Chemosensory receptors play key roles in insect behavior. Thus, genes encoding these receptors have great potential for use in integrated pest management. The hover fly Scaeva pyrastri (L.) is an important pollinating insect and a natural enemy of aphids, mainly distributed in the Palearctic and Nearctic regions. However, a systematic identification of their chemosensory receptor genes in the antennae has not been reported. In the present study, we assembled the antennal transcriptome of S. pyrastri by using Illumina sequencing technology. Analysis of the transcriptome data identified 60 candidate chemosensory genes, including 38 for odorant receptors (ORs), 16 for ionotropic receptors (IRs), and 6 for gustatory receptors (GRs). The numbers are similar to those of other Diptera species, suggesting that we were able to successfully identify S. pyrastri chemosensory genes. We analyzed the expression patterns of all genes by using reverse transcriptase PCR (RT-PCR), and found that some genes exhibited sex-biased or sex-specific expression. These candidate chemosensory genes and their tissue expression profiles provide information for further studies aimed at fully understanding the molecular basis behind chemoreception-related behaviors in S. pyrastri.
Citation: Li X-M, Zhu X-Y, He P, Xu L, Sun L, Chen L, et al. (2016) Molecular Characterization and Sex Distribution of Chemosensory Receptor Gene Family Based on Transcriptome Analysis of Scaeva pyrastri. PLoS ONE 11(5): e0155323. https://doi.org/10.1371/journal.pone.0155323
Editor: J Joe Hull, USDA-ARS, UNITED STATES
Received: December 23, 2015; Accepted: April 27, 2016; Published: May 12, 2016
Copyright: © 2016 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All of the chemosensory receptor genes of Scaeva pyrastri were submitted to the GenBank of the NCBI (accession number: KU291817-KU291876).
Funding: This work was supported by grants from the National Natural Science Foundation (NO. 31240075) of China, Forest Pest Census of Huaibei City (Huaibei Forestry Bureau, NO. 26700887), Natural Science Fund of Education Department of Anhui Province (NO. KJ2013A233).
Competing interests: The authors have declared that no competing interests exist.
An accurate and complex olfactory system helps insects find resources (e.g., suitable hosts, predators, oviposition sites, mates) . Previous studies have shown that insects tend to use their antenna—an efficient olfactory organ—to detect chemical signals from the external environment [2, 3]. The insect olfactory system involves several molecular components, including odorant binding proteins (OBPs) [4–6], chemosensory proteins (CSPs) [7, 8], and sensory neuron membrane proteins (SNMPs) [9, 10]. Additionally, 3 major chemosensory receptor families are involved: olfactory receptors (ORs) [1, 11, 12], gustatory receptors (GRs) [13–15], and ionotropic receptors (IRs) [4, 16, 17]. These receptors are located on the dendrites of neurons in antenna chemosensilla and other chemosensory tissues.
To explore the mechanisms underlying insect olfaction, the identification, sex distribution, and functional analyses of candidate chemosensory receptor genes are important initial steps. Compared with older gene cloning techniques such as rapid amplification of cDNA ends (RACE) and expressed sequence tag (EST) library construction [18–22], next-generation sequencing techniques such as RNA sequencing (RNA-seq) are now considered more efficient in generating data, less time-consuming, and more cost-effective. These recent technological advancements have allowed the large-scale identification of chemosensory genes from Diptera insects whose genomes are not yet sequenced, as is the case of Calliphora stygia , Bactrocera dorsalis , Mayetiola destructor , and the natural enemy insect Microplitis mediator . However, their exact functions are largely unknown, as these genes were mainly identified based on sequence similarity to reported genes. Their expression profiles, particularly those varying according to sex, and phylogenetic analyses could provide important information on the functions of chemosensory receptor genes [25, 27–30].
