Chem. Senses 40: 7–16, 2015
doi:10.1093/chemse/bju052 Advance Access publication October 25, 2014
Identification and Functional Characterization of Sex Pheromone Receptors in the Common Cutworm (Spodoptera litura) Jin Zhang1,2, Shuwei Yan1,2, Yang Liu1, Emmanuelle Jacquin-Joly3, Shuanglin Dong2 1 and Guirong Wang 1
Correspondence to be sent to: Guirong Wang, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China. e-mail: [email protected] Accepted September 13, 2014
Abstract Male moths can finely discriminate the sex pheromone emitted by conspecific females from similar compounds. Pheromone receptors, expressed on the dendritic membrane of sensory neurons housed in the long trichoid sensilla of antennae, are thought to be associated with the pheromone reception. In this study, we identified and functionally characterized 4 pheromone receptors from the antennae of Spodoptera litura (Lepidoptera: Noctuidae). A tissue distribution analysis showed that the expression of the 4 SlituPRs was restricted to antennae. In addition, SlituOR6 and SlituOR13 were specifically expressed in male antennae whereas SlituOR11 and SlituOR16 were male-biased. Functional investigation by heterologous expression in Xenopus oocytes revealed that SlituOR6 was specifically tuned to the second major pheromone component, Z9,E12-14:OAc, SlituOR13 was equally tuned to Z9,E12-14:OAc and Z9-14:OAc, with a small response to the major pheromone component Z9,E11-14:OAc, SlituOR16 significantly responded to the behavioral antagonist Z9-14:OH, whereas SlituOR11 did not show response to any of the pheromone compounds tested in this study. Our results provide molecular data to better understand the mechanisms of sex pheromone detection in the moth S. litura and bring clues to investigate the evolution of the sexual communication channel in closely related species through comparison with previously reported pheromone receptors in other Spodoptera species. Key words: pheromone receptor, Spodoptera littoralis, Spodoptera litura, Xenopus oocytes
Introduction The sense of smell plays a very important role for insect survival. Insects use their olfaction to find mates and food sources, locate oviposition site, and avoid enemies. Insects possess an acute and complex olfactory system to detect the volatile chemicals from the periphery where chemical messages are transformed into electrical signals, to the antennal lobes where information is integrated and sent to the brain, eventually generating a behavioral response (Leal 2013). Previous studies have shown that peripheral reception of semiochemicals involves several molecular components, including odorant-binding proteins (OBPs), chemosensory proteins, sensory neuron membrane proteins (SNMPs) and
2 classes of olfactory receptors: the odorant receptors (ORs) and the inotropic receptors (IRs) (Leal 2005; Rutzler and Zwiebel 2005; Benton et al. 2009; Touhara and Vosshall 2009; Gould et al. 2010; Grosjean et al. 2011; Leal 2013; Rytz et al. 2013). These receptors are functional when forming heteromers with a conserved subunit, named Orco in the case of ORs (Larsson et al. 2004; Benton et al. 2006). Moth sex pheromone detection has been for long time an excellent model system for studying the mechanisms of sensory perception at the molecular level because of the sensitivity and specificity of males detecting the pheromone emitted by conspecific females (Hildebrand 1995; Rutzler
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State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian District, 2 Beijing 100193, China, Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, No. 1 Weigang, 3 Xuanwu District, Nanjing 210095, China and INRA, UMR 1392 Institute of Ecology and Environmental Science iEES-Paris, Route de Saint-Cyr, 78026 Versailles cedex, France
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Figure 1. Different pheromone components and ratios in the 9 related Spodoptera species: S. litura (Tamaki et al. 1973; Sun et al. 2003), S. littoralis (Tamaki and Yushima 1974; MuÑOz et al. 2008), S. exigua (Dong and Du 2002), S. frugiperda (Mitchell et al. 1985), S. exempta (Cork et al. 1989), S. latifascia and S. descoinsi (Monti et al. 1995), S. eridania (Teal et al. 1985), S. sunia (Bestmann et al. 1988).
(OR6, OR11, OR13 and OR16) have been identified in each species through the analyses of an antennal transcriptome (Legeai et al. 2011) or by homology cloning (Liu et al. 2013a) with considerable similarity between the 2 species. One S. littoralis PR (OR6) was functionally characterized to be specifically tuned to the minor pheromone component Z9,E12-14:OAc using Drosophila antennae as a heterologous expression system (Montagne et al. 2012) and 2 S. exigua PRs were shown to be tuned to the pheromone components Z9,E12-14:OAc and Z9-14:OAc (OR13) and to Z9-14:OH (OR16) by expression in Xenopus oocytes (Liu et al. 2013a). Thus, investigating the molecular mechanisms of pheromone reception in S. litura would bring valuable information to better understand the mechanism of species isolation. In this study, we cloned 4 PR candidates from S. litura male antennae and mapped their expression patterns. Furthermore, we carried out functional characterization using Xenopus oocyte heterologous expression system combined with 2-electrode voltage clamp recording and identified ligand(s) for 3 of them. This study provides a basis for further investigation on the evolution of sexual communication channel in closely related moth species.
