-L. et al., 2018). This suggests that CSPs may play both olfactory and non-olfactory roles in insects.

The bean bug Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) is an important soybean pest in East Asia (Xu et al., 2021) and has a wide range of hosts. In addition to soybean, it can also harm Cruciferae, Gramineae, and other crops (Huang et al., 2021). R. pedestris damages soybeans by sucking, which results in poor growth and development of plants and insufficient pods (Chen J. H. et al., 2018). In recent years, R. pedestris has caused serious damage to the soybean plants in Huang-Huai-Hai region of China and has greatly reduced the yield of soybean, or lost the harvest. It has now become an important pest in China’s summer soybean producing areas (Chen J. H. et al., 2018; Li et al., 2019). However, the current treatment of pests is still dominated by the use of chemical insecticides and lacks efficient green prevention and control technologies. This has become a growing consensus that the development of green and efficient behaviour disruptors is a popular research direction based on the exploration of insect olfactory systems (Sun et al., 2011; Cui and Zhu, 2016; Qin et al., 2020). In this study, we identified 49 OBPs and 25 CSPs in the R. pedestris genome and found that these genes were clustered in highly conserved groups comprising OBP and CSP genes from other hemipteran species, respectively. The gene expression profiles of OBPs and CSPs showed that most RpedOBPs displayed antennal-biased expression, while many RpedCSPs were highly expressed in the antennae and non-antennal tissues, and some genes showed sex-biased expression. These results will help us identify the functions of RpedOBPs and RpedCSPs and develop environmentally friendly insecticides against this pest in the future.

Materials and Methods

Insect Rearing and Tissue Extraction

R. pedestris were fed with bean sprouts and maintained at a temperature of 26 ± 1°C under a 14:10 h light:dark photoperiod. Female and male adults, as well as larvae, were placed in insect cages for reproduction. Fifth instar larvae were collected and raised separately to obtain three-day-old virgin adults. The heads, thoraxes, abdomens, legs, wings, and antennae of virgin adults were collected. All collected samples were immediately frozen in liquid nitrogen and stored at ˗80°C for future use.

Sequence Data Collection and Analyses

Genome data, gene, protein, RNA and annotation files of R. pedestris were obtained from the Genome Warehouse (https://ngdc.cncb.ac.cn/gwh/Assembly/18849/show). We used the protein data of R. pedestris and blasted with different database of Nr (Non-Redundant Protein Sequence), Nt (nucleotide sequence database), UniProt (The Universal Protein Resource), KOG (Clusters of orthologous groups for eukaryotic complete genomes), eggNOG (evolutionary genealogy of genes: No-supervised Orthologous Groups), Interpro (the integrative protein signature), GO (Gene Ontology), and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases (Huang et al., 2021), so R. pedestris proteins were annotated based on homology (These data were derived from a previous genome paper). We acquired the genes and ORF sequences of OBPs and CSPs by corresponding protein ID using R. pedestris genomic database. To ensure the accuracy of gene sequences, we used OBP and CSP genes to blast with Nr (Non-Redundant Protein Sequence) database in NCBI BLAST (http://blast.ncbi.nlm.nih.gov/). We also selected some genes (RpedOBP8, RpedOBP37, RpedCSP5) to clone and sent to sequencing to verify the correctness of the sequences. A total of 49 OBPs and 25 CSPs were obtained based on the similarity analysis. Putative N-terminal signal peptides of all OBPs and CSPs were predicted using SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/), (Petersen et al., 2011).

RNA Isolation and cDNA Synthesis

Total RNA was extracted using a FastPure^® Cell/Tissue Total RNA Isolation Kit (Vazyme, Nanjing, China) following the manufacturer’s instructions, and RNA quality was checked using a spectrophotometre (NanoDrop^TM 2000; Thermo Fisher Scientific, United States). Single-stranded cDNA templates were synthesised from 1 μg of total RNA from various tissue samples using the MonScript™ RTlll Super Mix with dsDNase (Two-Step) (Monad, Shanghai, China).

