Candidate olfactory genes identified in Heortia vitessoides (Lepidoptera: Crambidae) by antennal transcriptome analysis

Abstract

Heortia vitessoides Moore is the most severe defoliating pest of Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae) forests. Olfaction in insects is essential for host identification, mating, and oviposition, in which olfactory proteins, including odorant-binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs), are responsible for chemical signaling. Here, we determined the transcriptomes of male and female adult antennae of H. vitessoides. We assembled 52,383 unigenes and annotated their putative gene functions based on the gene ontology (GO), eukaryotic ortholog groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Overall, 61 olfactory-related transcripts, including nine OBPs, 10 CSPs, 28 ORs, 12 IRs, and two SNMPs, were identified. Expression patterns of OBPs and CSPs in the female antennae, male antennae, and legs were performed using reverse transcription quantitative PCR (RT-qPCR). The results revealed that HvitOBP1, HvitOBP6, and HvitGOBP1 were enriched in the female antennae, while HvitOBP2, HvitOBP3, HvitOBP5, HvitGOBP2, and HvitPBP1 were enriched in the male antennae. HvitOBP4 was expressed at nearly the same level in the antennae of both males and females. Four CSPs (HvitCSP3, HvitCSP5, HvitCSP7, and HvitCSP10) and two CSPs (HvitCSP1 and HvitCSP4) were expressed at higher levels in the female and male antennae, respectively. HvitCSP6 was expressed at higher levels both in the female antennae and legs. Three CSP genes (HvitCSP2, HvitCSP8, and HvitCSP9) were expressed at higher levels in the legs. These results provide a basis for further studies on the molecular olfactory mechanisms of H. vitessoides.

Introduction

Insects detect chemical signals through their olfactory system, which obtains information from the environment and initiates behaviors such as habitat selection, mate selection, hunting, gathering, tropism, and communication (Field et al., 2000; Fatouros et al., 2008; Leal, 2013). Olfactory-related proteins are mainly located on the antennae, and to a lesser extent on other sensory appendages (Vosshall and Stocker, 2007; Sheng et al., 2017). There are many types of olfactory-related proteins, including odorant-binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs) (Fan et al., 2011; Wang et al., 2017).

Insect OBPs are soluble, acidic, and are highly concentrated in the lymph of the chemosensory sensilla of insect antennae (Vogt et al., 1991). OBPs are widely involved in olfactory perception and play a key role in transporting hydrophobic odorants to the ORs (Sanchez-Gracia et al., 2009). Based on the presence or absence of cysteine residues, OBPs can be divided into five groups: ‘Classical’ OBPs (with only one six-cysteine motif), ‘Dimer’ OBPs (with two six-cysteine motifs), ‘Plus-C’ OBPs (with extra cysteines), ‘Minus-C’ OBPs (with some cysteine residues missing), and ‘Atypical’ OBPs (with 9–10 cysteine residues) (Zhou et al., 2010). The classical OBPs are usually divided into subfamilies of pheromone-binding proteins (PBPs), general odorant-binding proteins (GOBPs), and antennal-binding protein X homologs (ABPXs) in lepidopteran species (Zhang et al., 2011). PBPs mainly bind to insect sex pheromones, which are a blend of compounds emitted by females to attract males, and GOBPs are usually expressed equally in the antennae of both sexes, consistent with a proposed role in the detection of both odorants and sex pheromones (Grosse-Wilde et al., 2006; He et al., 2010).

Insect CSPs are small, soluble, acidic proteins that have evolved to contain four conserved cysteine residues (Pelosi et al., 2014). Compared to OBPs, CSPs exhibit a wider expression profile, being found in olfactory tissues including the antennae, maxillary palps, and labial palps (Sheng et al., 2017). However, CSPs are also expressed broadly in non-olfactory tissues, including the pheromone glands, legs, and wings, suggesting that they participate in other physiological processes (Liu et al., 2015; Cui et al., 2017).

