Insects rely on their accurate olfactory systems to recognize chemical signals such as pheromones and plant volatiles, and thereby adapt to different environments and ecological niches (Gadenne et al., 2016; Haverkamp et al., 2018). The recognition of chemical cues in insects is a spectacularly complex process. When hydrophobic odorant or taste molecules diffuse into insect sensory lymph through epidermal pores, they are rapidly recognized, bound, and dissolved by odorant binding proteins (OBPs) or chemical sensory proteins (CSPs). Subsequently, they are transported to the lumen cilia of olfactory receptor neurons (ORNs), where chemical signals are converted into electrical signals (Leal, 2013), then these signals are transferred to the central nervous system, which manipulates insects to make corresponding responses. In the process of converting semiochemicals into electrical signals in the peripheral nerve system, at least three main chemosensory receptor families are involved, including odorant receptors (ORs), gustatory receptors (GRs), and ionotropic receptors (IRs) (Fleischer et al., 2018; Wicher, 2018). At the terminal of odorant recognition, odorant signals are inactivated or degraded by various odorant degrading enzymes (ODEs) (Younus et al., 2017; Wang et al., 2021).

Insect ORs, first reported in the Drosophila genome (Gao et al., 1999; Vosshall et al., 1999), are seven transmembrane domain (7-TMD) proteins consisting of 350–500 amino acids (Robertson, 2019). ORs are mainly expressed in the dendritic membranes of the ORNs, and there are two classifications: odorant receptor co-receptor (Orco) and odorant-specific olfactory receptor proteins (ORx) (Smart et al., 2008; Tian et al., 2022). Orco (formerly designated as OR83b) has a highly-conserved sequence, while common ORs, even in sibling species are incredibly variable (Wicher and Miazzi, 2021). For instance, the OR members in the fly are extremely divergent, with an average amino acid identity of ≈20% (Wetzel et al., 2001). Insect ORs recognize odorant molecules through a special heterodimer composed of an ORx and an Orco. Heterodimer ORx-Orco forms an ion channel that allows cations to pass through when it binds specific odorant molecules (Sato et al., 2008). In this complex, ORx is responsible for the specificity, while Orco is an obligate factor for the localization, stability, and protein folding of each ORx (Stengl and Funk, 2013). If Orco is knocked out, the olfactory-related behavior of an insect would be altered because of the disruption or even abolition of its OR functional repertoire (Fan et al., 2022). The essential function of Orco has been validated in numerous insects through RNA interference (RNAi) or gene editing technology. For example, RNAi-based silencing of Orco in the hemimetabolous blood-sucking insect Rhodnius prolixus results in losing the ability to find hosts, reducing the number of eggs laid and decreasing the survival rate (Franco et al., 2016). Moreover, mutations of Bombyx mori, whose Orco gene was knocked out by CRISPR-Cas9, are influenced on the aspects of their larval feeding and adult mating behavior (Liu et al., 2017).

Like ORs, insect GRs also contain 300–500 amino acids. However, GR family members are usually abundant in the taste organs and play key roles in sensing carbon dioxide, sugar, bitter compounds, and taste pheromones (Agnihotri et al., 2016; Fleischer et al., 2018). Insect IRs are related to the ionotropic glutamate receptors (iGluRs) family. They have been identified in both olfactory and gustatory organs and reported to be responsible for detecting acids, aromatics, and nitrogen-containing compounds (Agnihotri et al., 2016; Wicher and Miazzi, 2021; Dong et al., 2023). Several GRs and IRs are expressed in the antennae and have been characterized to mediate multiple olfactory-related capabilities, such as pheromone detection (Jones et al., 2007; Benton et al., 2009; Croset et al., 2010; Koh et al., 2014). GRs, IRs, and ORs together form a complex olfactory reception system and participate in olfactory responses.

The bean bug, Riptortus pedestris (Fabricius) (Hemiptera: Alydidae), is a polyphagous pest attacking legume plants in many East Asian countries (e.g., China, Japan, South Korea, and Thailand) (Mizutani et al., 2011; Lim, 2013). In the past 2 decades, this pest has become the dominant pest in soybean fields (Do et al., 2014; Li et al., 2019; Zhang et al., 2022). Both adults and nymphs of R. pedestris absorb soybean nutrients through the piercing-sucking mouthparts, and result in soybean stay-green, a phenomenon of a lack of leaf senescence, pod abortion, and abnormal seeds (Hill et al., 2006; Sakuraba et al., 2015; Li et al., 2019). Outbreak of R. pedestris could cause soybean yield losses, quality decrease, and potential germination decline of seeds (Rahman and Lim, 2017; Ahn et al., 2019; Park et al., 2023). It is convinced that R. pedestris population highly relies on chemicals cues in their aggregation, location of hosts, and avoidance of adverse environment (Xu et al., 2021). R. pedestris individuals of all developmental stages and sexes are reported to be attracted by the intraspecific aggregation pheromones, which were identified as a 1:5:1 mixture of (E)-2-hexenyl (Z)-3-hexenoate (E2HZ3H), (E)-2-hexenyl (E)-2-hexenoate (E2HE2H) and myristyl isobutyrate (MI) (Leal et al., 1995; Rahman et al., 2018). Meanwhile, R. pedestris can recognize soybean through a particular blend of plant volatiles, including (Z)-3-hexenol, (Z)-3-hexenyl acetate, 4-ethylbenzaldehyde, α-farnesene, and methyl salicylate (Song et al., 2022). The bean bugs also show the ability to distinguish hosts treated with chemical insecticides, especially bifenthrin (Maharjan and Jung, 2015). These odorants are recognized through its complicated and sophisticated olfactory system, with various olfactory-related proteins involved. A previous report claimed that 188 ORs, 6 GRs, and two IRs were identified from the antennal transcriptome of R. pedestris (Song et al., 2017), but the sequence information remains undisclosed and unavailable. More recently, 49 OBPs and 25CSPs were annotated from R. pedestris genome data (Li et al., 2022), which is significantly more than the transcriptome-based identification (eight OBPs and eleven CSPs) (Song et al., 2017). The results trigger a prediction that more chemosensory receptors would be explored from the R. pedestris genome.