Quantitative Real Time-Polymerase Chain Reaction

The qRT-PCR primers for 49 OBPs and 25 CSPs (Supplementary Table S1) were designed using Beacon Designer 7.9 (PREMIER Biosoft International, CA, United States). Expression profilings of RpedOBPs and RpedCSPs were performed using qRT-PCR in a LightCycler^® 96 (Roche, Switzerland) with a mixture of 5 μL MonAmp™ ChemoHS qPCR Mix (Monad, Shanghai, China), 0.2 μL of each primer (10 μM), 2.5 ng of sample cDNA, and 3.6 μL of sterilised ultrapure H₂O. The reaction program was as follows: 10 min at 95°C, 40 cycles at 95°C for 10 s, 60°C for 10 s, and 72°C for 30 s. The results were analysed using LightCycler® 96 SW 1.1. Then, fluorescence was measured over a 55–95°C melting curve to detect a single gene-specific peak and to check the absence of primer dimer peaks; a single and discrete peak was detected for all primers tested. Negative controls consisted of non-template reactions in which the cDNA was replaced with H₂O.

The expression levels of RpedOBPs and RpedCSPs were calculated relative to the reference genes RpedGAPDH (R. pedestris glyceraldehyde-3-phosphate dehydrogenase) and RpedEF (R. pedestris elongation factor) using the Q-Gene method in Microsoft Excel-based software Visual Basic (Muller et al., 2002; Simon, 2003). For each sample, three biological replicates were performed with three technical replicates per biological replicate.

Sequence Analyses

Based on sequence alignments, all phylogenetic trees in this study were constructed using the MEGA7 software (Kumar et al., 2016) using the neighbour-joining method, and each tree was tested by bootstrapping with 1,000 replicates. A phylogenetic tree was constructed based on the alignment results of cytochrome oxidase subunit I (COI) genes from different species (Aphis gossypii: KR017753.1, Nilaparvata lugens: AB325705.1, Sogatella furcifera: LC005703.1, Adelphocoris lineolatus: MZ608737.1, Adelphocoris suturalis: KY367052.1, Nysius ericae: KM022105.1, and R. pedestris: MG838422.1). The amino acid sequences of the RpedOBPs, RpedCSPs, and other hemipteran OBPs and CSPs were listed in Supplementary Table S2. The totals numbers of OBPs and CSPs in other insects have been reported in previous studies (Gu et al., 2011; Gu et al., 2013; Cao et al., 2014; He and He, 2014; Xue et al., 2014; Yang et al., 2014; Zhou et al., 2014; He et al., 2015; Zhou et al., 2015; Cui et al., 2017). Gene structure and exon/intron structure maps were generated using TBTools (version 1.098728) (Chen et al., 2020) based on R. pedestris annotated file (Gene Location Visualisation from GTF/GFF and Visualisation of Gene Structure). Pairwise similarity of sequences was also generated by TBTools based protein sequences (Protein Pairwise Similarity Matrix). Expression heat maps and bars were drawn using TBtools and GraphPad Prism 9.0, respectively, based on the qRT-PCR results.

Statistical Analysis

The qRT-PCR data (mean ± SE) of RpedOBPs and RpedCSPs from various samples were subjected to one-way nested analysis of variance (ANOVA) followed by a least significant difference test (LSD) to compare means using the SPSS Statistics software (version 22.0; SPSS Inc., Chicago, IL, United States).

Results and Discussion

Identification of OBP and CSP Genes in R. pedestris

Recent progress in the whole-genome sequencing provides insights into the molecular mechanisms of olfaction in insects (Cheng et al., 2017; Wan et al., 2019). Second- and third-generation sequencing methods have also been successfully used for R. pedestris genome assembly. Based on BUSCO completeness and contig length, the Wtdbg2 assembly was used for the draft assembly of the R. pedestris genome (Huang et al., 2021). We downloaded the R. pedestris genome, protein, RNA and annotation files using Genome Warehouse and further annotated them using the Nr, Nt, SwissProt, KOG, eggNOG, Interpro, GO, and KEGG databases. A total of 53 OBPs and 25 CSPs of R. pedestris were identified and corrected using the following correction through BLAST. Finally, 49 OBPs and 25 CSPs were identified and named RpedOBP1-49 (43 were new genes), RpedCSP1-25 (17 were new genes) (Table 1). The predicted results of the sequences analysis revealed that all OBPs and CSPs had full-length open reading frames (ORF), and 39 OBPs and 23 CSPs had a signal peptide, respectively. The 49 OBPs without signal peptides share 22.5–99.19% amino acid identities with each other, while the 25 CSPs share 22.5–99.07% amino acid identities with each other (Supplementary Table S3). Full-length sequences of the 49 RpedOBPs and 25 RpedCSPs were presented in the supplementary files.

