Identification and functional characterization of sex pheromone receptors in mirid bugs (Heteroptera: Miridae)

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Highlights

  • The odorant receptor (OR) repertoire was identified in three mirid bug species.

  • In each species, three ORs were predicted to have pheromone receptor (PR) function

  • The candidate PRs showed robust response to mirid bug sex pheromone components

Abstract

Mirid bugs are a group of important insect pests that cause large annual losses in agricultural production. Many studies have focused on the isolation and identification of sex pheromones in mirid bugs, and the components and biological activity of the sex pheromones have also been studied as a way to control these pests. However, few studies have focused on the mechanisms of pheromone perception. In this study, we identified the odorant receptor repertoire in three mirid bug species, Apolygus lucorum, Adelphocoris lineolatus, and Adelphocoris suturalis using antennal transcriptome sequencing and bioinformatics analysis. The candidate pheromone receptor (PR) genes were then identified by comparative transcriptomic and expression pattern analysis. Importantly, in vitro functional studies have shown that the candidate PRs have robust responses to the main mirid bug sex pheromone components (E)-2-hexenyl butyrate (E2HB) and hexyl butyrate (HB). Our study uncovered the mechanism of pheromone peripheral coding in these three species and elucidated the mechanism by which mirid bugs can specifically recognize a mate. Moreover, the results of our study will provide a theoretical basis for screening effective sex attractants or mating disturbance agents at the molecular and neural levels for enhanced control of these destructive pests.

Introduction

The Miridae (plant bugs) is one of the largest heteropteran families, with over 11,000 described species (Cassis and Schuh, 2012). Mirid bugs are distributed worldwide and have emerged as economically important pests of many crops. The resurgence of mirid bugs around the world, which is a negative consequence of the commercial adoption of Bt cotton, has attracted the attention of scientists (Lu et al., 2010; Wu et al., 2002). Insecticide use is the primary strategy to manage the various mirid bugs at present. Nevertheless, with large-scale and long-term use of insecticides, resistance in mirid bug populations has gradually increased (Zhang et al., 2015b). Accordingly, physical control, biological control, and other environmentally-friendly methods should be applied to the control of mirid bugs.

Insect sex pheromones are generally secreted by females, which can then be identified by heterosexual individuals and cause courtship, mating and other reactions (Karlson and Lüscher, 1959). Since the first insect pheromone components (Z,E)-10,12-hexadecadienol (bombykol) and (Z,E)-10,12-hexadecadienal (bombykal) were characterized in the silkworm moth Bombyx mori (Butenandt et al., 1959; Kaissling et al., 1978), hundreds of insect sex pheromones have been identified, many of which have been synthesized and applied artificially with good results (Witzgall et al., 2010). The study of sex pheromones in Miridae species has a history of almost 30 years since Smith et al. first isolated and identified the sex pheromones from the mullein bug, Campylomma verbasci, in 1991 (Smith et al., 1991). Since then, the sex pheromones of many species of mirid bugs distributed in America and Asia have been studied. Components of these sex pheromones are generally saturated and unsaturated short-chain esters and unsaturated ketoaldehydes, and the sex pheromone components of different mirid bugs are basically the same, but the proportions of the different components are quite different (Byers et al., 2013; Chinta et al., 1994; Drijfhout et al., 2000; Fountain et al., 2014; Innocenzi et al., 2005; Haedie and Minks, 1999; Millar and Rice, 1998). In particular, scientists have found that the sex pheromones of several species are composed of (E)-2-hexenyl butyrate (E2HB), hexyl butyrate (HB), and (E)-4-oxo-2-hexenal (4-OHE), but the ratios of the three compounds are different (Byers et al., 2013; Chinta et al., 1994; Drijfhout et al., 2000; Fountain et al., 2014; Innocenzi et al., 2005).

Because sex pheromone perception plays an important role in insect mating and speciation, a great deal of research has been conducted on the molecular and neurological mechanisms of insect sex pheromone perception, especially in Drosophila and several species of moths (Baker et al., 2004; Chang et al., 2016; Cosse et al., 1998; Kurtovic et al., 2007). The sensitive and specific detection of insects are mostly dependent on their outstanding olfactory system. Odorant receptors (ORs) play a predominant role in the odorant recognition process. In general, ORs always co-express with an conserved, nonconventional OR coreceptor (Orco); the OR-Orco complex forms a ligand-gate non-selective cation channel which opens directly upon activation of an appropriate ligand, and the complex is essential for the correct targeting of OR proteins to the dendritic membrane (Sato et al., 2008; Smart et al., 2008). In insects, a special subfamily of ORs, pheromone receptors (PRs), is directly involved in the recognition of sex pheromones (Krieger et al., 2005; Nakagawa et al., 2005). Male mirid bugs can perceive and respond to the specific and extremely low concentrations of sex pheromones released by conspecific females (Fountain et al., 2014; Ho and Millar, 2002; Smith et al., 1991; Yasuda and Higuchi, 2012; Yasuda et al., 2008; Zhang and Aldrich, 2008; Zhang et al., 2015c, 2015d). However, the molecular mechanism of sex pheromone recognition in mirid bugs is still unclear. Whether the bug has a highly specific pheromone recognition system similar to that in moths requires further study.