Insect ORs are seven transmembrane domain proteins that have a specific reversed membrane topology (intracellular N-terminus) compared to G-protein-coupled receptors (GPCRs) (Benton et al., 2006). Insect olfactory sensory neurons (OSNs) express two types of ORs: conventional ORs, a highly divergent family of receptors that are expressed in small subpopulations of OSNs; and a member of the olfactory co-receptor family, ORco (formerly called OR83b), a receptor without odor sensitivity, which is expressed in the majority of OSNs and is remarkably conserved across insect species (Larsson et al., 2004; Pitts et al., 2004; Smith, 2007). Based on chemical perception, ORs in the lepidopteran pheromone system are divided into two major groups: pheromone receptors (PRs) and general odorant receptors. PRs are considered to be connected to olfactory signal transduction of pheromone compounds in peripheral olfactory reception; and general odorant receptors are thought to detect plant volatiles (Jia et al., 2016). Insect ORs function via ORco, which transduces odorant signals by both heteromeric ligand-gated ion channels and cyclic nucleotide-activated cation channels, and therefore, can respond to signals rapidly, transiently, sensitively, and efficiently (Sato et al., 2008; Wicher et al., 2008).

IRs are a recently identified chemosensory receptor family and are another variant subfamily of the ionotropic glutamate receptors (iGluRs). The insect IRs contain structural regions that are conserved in iGluRs, namely, three transmembrane domains, a bipartite ligand-binding domain with two lobes, and one ion channel pore (Cao et al., 2014). IRs are expressed in coeloconic OSNs and may function in the detection of acids and ammonia (Zhao et al., 2016a). Insect IRs can be divided into two distinct subfamilies based on where they are expressed: the conserved “antennal IRs” and the species-specific “divergent IRs” (Croset et al., 2010; Abuin et al., 2011). Similar to ORco, IR25a and IR8a act as co-receptors, are broadly expressed, and play essential roles in regulating IR sensory cilia targeting and IR-based sensory channels (Abuin et al., 2011).

SNMPs, the only identified neuronal members of the CD36 family, belong to a larger gene family of receptors characterized by the human protein CD36. Insect SNMP/CD36 genes are divided into three major groups, and SNMPs belong to group three, which are divided into two subgroups, SNMP1 and SNMP2 (Nichols and Vogt, 2008). The SNMP1 subgroup is expressed in the antennae, and its members are related to pheromone-specific OSNs. Members of the SNMP2 subgroup are expressed in neurons, and to a greater extent, in sensilla support cells (Benton et al., 2007; Forstner et al., 2008; Vogt et al., 2009).

Heortia vitessoides Moore (Lepidoptera: Crambidae) is a serious defoliating pest of Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae), which produces valuable agarwood, a fragrant wood widely used in traditional medicine and the incense industry (Jin et al., 2016). The distribution of H. vitessoides ranges from India, Nepal, China, Sri Lanka, through South-East Asia and the East Indies to Queensland, the New Hebrides, and Fiji (Qiao et al., 2012). The biological characteristics, pesticide control methods, and behavior of H. vitessoides have been extensively studied for applications in insect control (Cheng et al., 2017). However, there have been no reports detailing an effective method of controlling this pest. Although the electroantennographic and behavioral responses of H. vitessoides to host plant volatiles have been studied (Qiao et al., 2012), little is known about the molecular basis of H. vitessoides olfaction. Therefore, the identification of olfactory genes will help expand our understanding of how this pest recognizes and locates its host, and may also facilitate the design of attractants for pest control. In this study, the female and male antennal transcriptomes of H. vitessoides were sequenced on a BGISEQ-500 platform, and 61 candidate olfactory genes were identified (nine OBPs, 10 CSPs, 28 ORs, 12 IRs, and two SNMPs). Furthermore, the sex expression profiles of nine OBPs and 10 CSPs were also determined by reverse transcription quantitative PCR (RT-qPCR). To our knowledge, this is the first report on the identification and characterization of multiple olfactory genes in this forest pest.