Scaeva pyrastri (L.) (Diptera: Syrphidae) is a pollinating and natural enemy insect found worldwide, although mainly distributed in the Palearctic and Nearctic . Adults are flower-visiting and larvae prey on aphids, a major agricultural pest . Previous studies have shown that some chemical cues (plant volatiles and the residues or secretions of aphids) play a key role in mediating many aspects of S. pyrastri behavior, such as parasitism and oviposition [33–35], but the specific molecular mechanisms of their chemosensory-guided behaviors are currently unknown. In the present study, we performed a transcriptome analysis based on adult S. pyrastri antennae, and identified 60 candidate chemosensory receptor genes comprising 38 ORs, 6 GRs, and 16 IRs. We further conducted a comprehensive analysis of their phylogeny and sex distribution and the results clearly demonstrated that some genes exhibit sex-biased or -specific expression. Thus, our data contribute to the overall understanding of chemoreception-based behavioral mechanisms in S. pyrastri.
Materials and Methods
S. pyrastri were collected in April 2015 from a Brassica campestris field in the Pollution-Free Planting Base of Huaibei City, Anhui Province, China. The field studies did not involve endangered or protected species, and no specific permissions were required for these research activities in these locations.
Insect rearing and collection
Adult S. pyrastri were separated into females and males, and reared on aphids. The rearing conditions were 25 ± 1°C, 12:12 light:dark photoperiod, and 70 ± 10% relative humidity. For transcriptome sequencing, the antennae of 600 adults (300 males and 300 females) were collected. For the tissue expression study, 150–200 female antennae (FA), 150–200 male antennae (MA), and 12–15 whole insect bodies without antennae (Bo) were also collected. All samples were immediately frozen in liquid nitrogen and stored at -80°C until use.
cDNA library construction, clustering, and sequencing
As previously described in detail [36–38], total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Construction of the cDNA library and Illumina sequencing were performed at Novogene Bioinformatics Technology Co., Ltd., Beijing, China. The mRNA was purified from 3 μg of total RNA using oligo (dT) magnetic beads and fragmented into short sequences in the presence of divalent cations at 94°C for 5 min. First-strand cDNA was then generated using random hexamer-primed reverse transcription, followed by second-strand-cDNA synthesis using RNaseH and DNA polymerase I. After adaptor end-repair and ligation, cDNA was amplified via PCR and purified using the QIAquick PCR Purification Kit to create a cDNA library. Library quality was assessed on an Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina), following the manufacturer’s protocol. After cluster generation, library preparations were sequenced on an Illumina Hiseq™ 2500 platform and paired-end reads were obtained.
De novo assembly of short reads and gene annotation
Raw reads were cleaned following the methods described in our previous studies [36–38], by removing reads with low-quality and/or containing adapters or poly-N tails. Transcriptome de novo assembly was performed based on clean short reads using the program Trinity (r20140413p1) [39, 40] with its default parameters. The Basic Local Alignment Search Tool (BLASTX) was used to search for sequence homology between unigenes > 150 bp resulting from the assembly and sequences deposited in the National Center for Biotechnology Information (NCBI) Non-redundant (Nr), Swiss-Prot, Kyoto Encyclopedia for Genes and Genomes (KEGG), and Clusters of Orthologous Groups (COG) databases (e-value < 10−5 for all databases). Proteins with the highest sequence similarity were retrieved, along with their functional annotations. We then used Blast2GO (e-value < 10−6)  for gene ontology (GO) annotation and functional classification of the unigenes.
The open reading frames (ORFs) of chemosensory genes were predicted using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Similarity searches were performed using the NCBI-BLAST network server (http://blast.ncbi.nlm.nih.gov/). The transmembrane domains of S. pyrastri ORs, IRs, and GRs (SpyrORs, SpyrIRs, and SpyrGRs, respectively) were predicted with the TMHMM Server Version 2.0 (http://www.cbs.dtu.dk/services/TMHMM).
