Specificity of the Receptor for the Major Sex Pheromone Component in Heliothis virescens

In a previous study, the Drosophila melanogaster OR67dGAL4;UAS system was used to functionally characterize the receptor for the major component of the sex pheromone in the tobacco budworm, Heliothis virescens Fabricius (Lepidoptera: Noctuidae), HvOR13. Electrophysiological and behavioral assays showed that transgenic flies expressing HvOR13 responded to (Z)-11-hexadecenal (Z11-16:Ald). However, tests were not performed to determine whether these flies would also respond to secondary components of the H. virescens sex pheromone. Thus, in this study the response spectrum of HvOR13 expressed in this system was examined by performing single cell recordings from odor receptor neuron in trichoid T1 sensilla on antennae of two Or67dGAL4 [1]; UAS-HvOR13 lines stimulated with Z11-16:Ald and six H. virescens secondary pheromone components. Fly courtship assays were also performed to examine the behavioral response of the Or67dGAL4[1]; UAS-HvOR13 flies to Z11-16:Ald and the secondary component Z9-14:Ald. Our combined electrophysiological and behavioral studies indicated high specificity and sensitivity of HvOR13 to Z11-16:Ald. Interestingly, a mutation leading to truncation in the HvOR13 C-terminal region affected but did not abolish pheromone receptor response to Z11-16:Ald. The findings are assessed in relationship to other HvOR13 heterologous expression studies, and the role of the C-terminal domain in receptor function is discussed. A third line expressing HvOR15 was also tested but did not respond to any of the seven pheromone components.


Introduction
Studies on pheromone processing by male moths have greatly contributed to the understanding of the mechanisms involved in animal sensory perception (Rützler and Zwiebel 2005;Touhara and Vosshall 2009; Kaupp 2010). Work in this area has been focused mainly on two moth species, Bombyx mori and Heliothis virescens Fabricius (Lepidoptera: Noctuidae), with the former having a simple two-component pheromone blend (Kaissling and Kasang 1978) and the latter a more complex pheromone blend (Vetter and Baker 1983). Molecular aspects of male pheromone reception in these two moths have been examined using different approaches (Sakurai et al. 2004(Sakurai et al. , 2011Nakagawa et al. 2005;Grosse-Wilde et al. 2007;Kurtovic et al. 2007) for which differences in the level of pheromone receptor response specificity and sensitivity have been observed.
The GAL4/UAS targeted gene expression system in Drosophila melanogaster (Brand and Perrimon 1993) has been used to functionally characterize odorant receptors (ORs) in insects (Dobritsa et al. 2003;Hallem et al. 2004a;Jones et al. 2005;Carey et al. 2010). The "empty neuron" in the ab3 basiconic sensilla (Δhalo;OR22a-Gal4/UAS) has been a useful tool for characterization of general odorant receptors as well as sex pheromone receptors (Hallem et al. 2004b;Hallem and Carlson 2006;Syed et al. 2006), while the knock-in mutant in trichoid T1 sensilla (OR67d GAL4 ;UAS) has been used to functionally characterize the D. melanogaster receptor for the pheromone cVA (Z11-18:OAc) (Ha and Smith 2006) and two moth sex pheromone receptors (Kurtovic et al. 2007). Recently, Syed et al. (2010) expressed a pheromone receptor from the silkworm moth, B. mori, BmOR1, in both of these expression systems (i.e., basiconic and trichoid) and found a more sensitive and specific receptor response in fly trichoid sensilla T1 than in the ab3 basiconic sensilla. The latter system not only needed higher doses of bombykol to stimulate the receptor, but was also unusually sustained, suggesting that this system is less suitable for testing pheromone receptors.