The number of RpedOBP and RpedCSP genes identified for R. pedestris is larger than those in other hemipterans (Figure 1). For example, 11 OBPs and 17 CSPs were identified in N. lugens (Xue et al., 2014; Yang et al., 2014; Zhang Y.-N. et al., 2016), 14 OBPs and 8 CSPs in A. lineolatus (Gu et al., 2011; Zhang Y.-N. et al., 2016), 16 OBPs and eight CSPs in A. suturalis (Cui et al., 2017), 28 OBPs and 16 CSPs in N. ericae (Zhang Y.-N. et al., 2016), nine OBPs and nine CSPs in A. gossypii (Gu et al., 2013; Zhang Y.-N. et al., 2016), and 12 OBPs and nine CSPs were identified in S. furcifera (He and He, 2014; Zhou et al., 2015). The differences in gene numbers may be explained by: 1) the different behaviours of different insects requiring distinct molecular mechanisms that have been developed over evolutionary time, and 2) the genomic data of R. pedestris that is more conducive to the comprehensive mining of OBP and CSP genes than other hemipteran species.

FIGURE 1

Localization of OBPs and CSPs in the R. pedestris Genome

To clarify the position of OBPs and CSPs in chromosomes, we carried out chromosome location analysis of all genes, and the results showed that 48 OBP genes were distributed across four chromosomes (Figure 2A); only RpedOBP49 was located on scaffold056, which could not be mapped to a chromosome based on the current genome assembly. Thirty OBPs clustered together on chromosome 2, followed by chromosome 5 (11 OBPs), 3 (four OBPs), and 1 (three OBPs). Twenty-five CSP genes were distributed across four chromosomes, with 21 CSPs clustered together on chromosome 3 and the others on chromosome X (two CSPs), 1 (one CSP), and 5 (one CSP) (Figure 2B). More than 60% OBP and 80% CSP genes are located within clusters, as seen in other insects (Gong et al., 2009; Gu et al., 2013), indicating a relatively recent expansion of the OBP and CSP families of R. pedestris and the diverse functions of genes have evolved in response to different odorants in the environment.

FIGURE 2

Genomic Structure of R. pedestris OBPs and CSPs

To further clarify the genomic structural characteristics of OBPs and CSPs, we obtained the gene lengths and intron numbers of OBPs and CSPs based on the genome annotation file of R. pedestris (Figure 3). The lengths of the OBP genes ranged from 3.065 to 46.888 kb, with 33 OBPs having six introns and the other 16 OBPs having four, five, seven, eight, nine, and 12 introns, respectively (Figure 3A). The CSP genes were much shorter, ranging from 2.114 to 34.628 kb, with one, two, or three introns (Figure 3B). The phylogenetic trees of OBPs and CSPs in R. pedestris showed that the genes clustered together tended to have similar genomic structures, which also implies that they may have similar functions. The sequences of RpedOBPs were longer and had more introns than those of RpedCSPs, indicating that they may have complex features of functional differentiation.

FIGURE 3

Phylogenetic Analyses of Hemipteran OBPs and CSPs

Two phylogenetic trees were constructed for the OBPs and CSPs using protein sequences from R. pedestris, A. lineolatus, Apolygus lucorum, Aphis gossypii, and other hemipteran species (Gu et al., 2011; Gu et al., 2013; Cao et al., 2014; He and He, 2014; Xue et al., 2014; Yang et al., 2014; Zhou et al., 2014; He et al., 2015; Zhou et al., 2015; Cui et al., 2017). Similar to that in other studies (Gu et al., 2011; Zhang Y.-N. et al., 2016; Cui et al., 2017), the OBP tree in this study showed that eight RpedOBPs (OBP1, 5–9, 13, and 42) could be divided into the Plus-C OBP subfamily, and the other 41 RpedOBPs clustered into the classic OBP subfamily (Figure 4). In the constructed CSP tree, our results indicated that all 25 RpedCSPs were distributed along various branches, and each clustered with at least one other moth orthologue (Figure 5). The diversity of the RpedOBPs and RpedCSPs families suggests a role for positive selection in the rapid evolution and functional diversification of these genes. We speculate that both RpedOBP and RpedCSP genes had some gene expansions, such as OBP1/5/6/7/8/13, OBP23/10/48/16/33/32/31/49/46/45/44/47/43, OBP25/26/27/28, OBP40/39/41/34/15/36/35/11, CSP13/8/19/22, and CSP12/15/14/16/17/19/18/6/7, indicating that these genes may be involved in the recognition of important odorants related to R. pedestris behaviour (Pelosi et al., 2005; Matsuo et al., 2007; Gu et al., 2012; Poivet et al., 2013; Martin-Blazquez et al., 2017; He et al., 2019).