The phylogenetic trees of SpyrORs, SpyrGRs, and SpyrIRs were reconstructed based on the sequences obtained here and on the amino acid sequences of ORs, GRs, and IRs reported for other insects. The OR data set contained 38 sequences from S. pyrastri, plus 204 combined from Drosophila melanogaster , C. stygia , Acyrthosiphon pisum [43, 44], and A. gossypii . The GR data set contained 6 sequences from S. pyrastri, plus 280 combined from D. melanogaster , Anopheles gambiae , and Bombyx mori . The IR data set contained 16 sequences from S. pyrastri, plus 154 combined from D. melanogaster , A. gossypii , Musca domestica, A. gambiae, and C. stygia . The amino acid sequences of S. pyrastri genes used for phylogenetic tree construction are listed in S1 Table. Amino acid sequences were aligned using ClustalX 1.83  and the phylogenetic trees were constructed in PhyML , based on the LG substitution model  with Nearest Neighbor Interchange(NNI); branch support was estimated with a Bayesian-like transformation of aLRT (aBayes). Dendrograms were created and colored in FigTree (http://tree.bio.ed.ac.uk/software/figtree/).
RNA isolation, cDNA synthesis, and reverse transcription-PCR analysis
As previously described in detail [36–38], total RNA was extracted with the SV 96 Total RNA Isolation System (Promega, Madison, WI, USA) following the manufacturer’s protocol, and including a DNaseI digestion to avoid genomic DNA contamination. RNA quality was verified with a NanoDrop™ 2000 (Thermo Fisher Scientific, USA). Single-stranded cDNA templates were synthesized using 1 μg of total RNA from both body and antennae tissue samples and the PrimeScript™ RT Master Mix (TaKaRa, Dalian, China). Gene-specific primers across the ORFs of predicted chemosensory genes were designed using Primer Premier 5.0 (PREMIER Biosoft International, CA, USA), and their sequences are listed in S2 Table. Reverse transcription (RT)-PCR (including negative controls with no cDNA template) profile was as follows: initial denaturation at 94°C for 4 min; 35–40 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s; and final incubation at 72°C for 10 min. Cycle number was reduced to 30 in the reference gene amplification. The reaction volume was 25 μL, containing 12.5 μL Premix Taq™ (TaKaRa Taq™ Version 2.0; TaKaRa, Dalian, China), 0.4 μM each primer, 1 μL sample cDNA (15 ng/μL), and 9.5 μL sterilized H2O. PCR products were analyzed via electrophoresis on 1.5% w/v agarose gels in TAE buffer (40 mmol/L Tris-acetate, 2 mmol/L Na2EDTA·H2O), and the resulting bands were visualized using SYBR Green I (Tiandz, Beijing, China). The gene encoding S. pyrastri glyceraldehyde-3-phosphate dehydrogenase (SpyrGAPDH) was used as reference to check the integrity of the cDNA template, and a reagents mix without cDNA template was used as the negative control (NC). Two independent biological replications were performed for each RT-PCR amplification, and each biological replication was repeated at least twice. The expected products of randomly selected genes were sequenced to confirm they corresponded to the originally identified sequence.
Transcriptome sequencing and sequence assembly
The transcriptome sequencing of S. pyrastri antennae provided about 51 million clean reads (5.1 Gb). After clustering and redundancy filtering, we acquired 63,672 unigenes with a N50 length of 1,130 bp (Table 1); unigenes > 500 bp accounted for 31.28% of the transcriptome assembly (Fig 1). As several recent publications have described [52, 53], these unigenes do not necessarily represent distinct genes.
Homology analysis and GO annotation
The BLASTX homology search performed for the 63,672 unigenes showed homology for 29,587 (46.46%) of them in the NCBI Nr protein database. The best match was to Ceratitis capitata sequences (26.20%), followed by M. domestica (21.40%), D. melanogaster (5.00%), D. willistoni (3.30%), and D. mojavensis (3.20%) sequences (Fig 2).
The S. pyrastri transcripts were searched by BLASTX against the non-redundancy protein database with a cutoff E-value 10−5. Species which have more than 1% matching hits to the S. pyrastri transcripts are shown.