Experimental procedures Insect rearing and tissue preparation
S. litura (Fabricius) were reared in our laboratory at 26 °C, 14:10 h light:dark (L:D) photoperiod and sexed as pupae. The tissues were dissected from 3-day-old adults and stored at −70 °C. Pheromone components
(Z9,E11)-tetradecadienyl acetate (Z9,E11-14:OAc), (Z9,E12)tetradecadienyl acetate (Z9,E12-14:OAc), (Z9)-tetradecenyl
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and Zwiebel 2005; Leal 2013; Liu et al. 2013a). A moth sex pheromone is usually a blend of pheromone components detected by specialized sensilla which house the dendrites of pheromone-responsive sensory neurons. These neurons are bathed in the sensillum lymph containing high concentrations of pheromone-binding protein (PBPs), a subclass of OBPs thought to be involved in pheromone components transport to the receptors (Vogt and Riddiford 1981; Steinbrecht and Gnatzy 1984; Nakagawa et al. 2005; GrosseWilde et al. 2007). Much progress has been made in understanding the pheromone processing by male moths over the past decade since the first identification of moth pheromone receptors (PRs) in the noctuid Heliothis virescens (Krieger et al. 2004). The earliest contribution in PR functional studies has been the characterization of the 2 PRs of Bombyx mori, BmorOR1 and BmorOR3, which respond to bombykol and bombykal, respectively (Sakurai et al. 2004; Nakagawa et al. 2005). Since then, an increasing number of studies have investigated the functional properties of PRs in a variety of moth species, including H. virescens, (Krieger et al. 2004; Grosse-Wilde et al. 2007; Krieger et al. 2009; Vasquez et al. 2010; Wang et al. 2011), Ostrinia spp. (Miura et al. 2009; Miura et al. 2010; Wanner et al. 2010), Helicoverpa armigera (Liu et al. 2013b), Plutella xylostella (Sun et al. 2013) and Agrotis segetum (Zhang and Lofstedt 2013). The armyworm genus Spodoptera (Noctuidae) contains 30 species which inhabit 6 continents (Pogue 2002). The sex pheromones of 9 species from genus Spodoptera have been characterized to date (Meagher et al. 2008). S. litura, is an important agricultural pest in the world and causes huge economic losses every year. Previous studies identified 4 components from female sex pheromone glands: Z9,E1114:OAc, Z9,E12-14:OAc, Z9-14:OAc, and E11-14:OAc in the ratio of 100:27:20:27 (Tamaki et al. 1973; Sun et al. 2003). The pheromone components and ratios of the related species have been summarized in detail in Figure 1. Spodoptera litura and Spodoptera littoralis (from Kenya) share similar sex pheromone components with a little difference (Tamaki and Yushima 1974; MuÑOz et al. 2008) whereas the pheromone blend of Spodoptera exigua contains a component, Z9-14:OH, which is recognized as a behavioral antagonist at least by males of S. littoralis (Campion et al. 1980; Ljungberg et al. 1993). The female sex pheromone composition of S. littoralis is highly dependent on the origin of the strain (MuÑOz et al. 2008). Previous studies have shown 2 types of trichoid sensilla on the antennae of this species: sensilla type I houses 2 neurons, one tuned to Z9,E12-14:OAc, another to Z9-14:OH. Sensilla type II contains a neuron tuned to the major pheromone component, Z9,Ell-14:OAc and a second one tuned still uncharacterized (Ljungberg et al. 1993; Ochieng et al. 1995). Although the molecular mechanisms of pheromone reception remains largely unknown in S. litura, PRs have been identified and functionally characterized in S. littoralis (Legeai et al. 2011; Montagne et al. 2012) and S. exigua (Liu et al. 2013a). Four sex PR candidate genes
Functional Characterization of Sex Pheromone Receptor in Spodoptera litura 9