FIGURE 4

FIGURE 5

Expression Profiles of R. pedestris OBP and CSP Genes

We used qRT-PCR to assess expression profiles of all R. pedestris OBPs and CSPs in the heads, thoraxes, abdomens, legs, wings, and antennae of the adults. The results showed that all OBPs and CSPs were expressed in the adult antennae of R. pedestris. Among the 49 RpedOBPs, 33 (approximately 67%) were significantly highly expressed in the antennae, including three male-biased (RpedOBP19, RpedOBP21, and RpedOBP32) and nine female-biased (RpedOBP2, RpedOBP6, RpedOBP9, RpedOBP17, RpedOBP24, RpedOBP34, RpedOBP36, RpedOBP48, and RpedOBP49). Among the 49 RpedOBPs, RpedOBP37 exhibited the highest expression level in male and female antennae (Figure 6). Compared to RpedOBPs, RpedCSPs were highly expressed in adult antennae as well as in non-antennal tissues. Of the 25 identified RpedCSP genes, only 11 RpedCSPs (approximately 44%) displayed antennal-biased expression; four RpedCSPs (RpedCSP3, RpedCSP12, RpedCSP20, and RpedCSP21) were male-biased and five RpedCSPs (RpedCSP4, RpedCSP9, RpedCSP11, RpedCSP13, and RpedCSP24) were female-biased in their expression (Figure 7). Several studies have shown that OBPs and CSPs are required for the correct recognition of some odorants from the external environment (Zhang et al., 2014; Liu et al., 2015; Chen G.-L. et al., 2018; Pelosi et al., 2018; Zhang et al., 2020a; Zhang et al., 2020b), therefore, we infer that the 33 RpedOBPs and 11 RpedCSPs highly expressed in adult antennae are likely to be involved in the crucial odorant reorganisation of R. pedestris (Krieger et al., 1996; Bohbot and Vogt, 2005; Zhang et al., 2014; Missbach et al., 2015; Chen G.-L. et al., 2018). The sex-biased RpedOBPs and RpedCSPs may be involved in the reorganisation of plant volatiles from oviposition sites or other sex-related odorants (He et al., 2010; Zhou et al., 2013; Zhang et al., 2019). Further analysis is needed to explore their exact roles, such as through fluorescence competitive binding assays (Liu et al., 2015; Ingham et al., 2020; Li et al., 2022), CRISPR/Cas9 mediated genome editing (Zhu G.-H. et al., 2016; Han et al., 2022), and gene mutations (Stowers and Logan, 2008; Zhang et al., 2020b).

FIGURE 6

FIGURE 7

Similar to the findings of previous studies (Zhang et al., 2013; McKenzie et al., 2014; Gu et al., 2015; Zhang L.-W. et al., 2016), we also found that there were 12 RpedOBP and six RpedCSP genes highly expressed in non-antennal tissues, including four leg-biased genes (RpedOBP14, RpedOBP35, RpedOBP44, and RpedCSP6), six head-biased genes (RpedOBP16, RpedOBP29, RpedOBP30, RpedOBP31, RpedOBP45, and RpedOBP46), one thorax-biased gene (RpedOBP39), two abdomen-biased genes (RpedOBP26 and RpedOBP28), and five wing-biased genes (RpedCSP1, RpedCSP2, RpedCSP8, RpedCSP10, and RpedCSP25), indicating that these genes may have other non-olfactory functions.

Conclusion

In conclusion, we identified 49 OBPs and 25 CSPs in the R. pedestris genome and found that these genes were clustered in highly conserved groups comprising OBP and CSP genes from other hemipteran species. To further understand the functions of these genes, we conducted comprehensive and comparative phylogenetic analyses and studied the gene expression profiles of OBPs and CSPs. We found that most RpedOBPs displayed antennal-biased expression, but many RpedCSPs were detected in the antennae and were highly expressed in non-antennal tissues, and some genes showed characteristics of sex-biased expression. Tissue- and sex-biased expression patterns will help us identify the functions of RpedOBPs and RpedCSPs, which will also aid in understanding the olfactory mechanism of R. pedestris and the development of environmentally friendly insecticides against this pest in the future.

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