Cloning and Expression Profiling of Odorantbinding Proteins in the Tarnished Plant Bug, Lygus Lineolaris

Insect bs_bs_banner Molecular Biology Insect Molecular Biology (2014) 23(1), 78–97 doi: 10.1111/imb.12064 Cloning and expression profiling of odorant-binding proteins in the tarnished plant bug, Lygus lineolaris J. J. Hull1*, O. P. Perera1† and G. L. Snodgrass† OBP, alternative splice variants, next-generation *USDA-ARS Arid Land Agricultural Center, Maricopa, sequencing. AZ, USA; and †Southern Insect Management Research Unit, USDA-ARS, Stoneville, MS, USA Introduction Abstract The tarnished plant bug, Lygus lineolaris, is a piercing- sucking polyphagous pest found throughout agricultural In insects, the perception and discrimination of regions in the USA and Canada. They have been reported odorants requires the involvement of odorant-binding to feed on >300 plant species, including a number of proteins (OBPs). To gain a better molecular under- important crops such as soybean, strawberry, alfalfa and standing of olfaction in the agronomic pest Lygus cotton (Young, 1986). Management practices have tradi- lineolaris (the tarnished plant bug), we used a tionally relied on broad-spectrum insecticides; however, transcriptomics-based approach to identify potential field resistance to many of the commonly used chemis- OBPs. In total, 33 putative OBP transcripts, including tries has been reported (Snodgrass, 1996; Snodgrass the previously reported Lygus antennal protein & Scott, 2000, 2002; Snodgrass et al., 2009). The (LAP), were identified based on the characteristic decreased efficacy of these traditional chemical OBP Cys signature and/or sequence similarity with approaches, coupled with the success of transgenic crops annotated orthologous sequences. The L. lineolaris against lepidopteran pests, has resulted in the elevation OBP (LylinOBP) repertoire consists of 20 ‘classic’ of Lygus pest status, in particular in the southeas- OBPs, defined by the spacing of six conserved Cys tern USA (http://www.entomology.msstate.edu/resources/ residues, and 12 ‘Plus-C’ OBPs, defined by the croplosses/2011loss.asp). Further compounding its pest spacing of eight conserved Cys and one conserved status, the development of alternative molecular-based Pro residue. Alternative splicing of OBP genes control strategies have been hampered by a lack of knowl- appears to contribute significantly to the multiplicity edge concerning the genetics governing Lygus biology of LylinOBP sequences. Microarray-based analysis of and physiology, in particular how this pest species inter- chemosensory tissues (antennae, legs and probos- acts with and responds to its external environment. cis) revealed enrichment of 21 LylinOBP transcripts in Like most insects, the foraging and reproductive behav- antennae, 12 in legs, and 15 in proboscis, suggesting iors of L. lineolaris and its sister species (e.g. Lygus hes- potential roles in olfaction and gustation respectively. perus, Lygus elisus, Lygus shulli, and Lygus rugulipennis) PCR-based determination of transcript abundance for are strongly influenced by environmentally defined chemi- a subset of the LylinOBP genes across multiple adult cal cues (Blackmer et al., 2004; Innocenzi et al., 2005; tissues yielded results consistent with the hybridiza- Frati et al., 2008) that trigger antenna-derived neuronal tion data. responses (Chinta et al., 1994; Ho & Millar, 2002; Keywords: Lygus lineolaris, odorant-binding pro- Innocenzi et al., 2004; Frati et al., 2009; Williams et al., teins, olfaction, real-time PCR, Plus-C OBP, classic 2010). As the primary sensory organs of olfaction, insect antennae typically contain, depending on the species, several hundred to several thousand sensilla hairs and First published online 14 November 2013. their associated sensory neurons (Zacharuk, 1980). The Correspondence: J. Joe Hull, USDA-ARS Arid Land Agricultural Research Center, 21881 N. Cardon Lane, Maricopa, AZ 85138, USA. Tel.: +1 520 316 antennae of L. lineolaris adults are four-segmented 6334; fax: +1 520 316 6330; e-mail: [email protected] appendages that contain ∼2000 sensilla hairs correspond- 1These authors contributed equally. ing to six sensillar types representing sensilla trichodea, 78 Published 2013. This article is a U.S. Government work and is in the public domain in the USA. Odorant-binding proteins in Lygus lineolaris 79 sensilla chaetica, and sensilla basiconica (Chinta et al., 2013). Consequently, to assess the role of OBPs in olfac- 1997). Activation of the neurons housed within these tion, in vitro binding assays have been used to examine sensilla triggers the stereotypical behavioural responses the binding capacity of recombinantly expressed OBPs associated with avoidance, host recognition, oviposition for specific odorant ligands (Zhou, 2010). In vivo studies, and mating (Martin et al., 