In the present study, we aimed to systematically identify chemosensory receptors from the genome of R. pedestris, explore their phylogenetic relationship with other Hemiptera insects, and determine their expression in the antennae of different development stages through RNA-seq and real-time quantitative PCR (qRT-PCR). We also verified the importance of RpedOrco in responding to aggregation pheromones using RNAi-based technology. Our results will provide a theoretical basis for further understanding of the olfactory recognition of R. pedestris.

Insects’ behavior highly relies on their accurate chemosensory system, in which chemosensory receptors play critical roles in detecting chemical signals. Particularly, ORs have been more widely studied. Since the first discovery of insect ORs in D. melanogaster (Clyne et al., 1999; Gao et al., 1999), ORs have been widely studied in a variety of insects, including dipterans, hymenopterans, lepidopterans, coleopterans, and hemipterans (Fan et al., 2022; Tian et al., 2022). The present study identified 310 candidate chemoreceptors from the reported R. pedestris genome, including 237 RpedORs, 42 RpedGRs, and 31 RpedIRs. Furthermore, their phylogenetic relationship, localization in chromosomes, and expression profile were also analyzed according to bioinformatics data.Moreover, our results of RNAi and behavioral bioassays demonstrated that RpedOrco is essential for R. pedestris detecting aggregation pheromones.

Genome and transcriptome were the mainstream method for identification and exploration of chemoreceptor genes. Previously, transcriptome annotation was the exclusive channel for identifying chemoreceptors in non-model species, mainly due to the lack of their genomic information. With the progress of sequencing technology, increasing numbers of insect genome data are unveiled and available, enabling the genome-wide identification of chemosensory genes. Generally, more ORs could be identified from the genome than transcriptome data because some insect ORs are highly diverse sequences and have low expression in specific issues (Tian et al., 2022). In Ap. lucorum, for instance, 155 ORs could be annotated from genome data (Tian et al., 2022), while only 110 ORs were reported in the antennal transcriptome (An et al., 2016). Similar to ORs, much more Rhynchophorus ferrugineus GRs were identified from the genome (65) than antennal transcriptome (16) (Engsontia and Satasook, 2021). These results indicate that genome-wide identification of chemosensory genes is more feasible and reliable compared with the transcriptome-based method (Tian et al., 2022). For R. pedestris, we also identified significantly more chemoreceptors than the previous report by Song et al. (2017), in which 188 ORs, 6 GRs, and two IRs are annotated from the antennal transcriptome.

The number of ORs is higher diverse among insects, ranging from as few as ten in Pediculus humanus to more than 400 in a social ant Camponotus foridanus (Zube and Rossler, 2008; Kirkness et al., 2010; Fan et al., 2022). In hemipteran species, the number of ORs also varies enormously. Tian et al. (2022) reannotated 887 OR from 11 species in nine hemipteran families and found that the OR numbers vary from 9 to 13 (Bemisia tabaci) to 155 (Ap. lucorum). Based on the whole-genome data, we found R. pedestris has a much larger odorant reception system than other hemipterans, with identification of up to 237 RpedORs. More RpedORs seemingly betoken that R. pedestris would have a broad host recipe because the number of ORs is reported to associate with the host breadth in many insects (Mitchell et al., 2019). In Hemiptera, however, Tian et al. (2022) demonstrated that OR number is not directly parallel to the diversity of the host. Thus, it is reasonable that R. pedestris has a considerably larger number of ORs (237) than polyphagous Aphis gossypii (47 from the genome data), which feeds on over 700 host plants worldwide (Cao et al., 2014; Tian et al., 2022).

