Functional Studies of Sex Pheromone Receptors in Asian Corn Borer Ostrinia furnacalis

Lepidopteran insects use sex pheromones for sexual communication. Pheromone receptors expressed on peripheral olfactory receptor neurons (ORNs) are critical part to detect the sex pheromones. In genus Ostrinia, several pheromone receptors were functional analyzed in O. nubilalis and O. scapulalis but the knowledge in O. furnacalis was rare. In this study, seven pheromone receptors were deorphanized by heterologous expression system of Xenopus oocytes. Functional types of sensilla trichoidea were classified by single sensillum recordings to interpret the response pattern of olfactory sensory neurons to Ostrinia pheromone components. OfurOR4 and OfurOR6 responded to the major sex pheromone Z/E12-14:OAc. OfurOR4 is the main receptor for both Z/E12-14:OAc and OfurOR6 mainly responded to E12-14:OAc. Functional differentiation of gene duplication were found between OfurOR5a and OfurOR5b. OfurOR5b showed a broad response to most of the pheromone components in O. furnacalis, whereas OfurOR5a was found without ligands. OfurOR7 showed a specific response to Z9-14:OAc and OfurOR8 mainly responded to Z11-14:OAc and E11-14:OAc. OfurOR3 did not respond to any pheromone components. Our results improved the current knowledge of pheromone reception in Ostrinia species which may contribute to speciation.


INTRODUCTION
Sex pheromone has been used by organisms for sexual communication, this remarkable trait is representative in insects especially for Lepidopterans (Symond et al., 2011). Male could detect and respond to female pheromone over long distance, e.g., 11 km for emperor moth Pavonia pavonia (Regnier and Law, 1968). Moth percept the sex pheromone via the pheromone sensitive trichoid sensilla distributed on their antennae. The entire olfactory system is heavily dependent on the types of receptors expressed in peripheral olfactory receptor neurons (ORNs; also called olfactory sensory neurons) which housed in the olfactory sensilla (Leal, 2013). This has been proved unambiguously by expressing an allospecific pheromone receptor PxylOR1 from the diamondback moth in the ORN that houses the bombykol receptor BmorOR1 in the silkworm moth, Bombyx mori. Electrophysiological and behavioral experiments showed that PxylOR1expressing male silkworm moths responded equally to bombykol (E10Z12-16:OH) and Z11-16:Ald (Sakurai et al., 2011).
Asian corn borer, O. furnacalis, is a grievous pest in China and causing serious damage on economic crop maize for 10-30% yield lost (Wang et al., 2000). In addition, this species fed on various host (over 27 species) belonging to nine families (Yuan et al., 2015). Females of O. furnacalis use Z12-14:OAc and E12-14:OAc with the ratio of 1:1 as their major sex pheromone components to attract males (Cheng et al., 1981;Huang et al., 1998b). Although the pheromone receptors were functionally characterized in the sibling species such as O. nubilalis and O scapulalis, only one pheromone receptor (OfurOR4) and an odorant receptor co-receptor (OfurOR2) has been deorphanized in O. furnacalis (Leary et al., 2012;Yang et al., 2015Yang et al., , 2016Zhang et al., 2015). The functional types of the sensilla have been described by Takanashi et al. (2006) and Domingue et al. (2007). In this study, all the pheromone receptors in O. furnacalis were functionally characterized using Xenopus oocytes system. In addition, single sensillum recordings were carried out to confirm the ORNs response for detecting the pheromones.

Insects
Ostrinia furnacalis was maintained under laboratory conditions with artificial diet at 28 • C, 14:10 (L:D), 60% relative humidity. Pupae were placed in tube individually for eclosion. Two-dayold adults were used in the present study. Male antennae were removed and frozen in liquid nitrogen immediately, then stored under −80 • C until use.

RNA Extraction and cDNA Synthesis
Total RNA was isolated from male antennae with TriZol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer's instruction. The cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) after a DNase I (Thermo Scientific) treatment. The quality of RNA was verified by Nanodrop ND-1000 spectrophotometer (NanoDrop Products, Wilmington, DE, United States) and gel electrophoresis.

Cloning of Pheromone Receptors in O. furnacalis
Full length of ORF encoding odorant receptors of O. furnacalis was obtained from antennal transcriptomic analysis and amplified by PCR using primeSTAR HS DNA polymerase following the manual (Takara, Dalian, China) . Primers used in this study were listed in Supplementary  Table S1. Transmembrane domains were predicted by TMHMM Server Version 2.0 1 and multiple sequence alignment and identity calculation were done by the DNAMAN 6.0 (Lynnon Biosoft, United States).