acetate (Z9-14:OAc), (E11)-tetradecenyl acetate (E11-14:OAc), (Z)-9-tetradecenol (Z9-14:OH), and (Z)-11-hexadecenyl acetate (Z11-16:OAc) (all 93–95% minimum purity) were chemically synthesized by Nimrod Inc. (Z)-9-hexadecenal (Z9-16:Ald) was purchased from Bedoukian. Stock solutions (1 M) were prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C. Before experiments, the stock solution was diluted in Ringer’s buffer (96 mM NaCl, 2 mM KCl, 5 mM MgCl2, 0.8 mM CaCl2, and 5 mM HEPES pH 7.6). 1× Ringer’s buffer containing 0.1% DMSO was used as a negative control. RNA isolation and cDNA synthesis
Gene cloning and vector construction
Fragment sequences of the 4 PR genes of S. litura were cloned using specific primers designed on the homologous sequences of S. littoralis (Legeai et al. 2011). To get the fulllength opening reading frame sequences of the candidate PRs, Rapid Amplification of cDNA End (RACE) polymerase chain reaction (PCR) was performed using SMARTer RACE cDNA Amplification kit (Clontech) for 3′ and 5′ ends amplification. The full-length sequences were confirmed by end-to-end PCR. The primers were designed using Primer Premier 5.0 software (PREMIER Biosoft International) and are listed in Supplementary Material S1. PCR reactions for the full-length sequences were carried out under the following conditions: 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 52 °C for 30 s, 72 °C for 1.5 min, 72 °C for 10 min. PCR products were run on a 1.0% agarose gel and verified by DNA sequencing. The expression plasmids were constructed by subcloning the PR cDNAs and the S. litura Orco cDNA (Wu et al. 2013) into the multiple cloning site of PT7Ts vector using the restriction enzymes NotI and ApaI. Bioinformatics and phylogeny
Protein sequences were aligned using ClustalW2 and transmembrane domains were predicted according to TMBase and the SFINX package (Hofmann and Stoffel 1993; Sonnhammer et al. 1998). The 4 S. litura PR sequences were used with other Lepidoptera ORs (GenBank accession numbers listed in Supplementary Material S2) to construct an unrooted neighbor-joining tree using MEGA 5.0. Quantitative real-time PCR
The expression profiles of the 4 PR transcripts in various adult tissues (antennae, proboscis, maxillary palps, legs,
Receptor expression in Xenopus oocytes and 2-electrode voltage-clamp electrophysiological
Capped RNAs were synthesized from linearized vectors with mMESSAGE mMACHINE T7 (Ambion) according to the manufacturer’s protocol. Template plasmids were fully linearized with SmaI, and capped cRNAs were transcribed using T7 RNA polymerase. Purified cRNAs were resuspended in nuclease-free water at a concentration of 2 µg/µL and stored at −80 °C. Mature healthy oocytes were treated with 2 mg/ml collagenase S-1 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. Oocytes were later microinjected with 27.6 ng SlituOR cRNAs and 27.6 ng SlituOrco cRNA. After 4 days of incubation at 18 °C in 1× Ringer’s solution (96 mM NaCl, 2 mM KCl, 5 mM MgCl2, 0.8 mM CaCl2, and 5 mM HEPES, pH 7.6) supplemented with 5% dialyzed horse serum, 50 mg/ mg/ml streptomycin and 550 mg/ml ml tetracycline, 100 sodium pyruvate, the whole-cell currents (nanoamperes/nA) were recorded from the injected oocytes with a 2-electrode voltage clamp. The experimental procedures were the same as described previously (Wang et al. 2011). Oocytes were exposed to the different compounds diluted at 10−4 M and doseresponse curves were performed from 10−8 to 10−3 M in ascending order of concentration, with an interval between exposures which allowed the current to return to baseline. Data acquisition and analysis were carried out with Digidata 1440A and Pclamp10.0 software (Axon Instruments Inc.). Dose–response data were analyzed using GraphPad Prism 5. All experiments were repeated 5 times on different oocytes and mean values of current ± standard error of the oocytes responding to 100 μM
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Total RNA was isolated from the different tissues using Trizol reagent (Invitrogen) following the manufacturer’s instruction and digested with DNase I (Fermentas). The single-stranded cDNAs were synthesized by First Strand cDNA Synthesis Kit (Fermentas).
