Identification and functional characterization of sex pheromone receptors in mirid bugs (Heteroptera: Miridae)
Graphical abstract
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
References (71)
- et al.
Functional characterization of one sex pheromone receptor (AlucOR4) in Apolygus lucorum (Meyer-Dur)
J. Insect Physiol.
(2020) - et al.
Identification and tissue distribution of odorant binding protein genes in the lucerne plant bug Adelphocoris lineolatus (Goeze)
Insect Biochem. Mol. Biol.
(2011) - et al.
Coding of odors by a receptor repertoire
Cell
(2006) - et al.
The molecular basis of odor coding in the Drosophila antenna
Cell
(2004) - et al.
Functional characterizations of one odorant binding protein and three chemosensory proteins from Apolygus lucorum (Meyer-Dur) (Hemiptera: Miridae) legs
J. Insect Physiol.
(2013) - et al.
Chemoreception to aggregation pheromones in the common bed bug, Cimex lectularius
Insect Biochem. Mol. Biol.
(2017) - et al.
Broadly and narrowly tuned odorant receptors are involved in female sex pheromone reception in Ostrinia moths
Insect Biochem. Mol. Biol.
(2010) - et al.
Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins
Insect Biochem. Mol. Biol.
(2008) - et al.
Proceeding from in vivo functions of pheromone receptors: peripheral-coding perception of pheromones from three closely related species, Helicoverpa armigera, H. assulta, and Heliothis virescens
Front. Physiol.
(2018) - et al.
Seasonal abundance of the mirids, Lygus lucorum and Adelphocoris spp. (Hemiptera Miridae) on Bt cotton in northern China
Crop Protect.
(2002)
An olfactory receptor from Apolygus lucorum (Meyer-Dur) mainly tuned to volatiles from flowering host plants
J. Insect Physiol.
Moth pheromone receptors: gene sequences, function, and evolution
Frontiers in Ecology and Evolution
Field resistance monitoring of Apolygus lucorum (Hemiptera: Miridae) in Shandong, China to seven commonly used insecticides
Crop Protection
A female-biased odorant receptor from Apolygus lucorum (Meyer-Dur) tuned to some plant odors
Int. J. Mol. Sci.
Silencing in Apolygus lucorum of the olfactory coreceptor Orco gene by RNA interference induces EAG response declining to two putative semiochemicals
J. Insect Physiol.
Artifacts and pheromone blends from Nezara spp. and other stink bugs (Heteroptera Pentatomidae)
Z. Naturforsch.
Insect olfaction and the evolution of receptor tuning
Frontiers in Ecology and Evolution
A comparison of responses from olfactory receptor neurons of Heliothis subflexa and Heliothis virescens to components of their sex pheromone
J Comp Physiol A Neuroethol Sens Neural Behav Physiol
Über den sexual-lockstoff des seidenspinners Bombyx Mori
Z. Naturforsch.
Sex pheromone component ratios and mating isolation among three Lygus plant bug species of North America
Naturwissenschaften
Identification of candidate olfactory genes in Chilo suppressalis by antennal transcriptome analysis
Int. J. Biol. Sci.
A de novo transcriptomic analysis to reveal functional genes in Apolygus lucorum
Insect Sci.
Odorant reception in the malaria mosquito Anopheles gambiae
Nature
Systematics, biodiversity, biogeography, and host associations of the Miridae (Insecta: Hemiptera: heteroptera: Cimicomorpha)
Annu. Rev. Entomol.
Sensillar expression and responses of olfactory receptors reveal different peripheral coding in two Helicoverpa species using the same pheromone components
Sci. Rep.
Olfactory reception of potential pheromones and plant odors by tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae)
J. Chem. Ecol.
Neurons discovered in male Helicoverpa zea antennae that correlate with pheromone-mediated attraction and interspecific antagonism
J. Comp. Physiol. A.
On-line thermal desorption-gas chromatography of intact insects for pheromone analysis
J. Chem. Ecol.
Pheromones mediating copulation and attraction in Drosophila
Proc. Natl. Acad. Sci. U. S. A.
Insect pheromone receptors-key elements in sensing intraspecific chemical signals
Front. Cell. Neurosci.
Further studies on sex pheromones of female Lygus and related bugs: development of effective lures and investigation of species-specificity
J. Chem. Ecol.
Sexual isolation of male moths explained by a single pheromone response QTL containing four receptor genes
P Natl Acad Sci USA
Full-length transcriptome assembly from RNA-Seq data without a reference genome
Nat. Biotechnol.
Pheromones of non-lepidopteran insects associated with agricultural plants
Smells like a new species: gene duplication at the periphery
Proc. Natl. Acad. Sci. U. S. A.
Cited by (0)
- 1
These authors contributed equally.