Functional Specificity of Sex Pheromone Receptors in the Cotton Bollworm Helicoverpa armigera

Male moths can accurately perceive the sex pheromone emitted from conspecific females by their highly accurate and specific olfactory sensory system. Pheromone receptors are of special importance in moth pheromone reception because of their central role in chemosensory signal transduction processes that occur in olfactory receptor neurons in the male antennae. There are a number of pheromone receptor genes have been cloned, however, only a few have been functionally characterized. Here we cloned six full-length pheromone receptor genes from Helicoverpa armigera male antennae. Real-time PCR showing all genes exhibited male-biased expression in adult antennae. Functional analyses of the six pheromone receptor genes were then conducted in the heterologous expression system of Xenopus oocytes. HarmOR13 was found to be a specific receptor for the major sex pheromone component Z11-16:Ald. HarmOR6 was equally tuned to both of Z9-16: Ald and Z9-14: Ald. HarmOR16 was sensitively tuned to Z11-16: OH. HarmOR11, HarmOR14 and HarmOR15 failed to respond to the tested candidate pheromone compounds. Our experiments elucidated the functions of some pheromone receptor genes of H. armigera. These advances may provide remarkable evidence for intraspecific mating choice and speciation extension in moths at molecular level.


Introduction
Male moth can accurately find and recognize mates through detection of the sex pheromone at extremely low concentration emitted from conspecific females. Notably, male moths can discriminate a subtle difference in stereochemistry or chirality of molecules or ratio change of two or a few components as a speciesspecific cue [1,2,3]. The outstanding sensitivity and specificity of moth pheromone detection has made the male moth antenna an attractive model system in animal olfactory research [4,5,6,7]. Since the first sex pheromone was discovered from the silkmoth, Bombyx mori [2], sex-pheromone components in more than 500 insect species, including many important agricultural pests, have been identified [8]. With the development of DNA sequencing technology, more and more sex pheromone receptor genes have been cloned, however, only a few have been functionally characterized.
Intensive studies have been focused on the silkworm moth B. mori since it has the simplest pheromone components and its whole genome sequence was released in 2004 [1,9,10,11,12,13]. The pheromone sensitive trichiod sensilla are in the majority amongst several different types of sensilla on male silkworm antennae [14,15]. Each trichiod sensilla houses two pheromone-sensitive neurons: one of which coexpresses BmOR1/BmOR2 that is activated by bombykol, the major pheromone that elicits the male mating behavior [16]. The other neuron coexpresses BmOR3/ BmOR2, which binds to bombykal, which has an inhibitory effect on male behavior response [1]. In addition to B.mori, much research on pheromone reception has been performed in the genus Ostrinia. Most species, including European corn borers (ECB), in the genus Ostrinia use varying ratios of Z11-and E11tetradecenyl acetate (Z11-and E11-14:OAc) as the two main components of their pheromone blend. At least five different sex pheromone receptor candidates have recently been identified and functionally characterized in vitro using Xenopus oocytes system. Except that ECB(Z) OR6 responded almost exclusively to Z11, the primary pheromone produced by ECB(Z) females, other receptors responded broadly to sex pheromone components in general [17,18,19].
Additionally, the genus Heliothis and closely related species including Helicoverpa armigera and Helicoverpa assulta are also good model systems for studying the evolution of the pheromone biosynthesis and perception systems since sex pheromone components of most species were identified and various species show distinct differentiation in sex pheromones components or ratios. All six sex pheromone receptor candidates in Heliothis virescens were identified by using a combination of genomic sequence analysis, cDNA-library screening as well as BAC library sequence and further functional characterization in heterologous expression systems, such as Xenopus oocytes or HEK293 cell culture [6,20,21]. The receptors' functional activity is closely associated with pheromone-sensitive neuronal function from single male sensillum electrophysiological recordings [22,23]. HvOR13 in A-type sensillum and HvOR6 in B-type sensillum specifically are tuned to Z11-16:Ald, the major pheromone component and Z9-14:Ald, the second pheromone component, respectively. HvOR14 and HvOR16 in C-type sensillum are, respectively, tuned to Z11-16:OAc and Z11-16:OH [21]. At this point, the function of pheromone receptors from the genus Heliothis and closely related species are still largely unknown.
Electrophysiological recordings from single male sensillum have demonstrated that there are at least two types of pheromoneresponsive sensilla in male antennae tuned to Z11-16:Ald and Z9-16:Ald, respectively [36]. At a molecular level, six Heliothishomologous pheromone receptor candidates were identified using transcriptomic analysis [37,38]. However, we know nothing about their functions.
In this study, we cloned six full-length pheromone receptor genes from H. armigera male antennae, which appeared to be orthologs of H. virescens pheromone receptor genes, respectively. Expression patterns of these pheromone receptor genes were evaluated by quantitative real-time PCR showing all genes exhibited male-biased expression in adult antennae. Finally, we characterized the functional properties of some of these pheromone receptor genes in the heterologous expression system of Xenopus oocytes.