The diversity of OR numbers among species is attributed to gain (via tandem duplication) and loss (via pseudogenization and deletion) events, which were considered as a consequence of random genomic drift or adaption to environment (Dong et al., 2009; Mendivil and Ferrier, 2012; Andersson et al., 2019; Tian et al., 2022; Zhang et al., 2023). The discovery of some tandem replications of the ORs of both ants and bees suggested the tandem replication in Hymenoptera is the main mechanism of OR amplification (Robertson and Wanner, 2006; Zhang et al., 2023). Moreover, the OR gene family in hemipteran insects has undergone rapid expansion, with the existence of gene replication as well (Nei et al., 2008; Tian et al., 2022). For instance, Acyrthosiphon pisum ORs form two large lineage-specific subfamily expansions which include some tandem arrays (ApOR20-22 on SCAFFOLD42, ApOR23-24 on SCAFFOLD6001, ApOR40-41 on SCAFFOLD150003), and most of the genes in two main clades have apparently undergone relatively recent duplications of genes (Smadja et al., 2009). In the present study, we found some RpedOR genes exist as genomic clusters, mostly concentrating on chr3 and chr4 (Figure 4). This result suggested that gene duplication also existed in R. pedestris that led to the increase of RpedORs number.The insect OR genes are mainly expressed in the antennae and other olfactory-related accessories, where volatile semiochemcials are recognized and subsequently trigger various olfactory-driving behaviors (Leal et al., 2013; Lombardo et al., 2017). To further explore the roles of ORs in olfactory recognition, we evaluated the RpedORs’ expression in antennae at different development stages of R. pedestris. Along with the development of nymphs, the majority RpedORs positively expressed in antennae of an increasing instar (Figure 5), suggesting that these RpedORs may constantly play roles in olfactory recognition during R. pedestris development. However, a few RpedORs (e.g., RpedOR30 and RpedOR33) are significantly expressed in the antennae of younger nymphs than adults, indicating that different RpedORs participate in specific olfactory responses, such as foraging or avoiding predators. In addition, many RpedORs showed significant expression differences between adults and nymphs (Figure 5), presumably because the adults shoulder more olfactory-related responsibilities, such as host shift, migration, finding mates, and location of optimal oviposition sites. These are the main reasons for the difference in OR expression in different insect stages (Harwood et al., 2009; Vaello al., 2017). For R. pedestris, additionally, nymphs and adults display conspicuous polymorphism in chemical production and emission, indicating they also smell different from conspecific volatiles (Xu et al., 2021). The identification and function of RpedORs associated with the recognition of conspecific secretions would be our further study focus.

Among all olfactory receptors, Orco is the most special and obligate one. Abundant literature has documented that insect Orcos are highly conserved among species, while specific ORs are relatively diverse with low similarity (Jones et al., 2005; Sun et al., 2020b). In this study, RpedOrco also shows high homology with Orcos from other hemipterans, with the highest similarity to HhalOrco (93.26%) in H. halys (Figure 7), which is consistent with previous findings in other insects (Hansen et al., 2014; Franco et al., 2016). In addition, the highly-conserved C-terminal region of Orco suggests that this region may associate with the functional interaction between ORx and Orco proteins (Butterwick et al., 2018; Zufall and Domingos, 2018; Sun et al., 2023). As an obligate unit of olfactory signal transmission in insect ORNs, Orco is predicted to assist specific ORs in recognizing chemical stimuli by forming a heteromeric ORx-Orco rather than singly responding to odorants (Stengl and Funk, 2013). Once Orco does not function properly, the insect olfactory transduction process is interrupted, impairing subsequent odorant detection. In light of its necessity in olfactory recognition, Orco attracts increasing focus as a target for developing pest control agents (Fan et al., 2022). To date, mutants of many insect species have been generated to characterize and investigate the function of Orco through gene silencing with RNA interference (RNAi) or gene editing with CRISPR-Cas9 (Tateishi et al., 2022; Wang et al., 2022). For example, RNAi-based silencing of the Orco gene in Protaetia brevitarsis adults significantly impaired their location of aggregation pheromones and food sources (Zhang et al., 2021). Knocking out of the Orco gene through CRISPR-Cas9 seriously affects the olfactory-related behaviors as well, which has been confirmed in Anopheles coluzzii (Sun Y. et al., 2020), A. sinensis (Wang et al., 2022), Bactrocera dorsalis (Xu et al., 2022), B. mori (Liu et al., 2017), Harpegnathos saltator (Yan et al., 2017), Helicoverpa armigera (Fan et al., 2022), Locusta migratoria (Li et al., 2016), Spodoptera frugiperda (Sun et al., 2023) and S. littoralis (Koutroumpa et al., 2016). Our results of silencing RpedOrco in R. pedestris reached the similar conclusion. The dsOrco-injected bugs, which showed a high knockdown rate of RpedOrco, lost the location activity to specific aggregation pheromones (Figure 8). We also tested the silencing efficiency of RpedOrco in nymphs, the results showed that it did not reach 85% within 3 days after injection. Based on the fact that the inter-age period lasts only about 3 days (Kim and Lim, 2010; Rahman and Lim, 2017), it cannot accurately define the instar during the behavioral bioassays. Nevertheless, the results that both RNA-seq and qRT-PCR showed the relatively high expression of RpedOrco in antennae of R. pedestris at different development stages (Figures 5, 6) also indicated its constant role in olfactory recognition.