and genitalia) isolated from males and females were evaluated in 3 independent tissue collections by qRT-PCR analysis on the ABI Prism 7500 Fast Detection System (Applied Biosystems). Two reference genes, GAPDH (GenBank No. HQ012003.2) and elongation factor 1-a (GenBank No. KC007373.1), were used to normalize the target gene expression and to correct for sample-to-sample variation. The qRTPCR primers were designed using the Beacon Designer 7.90 software (PREMIER Biosoft International) and are listed in Supplementary Material S1. The qRT-PCR reactions were performed (3 replicated wells for technical replicates) in 20 µL reactions containing 10 µL 2× Go Taq qPCR Master Mix (Promega), 0.5 µL of upstream and downstream primers (10 µM), 1 μL of the sample cDNA and 8 µL of sterilized ultrapure water. The PCR program was 95 °C for 2 min, 40 cycles at 95 °C for 30 s, 60 °C for 1 min. The PCR products were then heated to 95 °C for 15 s, cooled to 60 °C for 1 min and heated again to 95 °C for 15 s to measure the dissociation curves. The experiment was repeated 3 times using 3 independent RNA samples. The expression level of the PR −ΔΔCT method, where ΔCT = (CT, genes was analyzed using 2 PR gene − CT, reference gene), ΔΔCT = (ΔCT, different samples − ΔCT maximum) (Livak and Schmittgen 2001).
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stimuli were compared by One-Way ANOVA followed a least significant difference (LSD) test (significance level: P 250 nA,” Vertically striped circles represent “150–250 nA,” Right diagonal striped circles represent “100–150 nA,” Left diagonal striped circles represent “50–100 nA” and Filled circles represent “0–50 nA.” Triangle represents the response of SlitOR6.
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selectivity in Ostrinia PRs (Leary et al. 2012). Similarly in Agrotis ipsilon, 6 paralogs with high sequence similarity were shown to vary dramatically in their ligand selectivity and sensitivity (Zhang and Lofstedt 2013). It is thus possible that the 16 amino acid differences account for a different function for SexiOR6. SlituOR13 strongly responded to the minor pheromone components Z9-14:OAc and Z9,E1214:OAc. Based on EC50 values, SlituOR13 was approximately 5 times more sensitive to Z9-14:OAc compared with Z9,E12-14:OAc, just the opposite to what has been observed with SexiOR13 (Liu et al. 2013a). The 5 amino acid differences in the 7 transmembrane domains between SlituOR13 and SexiOR13 may account for these differences in selectivity. In our study, SlituOR13 only showed a small response to the major sex pheromone component Z9,E11-14:OAc at the concentration of 10−4 M. Several studies demonstrated that the presence of PBPs was necessary for Lepidopteran PRs correct functioning in in vitro expression systems (GrosseWilde et al. 2006; Grosse-Wilde et al. 2007; Sun et al. 2013). In addition, the presence of SNMP is required for detection of the pheromone component cis-vaccenyl acetate in Drosophila (Benton et al. 2007). Thus, the lack of SNMPs and PBPs in the Xenopus expression systems may account for the absence of SlituOR13 response to Z9,E11-14:OAc, or there may be other ORs responding to this pheromone. Next, we would add SlituPBPs and SlituSNMPs into this system to test if there were any influence on the reaction of PRs to the pheromone components. The transcriptome analysis of adult antennae from S. litura may be conducive to find new OR which tuned to the main pheromone component, Z9,E11-14:OAc. SlituOR16 presented the largest response to Z9-14:OH, as observed previously for the ortholog S. exigua OR16 (Liu et al. 2013a). Interestingly, this compound is part of the S. exigua pheromone blend, the mixture of Z9,E12-14:OAc and Z9-14:OH (9:1) providing the best efficiency to attract males of S. exigua (Dong and Du 2002), but the alcohol is not present in the S. litura pheromone blend. This compound may be detected by S. litura males as a behavioral antagonist to avoid cross-mating in
Functional Characterization of Sex Pheromone Receptor in Spodoptera litura 15
orphan. SlituOR6 responded to the second pheromone component, Z9,E12-14:OAc. SlituOR13 had a strong response to both Z9,E12-14:OAc and Z9-14:OAc. SlituOR16 responded robustly to the behavior antagonist, Z9-14:OH. Unfortunately, we did not find any PR strongly tuned to the main pheromone component, Z9,E11-14:OAc, as one would have expected. Nevertheless, the availability of additional characterized PRs in the Spodoptera complex makes it possible to investigate structure-function relationships and to propose residues which may contribute to the functional sites. Also, our work provides the bases for future investigation on the evolution of the sexual communication channel in closely related species.
Supplementary material can be found at http://www.chemse. oxfordjournals.org/
Funding This work was supported by the National Basic Research Program of China [2012CB114104]; the National Natural Science Foundation of China [31230062, 31071752] and Beijing Natural Science Foundation [6132028].
Acknowledgements
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We would like to thank Miss L. Yang for insect rearing, K. Yang and G. Zhu for the technical help in the experiment, P. He and Y.-N. Zhang for the advices in manuscript modification.