Electrophysiological Recordings Using Xenopus Oocyte System
Each receptor was first cloned into a blunt-vector (TransGen Biotech, China), subsequently subcloned into a PT 7 TS vector, and then took for cRNA synthesis using mMESSAGE mMACHINE TM T7 Kit (Thermo Fisher Scientific). Mature healthy Xenopus oocytes (stage V-VII) were prepared according the description from Liu et al. (2013). Briefly, the oocytes were separated and then treated with 2 mg/ml collagenase I in washing buffer (96 mM NaCl, 2 mM KCl, 5 mM MgCl 2 and 5 mM HEPES, pH 7.6) for 1-2 h at room temperature. The 1:1 mixture of pheromone receptor and OfurOrco (OfurOR2) cRNA (27.6 ng each) were microinjected into the oocytes. After an incubation for 4-7 days at 18 • C in incubation medium (1× Ringer's buffer, 5% dialysed horse serum, 50 mg/ml tetracycline, 100 mg/ml streptomycin, and 550 mg/ml sodium pyruvate), oocytes were recorded with a two-electrode voltage clamp. Currents induced by pheromone components (100 µM) were recorded using an OC-725C oocyte clamp (Warner Instruments, Hamden, CT, United States) at a holding potential of −80 mV. The data were acquired and analyzed with Digidata 1440A and pCLAMP 10.0 software (Axon Instruments Inc., Union City, CA, United States).

Single Sensillum Recordings
Sensilla trichoidea from 2-day-old male adults were used for the recordings. Individuals were restrained in a remodeled 1 ml plastic pipette tip with an exposed head fixed by dental wax, and antenna from one side was attached to a coverslip with doubleface tape. Two tungsten wire electrodes were used with one inserting into an compound eye and another into the sensilla. Ten individuals were recorded at basal (4), middle (3), and proximal (3) part of the antennae and ten sensilla were recorded for each individuals. Ten micrograms pheromone components (dissolved in hexane) were performed for each trial. Air flow was set at 1.4 L/min with a 300 ms stimulus air pulse controlled by Syntech Stimulus controller (CS-55, Syntech, Kirchzarten, Germany). AC signals were recorded (10 s, starting 1 s before stimulation) using a data acquisition controller (IDAC-4, Syntech, Kirchzarten, Germany) and analyzed with AUTOSPIKE v. 3.9 software (Syntech, Kirchzarten, Germany). The filter setting was 500 Hz at low cutoff and 3 kHz at high cutoff. Responses were calculated by counting the number of action potentials 1 s after stimulation.

Phylogenetic Analysis
Sequences of O. furnacalis were based on the transcriptome data . Sequences from other Ostrinia species were from the reported references (Miura et al., 2009(Miura et al., , 2010Yasukochi et al., 2011) and downloaded through NCBI. The amino acid sequences of pheromone receptors were aligned by MAFFT 2 . Phylogenetic tree was constructed and analyzed by bootstrap test with 1000-resampling through RAxML version 8 with the Jones-Taylor-Thornton amino acid substitution model (JTT) (Stamatakis, 2014).

Statistical Analysis
Data in the present study were normalized by log(X+1) and represented as mean ± SEM. The differences of responses to each pheromone components were analyzed by One-Way ANOVA and followed Duncan test (P < 0.05) by SPSS 20.0 (IBM, Endicott, NY, United States).

Gene Cloning and Sequence Analysis of Pheromone Receptors in O. furnacalis
All the pheromone receptor names in this study were followed Yang et al. (2015). The naming system of pheromone receptors among O. furnacalis, O. nubilalis, and O. scapulalis were shown in Table 1. Full length of amino acid sequences of the pheromone receptors (ranged from 421 to 474aa) and the predicted seven transmembrane domains were shown in Figure 1. The identity between all pheromone receptors was 58.66%. Among all the pheromone receptors, OfurOR5a and OfurOR5b shared high similarity and their identity was 88.21%. Identities among other receptors were significantly lower (e.g., OfurOR8/Ofur5a, 71.30%; OfurOR4/OfurOR6, 64.71%; OfurOR1/OfurOR3, 64.08% etc.). OfurOR1 was not cloned from the strain we used.