2011). Odorants gain access to however, provide more concrete evidence regarding the the neurons via cuticular pores that line the various physiological significance of putative OBPs. Mutational sensilla. Odorant-binding proteins (OBPs) facilitate the studies in Drosophila melanogaster have implicated movement of these predominantly lipophilic compounds an OBP termed LUSH in male detection of the sex through the aqueous sensillar lymph to specific olfactory pheromone, 11-cis-vaccenyl acetate (Xu et al., 2005; receptors (ORs) in the neuronal membrane (Leal, 2005; Laughlin et al., 2008), whereas studies using inter- Zhou, 2010). OBPs have also been implicated in indirect species hybrids of Drosophila showed that OBP57d- OR activation with the odorant-bound OBP conformation emediated Drosophila sechiella attraction to and D. serving as the ligand that interacts with the OR-binding melanogaster avoidance of odours from a D. sechiella pocket (Zhou, 2010; Sachse & Krieger, 2011). Conse- host plant (Matsuo et al., 2007). Systematic RNA quently, OBPs are generally thought to comprise the initial interference-mediated suppression of OBP expression signal filtering mechanism in olfaction. Some OBPs have has since demonstrated the relevance of OBPs in medi- also been implicated in odorant signal sequestration/ ating the detection and behavioural responses of D. termination (Steinbrecht, 1998). melanogaster to a host of ecologically relevant odours Insect OBPs are a family of relatively small (∼150 (Swarup et al., 2011). OBPs have also been linked to the amino acid; 15–20 kDa) water-soluble proteins character- detection of olfactory cues and blood-feeding behaviour ized by six highly conserved Cys residues (C1-C6) in in mosquitos (Biessmann et al., 2010; Pelletier et al., which the number of amino acids between C2-C3 (three 2010; Sim et al., 2012). Further evidence for the biologi- residues) and C5-C6 (eight residues) is invariant. OBPs cal importance of OBPs in olfaction was demonstrated by can be further subgrouped based on the presence or the significant enhancement in OR sensitivity in various absence of Cys residues: ‘classic’ OBPs are character- expression systems after the addition of specific OBPs ized by six Cys residues; ‘dimer’ OBPs are characterized (Syed et al., 2006; Forstner et al. 2009). by two ‘classic’ Cys signature motifs; ‘Plus-C’ OBPs have While the molecular basis of insect olfaction has been a highly conserved Pro residue and usually two addi- extensively examined in holometabolous insects (e.g. tional Cys residues; ‘Minus-C’ OBPs lack two of the six lepidopterans and dipterans), elucidation of the molecular highly conserved Cys; and ‘atypical OBPs’ have 9–10 components and mechanisms that comprise the hemip- Cys residues with an extended carboxyl terminus (Zhou, teran olfactory system has not been as fully developed. An 2010). Aside from the Cys spacing, OBPs are poorly initial effort using a novel computational algorithm with conserved across species and have no homology with deposited express sequence tag (EST) sequences from vertebrate OBPs (Zhou, 2010). Consequently, the multiple hemipteran species identified a relatively small identification and annotation of putative insect OBPs has number of putative OBPs (Xu et al., 2009). Those data relied extensively on the characteristic Cys signature have since been supplemented with recent genome and (Zhou et al., 2004, 2010, 2008; Li et al., 2005; Forêt & EST-based OBP projects in aphids (Zhou et al., 2010), the Maleszka, 2006; Jordan et al., 2008; Gong et al., 2009; lucerne plant bug (Adelphocoris lineolatus) (Gu et al., Liu et al., 2009; Xu et al., 2009, 2010; Gotzek et al., 2011a), the green plant bug (Apolygus lucorum) (Gu et al., 2011; Gu et al., 2011a; Mitaka et al., 2011). OBPs have 2011a; Hua et al., 2012), and the brown planthopper been identified from more than 40 insect species repre- (Nilaparvata lugens Stål) (Noda et al., 2008; Xu et al., senting 10 different orders with many of the transcripts 2009; He et al., 2011). Our understanding of the molecular predominantly expressed in olfactory tissues (Zhou, components comprising the Lygus olfactory system is 2010). Surprisingly, non-olfactory OBP transcripts, which even more incomplete with sequence and expression presumably have potential roles outside of olfaction, data currently limited to Lygus antennal protein (LAP), a have also been reported (Gong et al., 2009; Pelletier & ‘classic’ OBP preferentially expressed in adult male Leal, 2009, 2011; Vogel et al., 2010; Del Campo et al., L. lineolaris and L. hesperus antennae (Dickens et al., 2011; Sun et al., 2012). As elaborated by Leal (2005), 1995, 1998; Vogt et al., 1999), and the recently identified a major limitation to structural-based OBP gene annota- Lygus OR coreceptor

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