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doi:10.1093/chemse/bju052 Advance Access publication October 25, 2014
Identification and Functional Characterization of Sex Pheromone Receptors in the Common Cutworm (Spodoptera litura) Jin Zhang1,2, Shuwei Yan1,2, Yang Liu1, Emmanuelle Jacquin-Joly3, Shuanglin Dong2 1 and Guirong Wang 1
Correspondence to be sent to: Guirong Wang, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China. e-mail: [email protected] Accepted September 13, 2014
Abstract Male moths can finely discriminate the sex pheromone emitted by conspecific females from similar compounds. Pheromone receptors, expressed on the dendritic membrane of sensory neurons housed in the long trichoid sensilla of antennae, are thought to be associated with the pheromone reception. In this study, we identified and functionally characterized 4 pheromone receptors from the antennae of Spodoptera litura (Lepidoptera: Noctuidae). A tissue distribution analysis showed that the expression of the 4 SlituPRs was restricted to antennae. In addition, SlituOR6 and SlituOR13 were specifically expressed in male antennae whereas SlituOR11 and SlituOR16 were male-biased. Functional investigation by heterologous expression in Xenopus oocytes revealed that SlituOR6 was specifically tuned to the second major pheromone component, Z9,E12-14:OAc, SlituOR13 was equally tuned to Z9,E12-14:OAc and Z9-14:OAc, with a small response to the major pheromone component Z9,E11-14:OAc, SlituOR16 significantly responded to the behavioral antagonist Z9-14:OH, whereas SlituOR11 did not show response to any of the pheromone compounds tested in this study. Our results provide molecular data to better understand the mechanisms of sex pheromone detection in the moth S. litura and bring clues to investigate the evolution of the sexual communication channel in closely related species through comparison with previously reported pheromone receptors in other Spodoptera species. Key words: pheromone receptor, Spodoptera littoralis, Spodoptera litura, Xenopus oocytes
Introduction The sense of smell plays a very important role for insect survival. Insects use their olfaction to find mates and food sources, locate oviposition site, and avoid enemies. Insects possess an acute and complex olfactory system to detect the volatile chemicals from the periphery where chemical messages are transformed into electrical signals, to the antennal lobes where information is integrated and sent to the brain, eventually generating a behavioral response (Leal 2013). Previous studies have shown that peripheral reception of semiochemicals involves several molecular components, including odorant-binding proteins (OBPs), chemosensory proteins, sensory neuron membrane proteins (SNMPs) and
2 classes of olfactory receptors: the odorant receptors (ORs) and the inotropic receptors (IRs) (Leal 2005; Rutzler and Zwiebel 2005; Benton et al. 2009; Touhara and Vosshall 2009; Gould et al. 2010; Grosjean et al. 2011; Leal 2013; Rytz et al. 2013). These receptors are functional when forming heteromers with a conserved subunit, named Orco in the case of ORs (Larsson et al. 2004; Benton et al. 2006). Moth sex pheromone detection has been for long time an excellent model system for studying the mechanisms of sensory perception at the molecular level because of the sensitivity and specificity of males detecting the pheromone emitted by conspecific females (Hildebrand 1995; Rutzler
© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
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State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian District, 2 Beijing 100193, China, Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, No. 1 Weigang, 3 Xuanwu District, Nanjing 210095, China and INRA, UMR 1392 Institute of Ecology and Environmental Science iEES-Paris, Route de Saint-Cyr, 78026 Versailles cedex, France
8 J. Zhang et al.
Figure 1. Different pheromone components and ratios in the 9 related Spodoptera species: S. litura (Tamaki et al. 1973; Sun et al. 2003), S. littoralis (Tamaki and Yushima 1974; MuÑOz et al. 2008), S. exigua (Dong and Du 2002), S. frugiperda (Mitchell et al. 1985), S. exempta (Cork et al. 1989), S. latifascia and S. descoinsi (Monti et al. 1995), S. eridania (Teal et al. 1985), S. sunia (Bestmann et al. 1988).
(OR6, OR11, OR13 and OR16) have been identified in each species through the analyses of an antennal transcriptome (Legeai et al. 2011) or by homology cloning (Liu et al. 2013a) with considerable similarity between the 2 species. One S. littoralis PR (OR6) was functionally characterized to be specifically tuned to the minor pheromone component Z9,E12-14:OAc using Drosophila antennae as a heterologous expression system (Montagne et al. 2012) and 2 S. exigua PRs were shown to be tuned to the pheromone components Z9,E12-14:OAc and Z9-14:OAc (OR13) and to Z9-14:OH (OR16) by expression in Xenopus oocytes (Liu et al. 2013a). Thus, investigating the molecular mechanisms of pheromone reception in S. litura would bring valuable information to better understand the mechanism of species isolation. In this study, we cloned 4 PR candidates from S. litura male antennae and mapped their expression patterns. Furthermore, we carried out functional characterization using Xenopus oocyte heterologous expression system combined with 2-electrode voltage clamp recording and identified ligand(s) for 3 of them. This study provides a basis for further investigation on the evolution of sexual communication channel in closely related moth species.