Insect
The H. armigera colony was maintained at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. Larvae were reared on an artificial diet at 2761uC with a photoperiod of 14:10 (L:D). Pupae were selected by sex and placed in separate test tubes. Different tissues of male and female adults were excised at the base at 1-3 days after eclosion and immediately frozen in liquid nitrogen, and then stored at under 270uC until use.

RNA extraction and cDNA synthesis
The frozen tissue was transferred to a liquid nitrogen-cooled mortar and ground. The homogenate was covered with 1 mL of TriZol reagent (Invitrogen, Carlsbad, CA, USA). Further steps were performed according to the manufacturer's instruction. Total RNA was dissolved in RNA-free ddH 2 O. RNA quantity and integrity were determined on a Nanodrop ND-1000 spectrophotometer (Nano-Drop products, Wilmington, DE, USA) and gel electrophoresis. Prior to cDNA synthesis, RNA was treated with DNase I (Fermentas, Vilnius, Lithuania) to remove trace amounts of genomic DNA. The cDNA was synthesized by First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) and was used as a template in PCR reactions.
Cloning of full-length cDNA encoding pheromone receptors in H.armigera The sequences of six pheromone receptor genes and an Orco gene were identified through antennal transcriptomic analysis [38]. Four sequences including HarmOR11, HarmOR13, HarmOR16 and HarmOR2 were verified as full-length open-reading frames (ORFs) using RACE amplification in a previous report [37]. The full-length ORFs of the other three genes were verified in this study using SMARTer TM RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) following the user manual. Finally, full-length coding sequences of candidate pheromone receptor genes and Orco gene of H.armigera were PCR amplified from pools of total cDNA prepared from male antennae using primeSTAR HS DNA polymerase (Takara, Dalian, China). PCR reactions of 25 ml contained 0.25 ml primeSTAR HS DNA polymerase, 5 ml 56PrimerSTAR Buffer (Mg 2+ plus), 2 ml dNTP mixture (2.5 mM) and 0.5 ml of each primer (10 mM). All amplification reactions were carried out using a Veriti Thermal Cycler (Applied Biosystems, Carlsbad, CA, USA) under the following conditions: 94uC for 2 min; 34 cycles of 94uC for 30 s, 55-60uC for 30 s, 72uC for 1.5 min; and 72uC for 10 min. PCR amplification products were run on a 1.0% agarose gel and then verified by DNA sequencing. The primer sequences used in this study were listed in Material S1.

Sequence analysis
Multiple alignments and identity calculation were done with the ClustalW2 [39] software. The phylogenetic reconstruction implemented for the analysis of HarmPRs was performed based on the amino sequences of the six H. armigera PRs and the OR sequences identified in Lepidoptera (21 from H. virescens and 64 from B. mori) [20,40,41]. Unrooted trees were constructed by the neighborjoining with p-distance method, as implemented in MEGA5 software [42]. For transmembrane domain predictions the TMHMM Server Version2.0 (http://www.cbs.dtu.dk/services/ TMHMM) was used.

Quantitative real-time PCR
Total RNAs were extracted from antennae(A), heads without antennae (H), thoraxes (T), maxillary palps (MP), proboscises (PR), abdomens (AB), legs (L) and genitals (G). The qRT-PCR template were synthesized as described above and the negative reactions (all reagents without reverse polymerase) were sited to make sure there are no gDNA residual.. Primers were designed using the Primer Premier 5 software (PREMIER Biosoft International). The primer sequences are listed in Material S1. The annealing temperatures of the primers were controlled at 6263uC. The efficiency of primer was calculated by standard curve method. The efficiency of all the primers was ranging from 0.9-1.1. The qRT-PCR was performed following the manufacturer's instruction of the SYBR II Premix Ex Taq kit (Takara, Dalian, China) with a MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The PCR procedure was as followed: one cycle of 95uC for 30 s; 40 cycles of 95uC for 10 s, 55uC for 30 s (read); 95uC for 1 min, 55uC for 1 min. A dissociation curve was used to ensure primer specificity and lack of contamination. qRT-PCR products were analysed by 2% agarose gel electrophoresis and then verified by DNA sequencing. The housekeeping gene-actin was used as control. The experiment was repeated three times using three independent RNA samples. The expression level of the pheromone receptor genes was analyzed using th 2 2DDCT method. where DCT = (CT, PR gene-CT, actin gene), DDCT = (DCT, different samples 2DCT maximum) [43]. Statistical comparison of expressions of the pheromone receptor genes was assessed using a single classification ANOVA procedure. The data were presented as mean6SE and the images were optimized by GraphPad Prism 5.