The GO annotations resulting from the Blast2GO pipeline revealed that 33.24% (21,165) of all unigenes were successfully assigned to functional groups. The most well represented groups were: cellular, metabolic, and single-organism processes in the “biological process” category; cell, cell part, and organelle in the “cellular component” category; binding, catalytic activity, and transporter activity in the “molecular function” category (Fig 3).
The Y-axis shows the number of annotated GO terms in three categories: biological process, cellular component, and molecular function. The X-axis shows three areas of annotation, and in each area the sequences are further divided into subgroups.
Identification and phylogenetic trees of candidate OR, GR, and IR genes
According to the homology analysis, 60 transcripts belonging to chemosensory receptor families were newly identified in this study, including 38 ORs, 6 GRs, and 16 IRs (Table 2).
Thirty-eight different transcripts encoding candidate ORs were identified based on the S. pyrastri antennal transcriptome data, and 30 of these sequences contained a full-length ORF that encoded 361–476 amino acids. One OR sequence sharing high identity with the conserved Orco proteins of other insect species was obtained and thus named SpyrOrco. This amino-acid sequence had 90% identity with the co-receptor of M. domestica (XP_005175278.1), and the remaining SpyrORs had 22–69% identity with other insect ORs. Based on these comparisons, and on our previous predictions [23, 24, 54–57], full-length SpyrORs had 4–8 transmembrane domains (TMDs) (Table 2). Additionally, we identified 6 GR candidates in S. pyrastri, similar to the number of candidates reported in the recent antennal transcriptomic study of B. dorsalis . Among the 6 candidates, only 4 were likely to represent full-length ORFs (SpyrGR2, 3, 4, and 6), and each contained 6–7 TMDs.
The phylogenetic tree constructed using all SpyrOR sequences and the Orco sequences from 8 Diptera and 2 Hemiptera revealed the clustering of SpyrOrco with other Diptera Orco sequences (Fig 4). All identified SpyrORs had at least 1 dipteran orthologue. The tree constructed for GR sequences evidenced that 3 SpyrGRs (SpyrGR2, 3, and 6) were distributed in CO2 receptors, but no orthologues of sugar or fructose receptors were found (Fig 5). Although we identified 16 IR candidates in S. pyrastri, a similar number to those found for D. melanogaster , only 7 of these IR candidates likely represented full-length ORFs [SpyrIR3(75q2), 6(92a), 7(75d), 8(64a), 10(8a), 13(25a), and 16(84a)], encoding 645–1148 amino acids and containing 2–4 TMDs (Table 2). Phylogenetic analysis of the IRs revealed that these 16 candidates were clustered into antennal IRs, divergent IRs, and IR25a/IR8a clades (Fig 6).
The S. pyrastri translated genes are shown in blue. This tree was constructed using PhyML based on alignment results of ClustalX. Orco clade is marked in red. Sp: S. pyrastri, Ap: Acyrthosiphon pisum, Ag, A. gossypii, Dm: D. melanogaster, Sc: Stomoxys calcitrans, Md: Musca domestica, Cr: Chrysomya rufifacies, Cm: Chrysomya megacephala, Cs: C. stygia, Ls: Lucilia sericata, Cv: Calliphora vicina.
The S. pyrastri translated genes are shown in blue. This tree was constructed using PhyML based on alignment results of ClustalX. Sp: S. pyrastri, Dm: D. melanogaster, Ag: A. gambiae, Bm: B. mori.
The S. pyrastri translated genes are shown in black. This tree was constructed using PhyML based on alignment results of ClustalX. Sp: S. pyrastri, Dm: D. melanogaster, Cs: C. stygia, Md: M. domestica, Ag: A. gambiae, Ago: Aphis gossypii.