Experimental procedures Insect rearing and tissue preparation
S. litura (Fabricius) were reared in our laboratory at 26 °C, 14:10 h light:dark (L:D) photoperiod and sexed as pupae. The tissues were dissected from 3-day-old adults and stored at −70 °C. Pheromone components
(Z9,E11)-tetradecadienyl acetate (Z9,E11-14:OAc), (Z9,E12)tetradecadienyl acetate (Z9,E12-14:OAc), (Z9)-tetradecenyl
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and Zwiebel 2005; Leal 2013; Liu et al. 2013a). A moth sex pheromone is usually a blend of pheromone components detected by specialized sensilla which house the dendrites of pheromone-responsive sensory neurons. These neurons are bathed in the sensillum lymph containing high concentrations of pheromone-binding protein (PBPs), a subclass of OBPs thought to be involved in pheromone components transport to the receptors (Vogt and Riddiford 1981; Steinbrecht and Gnatzy 1984; Nakagawa et al. 2005; GrosseWilde et al. 2007). Much progress has been made in understanding the pheromone processing by male moths over the past decade since the first identification of moth pheromone receptors (PRs) in the noctuid Heliothis virescens (Krieger et al. 2004). The earliest contribution in PR functional studies has been the characterization of the 2 PRs of Bombyx mori, BmorOR1 and BmorOR3, which respond to bombykol and bombykal, respectively (Sakurai et al. 2004; Nakagawa et al. 2005). Since then, an increasing number of studies have investigated the functional properties of PRs in a variety of moth species, including H. virescens, (Krieger et al. 2004; Grosse-Wilde et al. 2007; Krieger et al. 2009; Vasquez et al. 2010; Wang et al. 2011), Ostrinia spp. (Miura et al. 2009; Miura et al. 2010; Wanner et al. 2010), Helicoverpa armigera (Liu et al. 2013b), Plutella xylostella (Sun et al. 2013) and Agrotis segetum (Zhang and Lofstedt 2013). The armyworm genus Spodoptera (Noctuidae) contains 30 species which inhabit 6 continents (Pogue 2002). The sex pheromones of 9 species from genus Spodoptera have been characterized to date (Meagher et al. 2008). S. litura, is an important agricultural pest in the world and causes huge economic losses every year. Previous studies identified 4 components from female sex pheromone glands: Z9,E1114:OAc, Z9,E12-14:OAc, Z9-14:OAc, and E11-14:OAc in the ratio of 100:27:20:27 (Tamaki et al. 1973; Sun et al. 2003). The pheromone components and ratios of the related species have been summarized in detail in Figure 1. Spodoptera litura and Spodoptera littoralis (from Kenya) share similar sex pheromone components with a little difference (Tamaki and Yushima 1974; MuÑOz et al. 2008) whereas the pheromone blend of Spodoptera exigua contains a component, Z9-14:OH, which is recognized as a behavioral antagonist at least by males of S. littoralis (Campion et al. 1980; Ljungberg et al. 1993). The female sex pheromone composition of S. littoralis is highly dependent on the origin of the strain (MuÑOz et al. 2008). Previous studies have shown 2 types of trichoid sensilla on the antennae of this species: sensilla type I houses 2 neurons, one tuned to Z9,E12-14:OAc, another to Z9-14:OH. Sensilla type II contains a neuron tuned to the major pheromone component, Z9,Ell-14:OAc and a second one tuned still uncharacterized (Ljungberg et al. 1993; Ochieng et al. 1995). Although the molecular mechanisms of pheromone reception remains largely unknown in S. litura, PRs have been identified and functionally characterized in S. littoralis (Legeai et al. 2011; Montagne et al. 2012) and S. exigua (Liu et al. 2013a). Four sex PR candidate genes
Functional Characterization of Sex Pheromone Receptor in Spodoptera litura 9