Receptor expression in Xenopus oocytes and electrophysiological recordings
Receptor expression and electrophysiological recording were performed as decribed in previous reports [44,45]. The full-length cDNA sequences of pheromone receptors were first cloned into pENTR/D-TOPO entry vectors (Invitrogen, Carlsbad, CA, USA) and then subcloned into pSP64T (converted from pSP64T-Oligo) destination vectors by means of the Gateway LR reaction. cRNAs were synthesized from linearized vectors with mMESSAGE mMACHINE SP6 (Ambion, Austin, TX, USA). Mature healthy Xenopus oocytes (stage V-VII) were separated and then treated with 2 mg/ml collagenase I in washing buffer (96 mM NaCl, 2 mM KCl, 5 Mm MgCl2, and 5 mM HEPES, pH 7.6) for 1-2 h at room temperature. The mixture of 27.6 ng pheromone receptor cRNA each and 27.6 ng HarmOrco (HarmOR2) cRNA Oocytes were later microinjected into the oocytes. The Xenopus oocytes were incubated for 4-7 days at 18uC in incubation medium (16 Ringer's buffer, 5% dialysed horse serum, 50 mg/ml tetracycline, 100 mg/ml streptomycin and 550 mg/ml sodium pyruvate) after injection. Whole-cell currents of the injected oocytes were recorded with a two-electrode voltage clamp. Odorant induced

Results
cDNA cloning and sequence analysis of pheromone receptor genes in the H. armigera Six cDNA sequences encoding H. armigera pheromone receptor genes were identified (GenBank accession number: KC538876-KC538881) and three of these sequences including HarmOR11, HarmOR13 and HarmOR16 were verified as full-length openreading frames in previous reports [37,38]. The full-length ORFs of other three genes including HarmOR6, HarmOR14 and HarmOR15 were obtained using RT-PCR and 59/39-RACE amplification. The lengths of amino acid residues encoded by these pheromone receptor genes ranged from 425 to 440 amino acid residues and were predicted to possess seven transmembrane domains. Multiple alignments of H. armigera PRs and their homologs in H. virescens showed the orthologous PRs in these two insects had high similarity. The identities between the two species of OR6, OR11, OR13, OR14, OR15, and OR16 were 80.3%, 95.8%, 90.9%, 84.1%, 83.6% and 88.2%, respectively ( Figure 1). The phylogenetic analyses showed that these PRs were clustered together with other lepidopteran PRs separated from general odor receptors (Figure 2).

Tissue-specific and male-biased expression of six sex pheromone receptor genes
The average values of expression levels of all sex pheromone receptor genes are from three biological replications by quantitative real-time PCR. All sex pheromone receptor genes were expressed in male antennae with higher levels compared to female antennae and the ratio (male: female) Figure 3A). In male antennae, HarmOR13 was detected as the most abundant, 2.2460.15, 5.9860.62, 8.3060.27 and 13.1561.86 times higher than HarmOR11, 16, 15 and 14, respectively. HarmOR6 was detected at the lowest level and was approximately two orders of magnitude less abundant than HarmOR13 ( Figure 3B). Generally speaking, all sex pheromone receptor genes were very weakly expressed, if at all, in other tissues such as heads, legs, abdomen and mouthparts except for HarmOR11 showing significant expression at male, but not female heads.
HarmOR13 is a specific receptor for the major sex pheromone component Each of the six pheromone receptors were co-expressed in Xenopus oocytes with the obligatory functional chaperone Har-mOrco for 5-7 days [1,21] followed by testing using a panel of candidate H. armigera pheromone compounds including the major and second sex pheromone components (Z11-16:Ald and Z9-16:Ald). The oocytes co-expressing HarmOR13 and HarmOrco robustly responded to 10 24 M Z11-16: Ald and yielded little if any response to Z9-14:Ald. In dose-response studies, 10 27 M Z11-16:Ald could elicit significant responses from oocytes that co- HarmOR16 is sensitively tuned to Z11-16: OH Xenopus oocytes co-expressing HarmOR16 and HarmOrco strongly responded to Z11-16:OH and have a mean amplitude response of 233nA. In the dose response studies, oocytes had measurable responses as low as 10 28 M and the EC 50 to Z11-16:OH was 3.0610 27 M. Xenopus oocytes co-expressing Har-mOR16 and HarmOrco also had significant response to 10 24 M Z9-14:Ald ( Figure 6). The other three candidate pheromone receptors including HarmOR11, HarmOR14 and HarmOR15 failed to respond to the tested candidate pheromone compounds (Figure 7).

