Identification and expression analysis of odorant binding proteins and chemosensory proteins from dissected antennae and mouthparts of the rice bug Leptocorisa acuta

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Highlights

  • 26 OBPs and 17 CSPs were identified in Leptocorisa acuta by generating transcriptomes from dissected antennae and mouthparts.

  • 11 OBPs and 9 CSPs were predominately expressed in antennae.

  • The sex-biased expression patterns of these antennae-predominant genes were characterized.

  • The phylogenetic relationships with known olfaction-related proteins from other hemipteran species were analyzed.

Abstract

The rice bug, Leptocorisa acuta (Tunberg) (Hemiptera: Alydidae), is a notorious pest in Asia, and it is significantly attracted by the volatiles derived from host plants. However, it remains unknown how L. acuta recognizes host volatile compounds at the molecular level. Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are thought to be responsible for the initial biochemical recognition during olfactory perception. Here, we followed the RNA Sequencing (RNA-Seq) approach to identify candidate genes encoding OBPs and CSPs from dissected antennae and mouthparts of L. acuta. In total, 26 unigenes were identified coding for OBPs (22 Classic OBPs and four Plus-C OBPs), and 17 unigenes coding for CSPs. Real-time quantitative PCR (RT-qPCR) revealed that 11 OBPs (LacuOBP1, 5, 6, 7, 9, 11, 13, 14, 17, 20 and 23) and nine CSPs (LacuCSP2, 3, 4, 5, 6, 8, 9, 10 and 12) were predominantly expressed in antennae, indicating that they might be essential for detection of general odorants and pheromones. Among these antennae-predominantly expressed genes, LacuOBP11 and LacuOBP13 showed male-biased expression and therefore may play crucial roles in the detection of sex pheromones. Seven LacuOBPs (LacuOBP4, 8, 10, 12, 21, 25 and 26) and two CSPs (LacuCSP7 and LacuOBP11) were predominantly expressed in mouthparts, suggesting that these genes might be involved in taste perception. Our work provides a starting point to facilitate functional study of these OBPs and CSPs in L. acuta at the molecular level in the future.

Introduction

The rice bug Leptocorisa acuta (Tunberg) (Hemiptera: Alydidae), is a notorious insect pest in rice-producing countries. This bug causes damage to rice by sucking out the contents of developing grains from pre-flowering spikelets to soft dough stage, causing unfilled or empty grains and discoloration (Hill, 2008). Nymph and adult bugs both feed on rice grains. L. acuta is also known to transmit Sarocladium oryzae and S. attenuatum (fungi), the casual agents for sheath rot disease, which causes plants to produce under-developed or damaged rice grains. Currently, the application of chemical insecticides is the primary strategy to control L. acuta (Kay et al., 1993). However, the extensive use of insecticides causes pest resistance, pest resurgence and environmental pollution of insecticide residues. L. acuta is significantly attracted to volatiles derived from host plants (Gunawardena and Ranatunga, 1989), indicating that olfaction-based approaches would be developed as a novel, environment-friendly strategy against this destructive pest. Towards this direction, the molecular mechanism for L. acuta to perceive these volatiles needs to be revealed.

Olfaction is the primary sensory modality in insects, guiding towards food sources, mating partners, and oviposition sites as well as to avoid natural enemies and other dangers (Field et al., 2000; Asahina et al., 2008). At the molecular level, olfactory perception starts with the penetration of odorant molecules into the cuticular pores in antennal sensilla in lymph. Odorants then bind to odorant-binding proteins (OBPs) or chemosensory proteins (CSPs) (Pelosi et al., 2006; Zhou, 2010). Subsequently, odorants are transported across the sensillar lymph and are delivered to odorant receptors (ORs) in the olfactory neuronal membrane. OBPs or a combination of OBPs and odorant molecules activates membrane-bound ORs, which are then transformed into electrical signals that are transmitted to the insect brain to elicit distinct behaviors (Vogt et al., 1999; Leal, 2013; Pelosi et al., 2018). Accordingly, OBPs and CSPs are very important because they help solubilize hydrophobic chemicals and may aid in odor detection, and discrimination (Pelosi et al., 2006; Leal, 2013). In addition, OBPs in mouthparts and legs might also participate in the discrimination and transportation of taste-related compounds (Swarup et al., 2014; Sun et al., 2016; Sun et al., 2017).

Insect OBPs are small soluble proteins that are highly abundant in the chemosensilla lymph, and can be categorized into five subfamilies based on the conservation of cysteine residues. The five subfamilies include ‘Classical’ OBPs each with six conserved cysteine residues (Hekmat-Scafe et al., 2002; Pelosi et al., 2006; Zhou, 2010), ‘Dimer’ OBPs each with two classical cysteine signature motifs (Hekmat-Scafe et al., 2002; Zhou et al., 2004), ‘Plus-C’ OBPs each with two extra cysteine residues in addition to the six conserved cysteine residue motif and one conserved proline residue (Zhou et al., 2004), ‘Minus-C’ OBPs each with a loss of two otherwise conserved cysteine residues, and ‘Atypical’ OBPs each with 9–10 cysteine residues and a long carboxyl terminus (Xu et al., 2003). CSPs belong to another type of small soluble proteins with four highly conserved cysteines (Pelosi et al., 2006). CSPs are highly expressed and widely distributed in chemosensory tissues. However, CSPs are also expressed broadly in non-olfactory tissues, which suggest that insect CSPs have other physiological and behavioral functions (Pelosi et al., 2006; Pelosi et al., 2014).