acetate (Z9-14:OAc), (E11)-tetradecenyl acetate (E11-14:OAc), (Z)-9-tetradecenol (Z9-14:OH), and (Z)-11-hexadecenyl acetate (Z11-16:OAc) (all 93–95% minimum purity) were chemically synthesized by Nimrod Inc. (Z)-9-hexadecenal (Z9-16:Ald) was purchased from Bedoukian. Stock solutions (1 M) were prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C. Before experiments, the stock solution was diluted in Ringer’s buffer (96 mM NaCl, 2 mM KCl, 5 mM MgCl2, 0.8 mM CaCl2, and 5 mM HEPES pH 7.6). 1× Ringer’s buffer containing 0.1% DMSO was used as a negative control. RNA isolation and cDNA synthesis
Gene cloning and vector construction
Fragment sequences of the 4 PR genes of S. litura were cloned using specific primers designed on the homologous sequences of S. littoralis (Legeai et al. 2011). To get the fulllength opening reading frame sequences of the candidate PRs, Rapid Amplification of cDNA End (RACE) polymerase chain reaction (PCR) was performed using SMARTer RACE cDNA Amplification kit (Clontech) for 3′ and 5′ ends amplification. The full-length sequences were confirmed by end-to-end PCR. The primers were designed using Primer Premier 5.0 software (PREMIER Biosoft International) and are listed in Supplementary Material S1. PCR reactions for the full-length sequences were carried out under the following conditions: 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 52 °C for 30 s, 72 °C for 1.5 min, 72 °C for 10 min. PCR products were run on a 1.0% agarose gel and verified by DNA sequencing. The expression plasmids were constructed by subcloning the PR cDNAs and the S. litura Orco cDNA (Wu et al. 2013) into the multiple cloning site of PT7Ts vector using the restriction enzymes NotI and ApaI. Bioinformatics and phylogeny
Protein sequences were aligned using ClustalW2 and transmembrane domains were predicted according to TMBase and the SFINX package (Hofmann and Stoffel 1993; Sonnhammer et al. 1998). The 4 S. litura PR sequences were used with other Lepidoptera ORs (GenBank accession numbers listed in Supplementary Material S2) to construct an unrooted neighbor-joining tree using MEGA 5.0. Quantitative real-time PCR
The expression profiles of the 4 PR transcripts in various adult tissues (antennae, proboscis, maxillary palps, legs,
Receptor expression in Xenopus oocytes and 2-electrode voltage-clamp electrophysiological
Capped RNAs were synthesized from linearized vectors with mMESSAGE mMACHINE T7 (Ambion) according to the manufacturer’s protocol. Template plasmids were fully linearized with SmaI, and capped cRNAs were transcribed using T7 RNA polymerase. Purified cRNAs were resuspended in nuclease-free water at a concentration of 2 µg/µL and stored at −80 °C. Mature healthy oocytes were treated with 2 mg/ml collagenase S-1 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. Oocytes were later microinjected with 27.6 ng SlituOR cRNAs and 27.6 ng SlituOrco cRNA. After 4 days of incubation at 18 °C in 1× Ringer’s solution (96 mM NaCl, 2 mM KCl, 5 mM MgCl2, 0.8 mM CaCl2, and 5 mM HEPES, pH 7.6) supplemented with 5% dialyzed horse serum, 50 mg/ mg/ml streptomycin and 550 mg/ml ml tetracycline, 100 sodium pyruvate, the whole-cell currents (nanoamperes/nA) were recorded from the injected oocytes with a 2-electrode voltage clamp. The experimental procedures were the same as described previously (Wang et al. 2011). Oocytes were exposed to the different compounds diluted at 10−4 M and doseresponse curves were performed from 10−8 to 10−3 M in ascending order of concentration, with an interval between exposures which allowed the current to return to baseline. Data acquisition and analysis were carried out with Digidata 1440A and Pclamp10.0 software (Axon Instruments Inc.). Dose–response data were analyzed using GraphPad Prism 5. All experiments were repeated 5 times on different oocytes and mean values of current ± standard error of the oocytes responding to 100 μM
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Total RNA was isolated from the different tissues using Trizol reagent (Invitrogen) following the manufacturer’s instruction and digested with DNase I (Fermentas). The single-stranded cDNAs were synthesized by First Strand cDNA Synthesis Kit (Fermentas).