In China, A. lucorum (Zhang, 2011), A. suturalis (Zhang, 2011), and A. lineolatus (Zhang et al., 2015c) are the three most common mirid bug pests that cause serious damage to agricultural crops (Jiang et al., 2015; Lu et al., 2010). All three of these Miridae species use E2HB, HB, 4-OHE, and some other minor components as their sex pheromones, which makes them an ideal model for studying the mechanism of sex pheromone recognition in mirid bugs (Yang et al., 2015; Zhang, 2011; Zhang et al., 2015c; Zhang et al., 2016b). There are previous reports of studies on odorant binding proteins (OBPs) in these three species, and our laboratory has also cloned and studied the function of some general ORs from A. lucorum (Gu et al., 2011; Hua et al., 2012, Hua et al., 2013; Li et al., 2020; Yan et al., 2015; Zhang et al., 2016a; Zhou et al., 2014). However, there are few reports on the molecular mechanism of sexual information perception in these three species of mirid bugs.

In this study, we identified the OR repertoires in the three mirid bug species by antennae transcriptome sequencing and analysis. The expression of all candidate AlucOR genes in male and female antennae was analyzed by semi-quantitative reverse transcription PCR (RT-PCR). Three AlucORs, three AlinORs, and three AsutORs were predicted to be candidate PRs in the three mirid bugs for two reasons: 1) the nine predicted ORs proteins clustered together to form an independent clade in the phylogenetic tree, 2) all three AlucORs genes in this clade showed biased expression in male antennae. The full length open reading frames (ORFs) of the nine PR genes were then verified by rapid amplification of cDNA ends (RACE) and cloned. Finally, we functionally characterized the nine PRs in vitro via heterologous expression in Xenopus oocytes with two-electrode voltage-clamp recordings, and found that eight of the PRs were activated by E2HB and HB. This work provides a basis for understanding the molecular mechanism of olfactory recognition in mirid bugs and establishes a foundation for developing novel strategies to control these destructive pests.

Section snippets

Insect rearing and RNA isolation

The A. lucorum individuals used in our experiments were obtained from a laboratory colony established and maintained at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. A. suturalis was obtained from a laboratory colony established and maintained at Huazhong Agricultural University. A. lucorum and A. suturalis were reared on fresh corn and green beans and maintained at 28 ± 1 °C, with 60% ± 5% relative humidity (RH), and a 14 h:10 h light:dark

Antennal transcriptome analysis and identification of candidate ORs

A. lucorum, A. lineolatus, and A. suturalis antennal transcriptomes were sequenced using the Illumina HiSeq 2000 platform combined with Trinity assembly. The final transcript datasets containing 99,926, 95,262, and 56,126 unigenes were obtained for A. lucorum, A. lineolatus, and A. suturalis, respectively. Due to the higher quality of the A. suturalis antennal transcriptomes, the dataset was much smaller than the other two datasets. Based on our analysis of the antennal transcriptomes in the

Discussion

In the three closely related Miridae species A. lucorum, A. lineolatus, and A. suturalis, the female bugs produce and release similar blends of ternary sex pheromone components in different ratios, and this is vitally important for interspecific isolation. Through a series of systematic studies and analyses, we predicted and verified the PRs in these three mirid bug species, which will allow us to further define the molecular mechanisms of sex pheromone recognition in mirid bugs. To predict and

Conclusions

In conclusion, the candidate PRs and their functions in three species of mirid bugs (A. lucorum, A. lineolatus, and A. suturalis) have been identified and studied. Our detailed characterization of the sex pheromone recognition system in mirid bugs has revealed the olfactory recognition pattern in Miridae bugs differs from that in moths, because the PRs exhibited obvious functional overlap. Our results will help uncover the mechanisms of sex pheromone recognition in detail. Furthermore, this

Availability of data and materials

All the data and resources generated for this study are included in the article and the supplemental materials. The cDNA sequences of the functional identified PRs and Orcos have been submitted in Genbank under the accession MN657161, MN657162, MN657163, MN657165, MN657166, MN657167, MN657168, MN657171, MN657172, MN657173, MN657174 and MN657177. We are willing to share all the data and resources in this study with the public.

Author contributions

Y.L. and GW conceived the idea and designed the study. S.Z., S.Y., Z.Z., S.C., B.L. and Y.L. performed the experiments and analyzed the data. S.Z., Y.L. and G.W. wrote the manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgments

We thank Dr. Xiangdong Mei from Institute of Plant Protection, Chinese Academy of Agricultural Sciences for gifting the pheromone components. We thank Zhenjie Qin for helping rear the experimental mirid bugs for the study. We thank Dr. Wei Liu from Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences for providing pictures in graphical abstract. This work was funded by National Natural Science Foundation of China (31672095, 31621064), National Key R&D Program of

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