The first essential step towards understanding the molecular mechanism of chemosensory reception of insect is to investigate chemoreception-related genes, which encode the proteins that function in chemical detection. Genome or transcriptome analyses has led to the discovery of numerous OBP and CSP genes from important hemipteran pest, including aphids Acyrthosiphon pisum (Zhou et al., 2010), Aphis gossypii (Gu et al., 2013), Sitobion avenae (Xue et al., 2016), Myzus persicae (Wang et al., 2019); planthopper Nilaparvata lugens (Zhou et al., 2014; He et al., 2019), Sogatella furcifera (He and He, 2014; He et al., 2015; He et al., 2019), Laodelphax striatellus (He et al., 2019); psyllid Diaphorina citri (Wu et al., 2016) and Cacopsylla chinensis (Xu et al., 2019); plant bugs Adelphocoris lineolatus (Gu et al., 2011), Lygus lineolaris (Hull et al., 2014), Apolygus lucorum (Yuan et al., 2015), Apolygus lucorum (An et al., 2016), Adelphocoris suturalis (Cui et al., 2016), Halyomorpha halys (Paula et al., 2016), Tessaratoma papillosa (Wu et al., 2017), Cyrtorhinus lividipennis (Wang et al., 2017; Wang et al., 2018) and Tropidothorax elegans (Song et al., 2018). Furthermore, the functions of some OBP and CSP genes in the chemosensory perception process of hemipteran have been predicted and verified. Typically, two OBPs (ApisOBP3 and ApisOBP7) in A. pisum have been identified that regulate the behavioral response to the alarm pheromone (E)-β-farnesene (Sun et al., 2012; Zhang et al., 2017). In addition, the OBPs and CSPs in hemipteran species have been found that participate in mediating responses to plant volatiles. For example, three homologous OBPs (NlugOBP8, SfurOBP11 and LstrOBP2) in three rice planthoppers (N. lugens, S. furcifera and L. striatellus) responsible for host volatiles binding and be involved in seeking rice plants (He et al., 2019). NlugCSP8 in N. lugens have been found that mediates behavioral responses to representative host attractants (Waris et al., 2018). SfurCSP5 in S. furcifera has high binding affinities to host volatile mixtures that can attract rice planthoppers (Chen et al., 2018). Therefore, some specific OBPs and CSPs in hemipteran species play a key role in the host and pheromone recognition. In addition, in A. lineolatus, AlinOBP11 with high expression in mouthparts likely function in perceiving non-volatiles and other phytochemicals from host plants (Sun et al., 2016).

Our long-term goal is to elucidate the molecular basis for chemoreception of L. acuta and to facilitate the design and implementation of novel intervention strategies against this rice pest. As the first step, the objective of the present study is to systematically identify and characterize genes encoding OBPs and CSPs. Specifically, we generated transcriptomes from dissected antennae and mouthparts from L. acuta, and annotated all unigenes coding for OBPs and CSPs from the transcriptomes. Phylogenetic analyses were then undertaken on hemipteran OBPs and CSPs. We also conducted real-time quantitative PCR (RT-qPCR) to quantify the tissues expression patterns of the identified genes. We believe that our work provides a starting point to facilitate functional characterization of OBPs and CSPs in L. acuta.

Section snippets

Insect tissue collection and RNA isolation

The colony of L. acuta used in this study was derived from insects originally collected from rice fields in the environs of Leizhou, Guangdong, China (110°05′E, 20°34′N). The colony was maintained in the laboratory on rice seedlings in climate-controlled chamber under 28 °C ± 1 °C with 85% relative humidity and a photoperiod of 14 h light versus 10 h.

Various tissues from adults were dissected under a microscope and the tissues were homogenized to powder immediately in liquid nitrogen. The

Transcriptome assembly

To identify OBPs and CSPs from L. acuta, cDNA libraries made from adult antennae and mouthparts were sequenced using the Illumina HiSeq 2500 sequencing platform. A total of 41,574,598 and 46,815,184 raw reads were obtained from antenna and mouthpart libraries, respectively. After removing adaptor sequences, low quality sequences and N-containing sequences, approximately 41.6 million and 46.8 million clean reads were retained for these two samples. The assembly of all clean reads together led to

Discussion

In this study, we generated transcriptomes with dissected antennae and mouthparts from L. acuta adults of both females and males. A total of 26 OBP-encoding and 17 CSP-encoding unigenes were identified from the transcriptomes for the first time. RNA-seq data indicated that all OBP- and CSP-encoding genes were expressed in antennae. The total number of OBP-encoding genes in L. acuta was less than those reported from several other hemipterans. For example, A. lucorum has 38 OBP-encoding genes (

Declaration of competing interest

The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Acknowledgments

This work was supported by Guang Zhou City Key Laboratory of Subtropical Fruit Tree Outbreak Control (Grant No. 201805010008), Serious Pest and Disease Mechanism and Sustainable Control Innovation Team of Guangdong Province (Grant No. 2017KCXTD018) and Postgraduate Science and Technology Innovation Fund of Zhongkai University of Agriculture and Engineering (Grant No. KJCX2019006).

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