In male moths, neurons expressing pheromone receptors are housed in trichoid sensilla dedicated to pheromone reception (Christensen and Hildebrand 2002). Therefore, the higher sensitivity and specificity of the D. melanogaster trichoid T1 sensilla system expressing BmOR1 may be due to the innate biochemical machinery and structural features of sensilla for detection of the sex pheromones, in which case a similarly sensitive and specific response is expected for other moth sex pheromone receptors. For example, the H. virescens major pheromone component receptor, HvOR13, expressed in D. melanogaster trichoid T1 sensilla was found to respond to its putative ligand (Z)-11hexadecenal (Z11-16:Ald) (Kurtovic et al. 2007). However, it is unclear whether this receptor, expressed in D. melanogaster, has high specificity for Z11-16:Ald or could also respond to some or all of the secondary components of the H. virescens sex pheromone. Functional analyses of other moth pheromone receptors by Wanner et al. (2010) and Miura et al. (2010) both found that some pheromone receptors of Ostrinia nubilalis were broadly tuned, while one receptor appeared to be highly specific for one pheromone component. Our study was therefore designed to determine the degree of specificity of HvOR13 expressed in this system by performing single cell recordings from odor receptor neurons in trichoid T1 sensilla on antennae of two Or67d GAL4 [1] ;UAS-HvOR13 lines and a control line (Or67d +[1] ) stimulated with Z11-16:Ald and six H. virescens secondary pheromone components. The electrophysiological response of another construct, Or67d GAL4 [1] ;UAS-HvOR15, to the seven H. virescens pheromone components was also examined. HvOR15 was considered a candidate receptor for Z9-14:Ald (Baker 2009;Krieger et al. 2009, Gould et al. 2010; however, it has been shown that it does not respond to this pheromone component in a Xenopus laevis oocyte system (Wang et al. 2011), a finding that we expect to corroborate in the D. melanogaster trichoid T1 sensilla system. In addition, fly courtship assays were performed to examine the behavioral response of the Or67d GAL4 [1] ;UAS-HvOR13 flies to Z11-16:Ald and Z9-14:Ald. Expression levels of HvOR13 in both Or67d GAL4 [1] ;UAS-HvOR13 lines were measured by qRT-PCR to determine if HvOR13 expression was associated to differential electrophysiological and behavioral responses between the two Or67d GAL4 [1] ;UAS-HvOR13 lines. The combined electrophysiological and behavioral studies indicated that HvOR13 showed high specificity and sensitivity for Z11-16:Ald, with results comparable to those observed for HvOR13 heterologously expressed in X. laevis oocytes (Wang et al. 2011), a useful finding considering that expression of other insect odorant receptors in these two systems do not always produce similar results Wang et al. 2010). Interestingly, a truncation of the HvOR13 C-terminal region appeared to affect, but did not completely abolish, pheromone receptor function, a finding that could be linked to the functional importance of the Cterminal domain in the formation of the odorant receptor/Orco heteromeric complex (Benton et al. 2006;de Bruyne et al. 2009;Vosshall and Hansson 2011).

Single sensillum recordings
Recordings were performed as described by Syed et al. (2006). In brief, a D. melanogaster adult was restrained, a glass reference electrode was placed in the eye, and the recording electrode was brought into contact with the base of the trichoid sensillum. Recorded extracellular action potentials (spontaneous and upon stimulation) were amplified, fed into an IDAC4-USB box (Syntech, www.syntech.nl), recorded, and analyzed with Auto Spike version 3.7 (Syntech). AC signals (action potentials or spikes) were band-pass filtered between 100 and 10,000 Hz. The preparation was held in a humidified air stream delivered at 20 mL/sec, to which a stimulus pulse of 2 mL/sec was added for 500 msec. Unless specified otherwise, signals were recorded for 10 sec starting 2 sec before stimulation, and spikes were counted off-line in a 500 msec period before and during the stimulation. Responses from individual neurons were calculated as the increase in spike frequency (spikes/sec) relative to the pre-stimulus frequency. At least three flies of each of the four genotypes (Or67d +[1] , Or67d GAL4 [1] ;UAS-HvOR13, Or67d GAL4 [1] ;UAS-HvOR13*, and Or67d GAL4 [1] ;UAS-HvOR15) were examined, and recordings were made from up to five sensilla from each fly tested. Data were pooled after observing no significant differences between sensilla, sexes, or age groups (1-to 5-day-old flies) were observed.