Sex distribution and tissue expression of candidate OR, GR, and IR genes
Based on recent studies, including our own, RT-PCR is a reliable method for analyzing the tissue expression of chemosensory genes in many insects [28, 37, 59–62]. Therefore, we also used this method to investigate the chemosensory receptor genes expressed in S. pyrastri antennae and body, using SpyrGAPDH as the reference gene. RT-PCR showed that 37 candidate ORs (except for SpyrOR38) were expressed in the antennae. Of the candidate ORs, 1 (SpyrOR13) exhibited male-biased expression, while 11 (SpyrOR1, 3, 4, 7, 15, 19, 23, 26, 29, 30, and 36) exhibited female-biased expression. Remarkably, SpyrOR9 was the only gene exhibiting male-specific expression. The remaining ORs were expressed fairly equally in both male and female antennae (Fig 7). Candidate GR and IR genes did not exhibit significant sex biased expression, except for SpyrIR16(84a) that showed male-biased expression in the antennae (Fig 7). No candidate receptor genes were expressed in S. pyrastri body tissues (Fig 7).
In comparison with our understanding of insect pests, the molecular basis of chemoreception in natural enemies is poorly understood. Here, we sequenced and analyzed the transcriptome of S. pyrastri using samples from antennae. Among the 63,672 unigenes, only 46.46% had homologous matches to the NCBI Nr protein database and 33.24% were annotated to 1 or more GO terms. Although these percentages are similar to those found in other species [63–65], they indicate that most S. pyrastri genes are either non-coding or homologous with genes that have not been annotated to GO terms. Importantly, we identified 60 novel chemosensory receptor genes in S. pyrastri. Our results not only provide an important basis for further elucidation of the molecular mechanisms underlying chemoreception, but also provide insights into insect physiology and natural enemy-based control strategies [66–68].
Previous studies found that chemical cues (e.g., sex pheromones, aphid secretions, and plant volatiles) play a major role in mediating many S. pyrastri behaviors, including mating, parasitism, and oviposition [33–35], suggesting that consistently expressed ORs, GRs, and IRs are likely involved in these behaviors. However, S. pyrastri chemosensory genes have not been identified before this study. The number of chemosensory receptor transcripts identified in S. pyrastri in the present study (60) was greater than that reported for B. dorsalis (40) , but 2.5 times lower than that found in D. melanogaster (153) and 2.9 times lower than that of A. gambiae (177) [42, 58, 69] whose genomes have been sequenced. These differences suggest a high probability of identifying more S. pyrastri chemosensory receptor genes once its genome is fully sequenced.
Furthermore, 59 of the 60 chemosensory receptor genes were expressed in adult antennae, which mirror the numbers found in D. melanogaster , indicating their importance in S. pyrastri olfaction. We found fewer ORs in the antennal transcriptome of S. pyrastri (38) than in the complete genome of D. melanogaster (62). However, our S. pyrastri OR count was closer to that in the antennal transcriptome of C. stygia (50), suggesting that we may have missed larvae-biased or lowly expressed ORs. SpyrOR38 expression, for instance, was not detected in RT-PCR but was identified in RNA-Seq, suggesting that the latter method may be more sensitive than RT-PCR for detecting low expression levels. Despite these limitations, the patterns evidenced in the present and previous studies are consistent with the evolution of species-specific plant-host adaptation and odorant perception in Diptera. Remarkably, and similar to that observed in other insects [23, 26, 64, 71], a species-specific expansion of ORs was found in S. pyrastri, as evidenced by the number of SpyrORs with no orthologues distributed in the several clusters of the phylogenetic tree (SpyrOR31/38/35/23, SpyrOR7/36/22/26, SpyrOR32/19/28/29, and SpyrOR27/3/34/1/4, Fig 4). In addition, 7 of these ORs (Spyr36/26, Spyr19/29, and Spyr3/1/4) exhibited female-biased expression, suggesting they might be related to specific odor-oriented female behaviors, such as selecting conspecific males and the oviposition substrate. Thus, these receptor-mediated behaviors might be species-specific behaviors, as the cues triggering them are only perceived by hover fly females cues, meaning these 7 female-biased ORs might be species-specific receptors.