Electrophysiological Analysis of the Male s. trichoidea
The single sensillum recordings were performed on the s. trichoidea of male antennae. In total 95 s. trichoidea were  successfully recorded, among them, 82 sensilla responded to the provided pheromone components. Spontaneous activity often indicated more than one class of spike amplitudes that suggested that spikes from more than one neuron were recorded. But it was difficult to discriminate how many neurons in one sensillum or which neuron was responsible for the stimuli because the boundary between spikes was unclear. Four types (A-D) of sensilla were observed in which most of them were Type A (79.2%, 76/96) and they responded to all the provided pheromones except E11-14:OH. The mean responses to Z/E12-14:OAc were relatively higher than other components but no significant difference between them in Type A sensilla (Figure 3). Other types were also observed but the abundance was very low, with the number of 2 (Type B), 3(Type C), and 1(Type D). Type C sensilla responded to three components: E11-14:OAc, Z/E12-14:OAc. Type B and Type D showed specific response to Z/E12-14:OAc and Z9-14:OAc, respectively (Figure 3).

DISCUSSION
The genus Ostrinia has been treated as the model system to study sex pheromone communication because sex pheromone components have been identified in nine species and many species use same pheromone components with different proportion. We cloned seven sex pheromone receptors based on the previous transcriptomic study  and reviewed the names of the deorphanized pheromone receptor system in different Ostrinia research articles (Table 1). Unlike Bombyx mori (Sakurai et al., 2004;Nakagawa et al., 2005), in which the main pheromone receptors were narrowly tuned, most of pheromone receptors in O. furnacalis were broadly tuned to the pheromone components in Xenopus oocyte system. The result was basically consistent with the previous studies (Miura et al., 2009;Wanner et al., 2010;Leary et al., 2012). Among all pheromone receptors, OfurOR4 had significantly stronger response than the other tested receptors. The possible reason might be the system we used was heterologous expression system. When the pheromone receptor expressed in vivo there are other factors which affect the odor perception like OBPs, SNMP, etc. It was reported that the PBPs could increase the sensitivity of PRs to pheromones (Chang et al., 2015). Other receptors we tested might need OBP or SNMP to achieve higher sensitivity. It was also possible that other receptors need to expressed together to form a channel to achieve higher sensitivity coordinately. In O. nubilalis, different ORs could be observed in one neuron by in situ hybridization (Koutroumpa et al., 2014). OfurOR4 has been identified as the receptor which could equally response to main components Z12-14:OAc and E12-14:OAc (Leary et al., 2012). Our results were basically consistent with the previous study. Z/E12-14:OAc might share same binding sites and could interfere with each other thus stimulate order could affect the results of the recording. That might cause the difference in response of the different stimuli order for OfurOR4. We found the additional receptor (OfurOR6) for main component E12-14:OAc. It seems that O. furnacalis need OfurOR4 and OfurOR6 to perceive its pheromone components coordinately, but the mechanism need to be further studied. O. furnacals use Z12-14:OAc and E12-14:OAc with ratio of 1:1 (Cheng et al., 1981;Huang et al., 1998b). In the field test, any trap lure loaded with a ratio other than 1:1 of Z/E12-14:OAc (more Z12 or E12) will cause the reduced captures (Cheng et al., 1982). Thus OfurOR4 might receive specific ratio of 1:1 Z/E components to initiate mating behavior. If the ratio deviates from 1:1 like more E12, OfurOR6 might have specific response to this redundant part of E12 and initiate antagonistic behavior together with OfurOR4.
It is interesting that the phenomenon of gene duplication for pheromone receptors in Ostrinia is very common. Various duplicates for pheromone receptors could be observed in each OR group (Yasukochi et al., 2011). In O. furnacalis, functional differentiation of gene duplication was found in OfurOR5. Similar phenomenon was found in other Lepidopterans. In Helicoverpa armigera, HarmOR14 and HarmOR14b shared high degree of identity but with different function in vitro. HarmOR14b responded to Z9-14:Ald whereas HarmOR14 did not response to any of H. armigera pheromone components (Liu et al., 2013;Chang et al., 2016). In Agrotis segetum, AsegOR1, AsegOR6-10 share high levels of amino acid sequence identity with each other (>70%), whereas their function were dramatically different (Zhang and Löfstedt, 2013).