Courtship assays
Single pair courtship assays were performed following Kurtovic et al. (2007). In brief, ;UAS-HvOR13* male flies (5-8 days old). A clear acetate sheet prevented contact between female and male flies. Flies were allowed to recover for 1 hr before behavioral assays were performed. Courtship index, the percentage of time for which the male courts the female during a 10-min assay, was used to quantify male courtship behavior. In these assays, Or67d +[1] male flies were expected to be avid courters in greater than 70% of all treated female flies. In the courtship ritual, the male orients toward and follows the female, taps her with his forelegs, sings a courtship song by extending and vibrating one wing, licks her genitalia, and finally curls his abdomen for copulation (Demir and Dickson 2005 Random hexamers (Applied Biosystems, Invitrogen) were used as cDNA synthesis primers in a reaction mix that included 10X Array Script buffer (Ambion), RNAseOUT (Invitrogen), and 10 mM dNTP (Invitrogen). Primers targeting exons of HvOR13 and the housekeeping gene RP49 were designed with PRIMER EXPRESS 2.0 software (Applied Biosystems) set to select for an optimal primer annealing temperature of 59° C (58-60° C), amplicon sizes of 40-150 bp, a -3'GC clamp = 0, and a minimum and maximum GC content of 30% and 80%, respectively. Primers were designed based on H. virescens OR13 and B. mori RP49 mRNA sequences obtained from GenBank database. Quantitative RT-PCR was performed with an ABI Prism 7900 sequence detector and 96-well optical reaction plates (Applied Biosystems). All reactions were performed in triplicate in a total volume of 10 µL containing 5 µL of SYBR Green PCR Master Mix (Applied Biosystems) and 10 µM of each primer under the following conditions: 50° C for 2 min, 95° C for 10 min followed by 40 cycles of denaturation at 95° C for 15 sec, annealing and extension at 60° C for 1 min, followed by 95° C for 15 sec and 60° C for 15 sec. A dissociation curve and negative control (cDNA reaction without reverse transcriptase enzyme) were used to ensure primer specificity and lack of contamination. Six samples per genotype were examined, and the same samples were run on separate plates twice (two runs). A standard curve was generated for each primer set using dilutions of genomic DNA to calculate the relative quantities of mRNA levels. For each sample, the ratio of the expression level of the target gene to that of the control gene (RP49) was calculated (ABI User Bulletin 2, Applied Biosystems) and used for data analysis.  Significant differences among treatments within a column are indicated with different alphabetic letters (LSMeans, α = 0.05). Courtship index is defined as the percentage of time the male courts the female during a 10-min assay.
the following conditions: 94° C for 3 min followed by 19 cycles of denaturation at 94° C for 1 min, annealing at 57° C for 1 min with 0.5° C decreasing per cycle, extension at 72° C for 2 min followed by 19 cycles of denaturation at 94° C for 1 min, annealing at 47° C for 1 min and extension at 72° C for 2 min, followed by 72° C for 7 min. PCR products were purified and sequenced by Genewiz (www.genewiz.com). Nucleotide and translated sequences were aligned using ClustalW2 (EMBL-EBI, www.ebi.ac.uk).