Discussion
Sex pheromone communication between male and female moths is believed to have contributed to successful intraspecific mating choice and interspecific isolation [46]. The behavioral response of male moths to sex pheromone has been proven to be closely linked to the activity of the peripheral olfactory receptor neurons (ORNs) [1,17]. The peripheral ORNs of male moths, which are tuned to sex pheromone components, are housed within long sensilla trichodea in the antennae usually in groups of two or three ORNs in each sensillum. Recent studies revealed that in many, if not all, cases the odor receptor was the primary determinant of the odor response spectrum of the peripheral ORN [21,47,48]. The cotton bollworm, H. armigera and its closely related species, such as H. assulta and Heliothis spp. have similar sex pheromones components with tiny differentiations in minor components or ratios. Therefore, elucidating the functions of pheromone receptor genes will provide remarkable evidence for intraspecific mating choice and speciation extension in moths at molecular level.
In this study, we identified and cloned the full-length ORFs of six sex pheromone receptor genes from H. armigera antennae by RT-PCR and RACE techniques based on previous transcriptomic information [38]. Each gene has a corresponding homolog in H. virescens and the protein sequences encoded by these genes possess putative seven-transmembrane domains, the typical characteristic of insect odor receptors. Not surprisingly, all these sex pheromone receptor genes showed male-biased expression patterns, since long trichodea sensilla, tuned to sex pheromone components emitted from conspecific females, are significantly abundant in male antennae. Notably, OR13 from H. virescens and H. armigera that shared identity up to 91% and specifically tuned to the major sex pheromone component (Z11-16:Ald), were detected as the most abundant transcript in male antennae of both species [6,21,49]. The function of OR6 was distinct between species even though they were 88% identical. HvOR6 is narrowly tuned to the second sex pheromone component, Z9-14:Ald, emitted from H.virescens female. And HarmOR6 is equally tuned to both Z9-16:Ald and Z9-14:Ald, which recently has been thought to be minor sex pheromone components in H.armigera. The function of OR16 has slightly diverged in the two species. Both OR16 genes are sensitively tuned to Z11-16:OH, an attraction inhibitor when added to attraction blends [27,29]. HarmOR16 also significantly responded to Z9-14:Ald, which may explain why Z9-14:Ald acted as an attraction inhibitor at high concentrations. As in H.virescens, no ligand for OR11 and OR15 was identified. The neuron which houses OR11 or OR15 in H.virescens didn't respond to tested sex pheromone components in previous electrophysiological recording [22]. The possible explanation is that those genes lost their function during evolution or their ligands aren't included in tested odor panel. OR14 from H.virescens has reasonable but weak response to Z11-16:Ac, however, the oocyte that coexpressed HarmOR14/HarmOrco didn't respond to any odorants tested in this study. Our studies didn't involve PBPs, further testing of OR function with PBPs is certainly needed. To date, although no solid experiment has shown that any PBP, with the exception of lush in Drosophila [50], are indispensable for corresponding pheromone receptor's function either in vitro or in vivo, several publications indicated that PBPs could remarkably increase the specificity or sensitivity for some pheromone receptors [6,51,52].
In H.virescens, in situ hybridization studies for all pheromone receptor genes except for HvOR6 have been carried out and single sensillum electrophysiological recordings were available. Thus, these pheromone receptor genes could be specifically assigned to six neurons of three types of long trichodea sensilla. Further studies in H.armigera on colocalization of pheromone receptor genes and electrophysiological function of pheromonesensitive receptor neurons will make integrated comparison of sex pheromone perception in the two closely related species possible and also significantly improve the importance of this study.

Supporting Information
Material S1 Primers for gene clone and qRT-PCR expression analyses of HarmPRs. (XLSX)