OfurOR7 showed a specific response to Z9-14:OAc which is the sex pheromone component of O. zaguliaevi and O. zealis (Huang et al., 1998a;Ishikawa et al., 1999a) and a behavioral antagonist (Takanashi et al., 2006). OfurOR8 mainly responded to Z11-14:OAc and E11-14:OAc which were the sex pheromone components of O. nubilalis sex pheromone (Roelofs et al., 1985). Thus, OfurOR7 and OfurOR8 might be involved with interspecific recognition in Ostrinia species. Besides, OfurOR7 is the only one of pheromone receptors which highly expressed in male and female simultaneously , indicate that Z9-14:OAc might be an important pheromone clue for both sexes. Those receptors might contribute to reproductive isolation between Ostrinia species.
Phylogenetic relationship showed that each OR group (OR1, OR3-8) in Ostrinia formed a clade and shared high degrees of identity (81.35-97.44%) (Figure 4). But most of the response pattern, especially for receptor responsible to the main pheromone components, was quite different among those closely related Ostrinia species when compare with previous studies. In genus Ostrinia, the ratio of the Z/E main pheromone components was usually considered to regulate the mating behavior. Those different response patterns make that mechanism more complex and need to be solved case by case. O. nubilalis and O. scapulalis used same pheromone components with same ratio (Z/E11-14:OAc, 97:3-Ztype, and 1:99-Etype) (Glover et al., 1987;Ishikawa et al., 1999b), OnubOR4 mainly responded to E11-14:OAc and OnubOR6 responded to Z11-14:OAc, the response values were equal in this two main receptors (Wanner et al., 2010). OscaOR4 showed a similar response compared to OnubOR4, but no response of OscaOR6 to any pheromones (Miura et al., 2010). O. furnacalis used Z/E12-14:OAc with 1:1 ratio which was quite different from O. nubilalis and O. scapulalis, OfurOR4 equally responded to Z12-14:OAc and E12-14:OAc, OfurOR6 mainly responded to E12-14:OAc different from OnubOR6 which responded to Z components. Comparisons of other pheromone receptors in Ostrinia were listed in Figure 4 in detail.
The results of single sensillum recordings were basically similar to the previous studies (Takanashi et al., 2006;Domingue et al., 2007), most of the sensilla (Type A) responded to five pheromone components but we failed to distinguish the exact neurons. Corresponding to the results from Xenopus oocyte  Table 1, OnubOR1 = OnubOR5, OnubOR3 = OnubOR4, OnubOR4 = OnubOR3, OnubOR5a = OnubOR1, The former was the name we use for phylogenetic analysis, the later was the name reported from reference in Table 1; triangles represented the response was very weak).
system, it seems that multiple the pheromone receptors were expressed on the neurons in Type A sensilla. We found three other types in which Type B sensilla only responded to Z/E12-14:OAc, indicated that the neuron in these sensilla might specifically express OfurOR4 and OfurOR6. Similarly, Type D of which specifically responded to Z9-14:OAc are possibly associated with the expression of OfurOR7. Type C responded to E11-14:OAc, Z/E12-14:OAc, which similar to Type A but difficult to speculate the expressed receptors in these sensilla according the results from Xenopus oocyte system. Possibly because the pheromone receptors co-expressed in O. furnacalis, that pattern has been reported in its closely related specie O. nubilalis (Koutroumpa et al., 2014). We did not find any neuron that responded E12-14:OH. In Ostrinia, OscaOR1, and OlatOR1 has been reported for responding E12-14:OH (Miura et al., 2009). Thus OfurOR1 might has same response profile. OfurOR1 could not be cloned in our strain and also not exist in the transcriptome  might indicated the degeneration of OfurOR1 in the colony we used. And it can also be that expression level of OfurOR1 is too low. Utilization of in situ hybridization and CRISPR-Cas9 might further elucidate the neuron distribution and receptor expression pattern in single sensillum.

AUTHOR CONTRIBUTIONS
WL, BY, and G-rW designed the research, analyzed the data, and wrote the paper. WL and SC performed the research. X-cJ provided biological samples.

ACKNOWLEDGMENTS
We thank Sai Zhang and Yi Lin for rearing the experimental moth for the study.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.00591/full#supplementary-material FIGURE S1 | Current traces of OfurOR4/OfurOR2 in response to pheromone compounds (100 µM) with different order.
TABLE S1 | Primers of pheromone receptors used for PCR.