and genitalia) isolated from males and females were evaluated in 3 independent tissue collections by qRT-PCR analysis on the ABI Prism 7500 Fast Detection System (Applied Biosystems). Two reference genes, GAPDH (GenBank No. HQ012003.2) and elongation factor 1-a (GenBank No. KC007373.1), were used to normalize the target gene expression and to correct for sample-to-sample variation. The qRTPCR primers were designed using the Beacon Designer 7.90 software (PREMIER Biosoft International) and are listed in Supplementary Material S1. The qRT-PCR reactions were performed (3 replicated wells for technical replicates) in 20 µL reactions containing 10 µL 2× Go Taq qPCR Master Mix (Promega), 0.5 µL of upstream and downstream primers (10 µM), 1 μL of the sample cDNA and 8 µL of sterilized ultrapure water. The PCR program was 95 °C for 2 min, 40 cycles at 95 °C for 30 s, 60 °C for 1 min. The PCR products were then heated to 95 °C for 15 s, cooled to 60 °C for 1 min and heated again to 95 °C for 15 s to measure the dissociation curves. The experiment was repeated 3 times using 3 independent RNA samples. The expression level of the PR −ΔΔCT method, where ΔCT = (CT, genes was analyzed using 2 PR gene − CT, reference gene), ΔΔCT = (ΔCT, different samples − ΔCT maximum) (Livak and Schmittgen 2001).
10 J. Zhang et al.
stimuli were compared by One-Way ANOVA followed a least significant difference (LSD) test (significance level: P 250 nA,” Vertically striped circles represent “150–250 nA,” Right diagonal striped circles represent “100–150 nA,” Left diagonal striped circles represent “50–100 nA” and Filled circles represent “0–50 nA.” Triangle represents the response of SlitOR6.
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selectivity in Ostrinia PRs (Leary et al. 2012). Similarly in Agrotis ipsilon, 6 paralogs with high sequence similarity were shown to vary dramatically in their ligand selectivity and sensitivity (Zhang and Lofstedt 2013). It is thus possible that the 16 amino acid differences account for a different function for SexiOR6. SlituOR13 strongly responded to the minor pheromone components Z9-14:OAc and Z9,E1214:OAc. Based on EC50 values, SlituOR13 was approximately 5 times more sensitive to Z9-14:OAc compared with Z9,E12-14:OAc, just the opposite to what has been observed with SexiOR13 (Liu et al. 2013a). The 5 amino acid differences in the 7 transmembrane domains between SlituOR13 and SexiOR13 may account for these differences in selectivity. In our study, SlituOR13 only showed a small response to the major sex pheromone component Z9,E11-14:OAc at the concentration of 10−4 M. Several studies demonstrated that the presence of PBPs was necessary for Lepidopteran PRs correct functioning in in vitro expression systems (GrosseWilde et al. 2006; Grosse-Wilde et al. 2007; Sun et al. 2013). In addition, the presence of SNMP is required for detection of the pheromone component cis-vaccenyl acetate in Drosophila (Benton et al. 2007). Thus, the lack of SNMPs and PBPs in the Xenopus expression systems may account for the absence of SlituOR13 response to Z9,E11-14:OAc, or there may be other ORs responding to this pheromone. Next, we would add SlituPBPs and SlituSNMPs into this system to test if there were any influence on the reaction of PRs to the pheromone components. The transcriptome analysis of adult antennae from S. litura may be conducive to find new OR which tuned to the main pheromone component, Z9,E11-14:OAc. SlituOR16 presented the largest response to Z9-14:OH, as observed previously for the ortholog S. exigua OR16 (Liu et al. 2013a). Interestingly, this compound is part of the S. exigua pheromone blend, the mixture of Z9,E12-14:OAc and Z9-14:OH (9:1) providing the best efficiency to attract males of S. exigua (Dong and Du 2002), but the alcohol is not present in the S. litura pheromone blend. This compound may be detected by S. litura males as a behavioral antagonist to avoid cross-mating in
Functional Characterization of Sex Pheromone Receptor in Spodoptera litura 15
orphan. SlituOR6 responded to the second pheromone component, Z9,E12-14:OAc. SlituOR13 had a strong response to both Z9,E12-14:OAc and Z9-14:OAc. SlituOR16 responded robustly to the behavior antagonist, Z9-14:OH. Unfortunately, we did not find any PR strongly tuned to the main pheromone component, Z9,E11-14:OAc, as one would have expected. Nevertheless, the availability of additional characterized PRs in the Spodoptera complex makes it possible to investigate structure-function relationships and to propose residues which may contribute to the functional sites. Also, our work provides the bases for future investigation on the evolution of the sexual communication channel in closely related species.
Supplementary material can be found at http://www.chemse. oxfordjournals.org/
Funding This work was supported by the National Basic Research Program of China [2012CB114104]; the National Natural Science Foundation of China [31230062, 31071752] and Beijing Natural Science Foundation [6132028].
Acknowledgements
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We would like to thank Miss L. Yang for insect rearing, K. Yang and G. Zhu for the technical help in the experiment, P. He and Y.-N. Zhang for the advices in manuscript modification.
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