Discussion
The combined electrophysiological and behavioral data showed that the response of HvOR13 expressed in D. melanogaster T1 sensilla (OR67d GAL4 ;UAS) was highly sensitive and specific and in agreement with the findings of Syed et al. (2010) for BmOR1 in the same heterologous expression system. Expression of HvOR13 and other H. virescens pheromone receptors in Flp-In T-Rex293/Gα 15 cells (Grosse-Wilde et al. 2007) indicated that this heterologous expression system is not as specific as the D. melanogaster system that we used. Moreover, pheromone binding proteins and an organic solvent had to be used in the cell system to increase sensitivity and specificity of the H. virescens receptors tested. It is possible that D. melanogaster biochemical components involved in cVA detection may increase the sensitivity and specificity of moth pheromone receptors expressed in the OR67d GAL4 ;UAS system.
Recently, Wang et al. (2011) expressed HvOR13 in X. laevis oocytes and found a level of specificity of the receptor response based on their electrophysiological results that is comparable to the results reported in our study. In the same study, it was found that HvOR15 expressed in X. laevis oocytes did not respond to H. virescens pheromone components and 50 general odorants. Thus, the lack of response of Or67d GAL4 [1] ;UAS-HvOR15 flies to the seven pheromone components tested in our study supports the findings of Wang et al. (2011). Despite the technical differences between the D. melanogaster and the X. laevis systems, both are useful for characterization of pheromone receptors in moths and possibly other insect taxa. It is important to note that when X. laevis oocytes and D. melanogaster empty neurons were used for functional characterization of a large set of Anopheles gambiae odorant receptors, the two methods did not always produce similar results Wang et al. 2010). This emphasizes the need to conduct both types of assays in order to make firm conclusions about functional specificity of important receptors. In addition, expressing moth odorant receptors in fly trichoids offers a system to test the behavioral output in response to cognate pheromone ligands.
The results of our study also suggest that a point mutation leading to a major amino acid change at position 316 (L Q), and another resulting in an early termination codon at 384 (Q stop codon), in Or67d GAL4 [1] ;UAS-HvOR13* flies may have affected HvOR13 structure and function, which could be associated with differences in electrophysiological and behavioral responses observed between Or67d GAL4 [1] ;UAS-HvOR13 lines. It has been shown that the C-terminal domain of odorant receptors plays a an important role in the formation of the odorant receptor/Orco heteromeric complex (Benton et al. 2006;de Bruyne et al. 2009), and that the three conserved motifs (A, B, and C) within the last 70-90 amino acid residues of this region ap-pear to have major functional importance (Miller and Tu 2008). As shown in Figure 1, Or67d GAL4 [1] ;UAS-HvOR13* flies have an incomplete HvOR13 C-terminal region. It is possible that the lack of motif C and the partial motif B may be affecting heterodimer formation and the localization and stability of HvOR13 in OR neuron dendrites (Benton et al. 2006;de Bruyne et al. 2009). However, HvOR13 response was not completely obliterated, indicating that a C-terminal missing the last 42 amino acids could affect but not necessarily abolish receptor function. Also, the Nterminal half of odorant receptors has been suggested to be involved in odor binding (de Bruyne et al. 2009), which may explain how this mutated pheromone receptor could possibly bind to its ligand, Z11-16:Ald. Thus, we suggest that the Or67dGAL4-UAS system is not only a powerful tool for characterization of insect pheromone receptors, but is also very useful for testing mutations that could affect pheromone receptor function.  ;UAS-HvOR13 but not from one expressing HvOR13* in a trichoid sensillum of D. melanogaster Or67d GAL4 [1] ;UAS-HvOR13* . A. Traces of the excitatory responses recorded from Or67d GAL4 [1] ;UAS-HvOR13 T1 sensilla to increasing pheromone dose. B. Dose-response curve for Or67d GAL4 [1] ;UAS-HvOR13 (n = 11) and response to 10 μg for Or67d GAL4 [1] ;UAS-HvOR13* (n = 9). No differences between males and female responses were recorded. High quality figures are available online.   16:Ald, were tested at 10 μg source dose, except Z11-16:Ald, which was tested at 0.01, 0.1, 1, and 10 μg source doses (n = 11).High quality figures are available online.