One gene (SpyrOrco) displayed high identity with Orco genes known for other insects, suggesting that Orco also acts in S. pyrastri. This protein is more highly conserved than other ORs [1, 42, 72, 73] and might act as a chaperone and dimerization partner for other insect ORs, forming a ligand-gated ion channel to specific ligands [56, 73–75] and having a similar function in different insects . Notably, only 2 ORs were male-biased (SpyrOR13) and male-specific (SpyrOR9). These 2 genes might function as insect pheromone receptors (PRs), a well-studied group [77–80], similar to DmelOR67d in D. melanogaster and BmorOR1 in B. mori, which are essential for detecting the male-specific pheromone 11-cis-vaccenyl acetate (VA) [81, 82] and to respond to the sex pheromone component bombykol , respectively. Additionally, we identified 11 genes displaying female-biased expression (SpyrOR1, 3, 4, 7, 15, 19, 23, 26, 29, 30, and 36). These SpyrORs may be responsible for detecting oviposition-related cues or male-produced courtship pheromones. Still, the putative functions of male- and female-biased genes require verification with in vitro and in vivo studies.
To distinguish candidate IRs from ionotropic glutamate receptors (iGluRs), SpyrIRs were aligned with iGluRs from D. melanogaster and IR orthologues from several other insect species before BLASTX and phylogenetic analyses. Overall, we identified 16 IRs in the antennal transcriptome of S. pyrastri and verified these were distinct from iGluRs, suggesting S. pyrastri has fewer antennal-expressed IR genes than D. melanogaster (18)  and A. gambiae (22) . Although we might have missed some transcripts in our antennal transcriptome, the available SpyrIR genes provided some insight into their function. Specifically, sequence alignments and phylogenetic analyses revealed that SpyrIR10(8a) and SpyrIR13(25a) belong to the co-expression IR group. Receptors in this group are similar to Orco as their co-expression with other IRs implies they play a role as co-receptors . Thus, SpyrIR10(8a) and SpyrIR13(25a), as well as other SpyrIRs, may serve the same function in chemical communication as their orthologues in D. melanogaster [17, 84, 85].
Although many GRs have been identified in a variety of insect species [23, 24, 55, 65, 86, 87], we only identified 6 GRs in S. pyrastri. However, this low number was expected because GRs are primarily expressed in gustatory organs such as the proboscis and not in the antennae . Despite the low number of GRs, phylogenetic analysis revealed that 3 GRs (SpyrGR2, 3, and 6) were clustered into the “CO2 Receptors” group with DmelGR21a and DmelGR63a from D. melanogaster, indicating they might be involved in CO2 detection [88, 89]. Like SpyrIRs, SpyrGRs were equally expressed in male and female antennae. Therefore, the function of GRs in olfactory progresses appears not to differ between sexes.
Based on RNA-seq and RT-PCR data, sequence analysis of the antennal transcriptome data allowed successfully identifying an extensive set of candidate OR, GR, and IR genes that might be related to the odorant perception of S. pyrastri. As the first step towards understanding their function, we performed a comprehensive and comparative analysis of ORs, GRs, and IRs phylogeny and expression patterns according to sex. These analyses evidenced the species-specific expansion and the sex-specific or -biased expression of some genes, respectively. Therefore, this study contributes to an increased understanding of the molecular mechanisms underlying chemosensory-guided behaviors in S. pyrastri, providing data for further functional analyses of chemosensory receptors.
S1 Table. Amino acid sequences of S. pyrastri used in phylogenetic analyses.
We thank Bachelor students Geng Chen, Wei-Wei Zhou, Tian Tian, Xiao-Tong Zhu, and Xiao-Xue Xu (Huaibei Normal University, China) for help in collecting insect. This work was supported by grants from the National Natural Science Foundation (NO. 31240075) of China, Forest Pest Census of Huaibei City (Huaibei Forestry Bureau, NO. 26700887), Natura Science Fund of Education Department of Anhui Province (NO. KJ2013A233).
Conceived and designed the experiments: XML YNZ. Performed the experiments: ZQW LC. Analyzed the data: XYZ PH LX LS DGD YNZ. Contributed reagents/materials/analysis tools: XML YNZ DGD. Wrote the paper: XML YNZ.
- 1. Leal WS. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annu Rev Entomol. 2013;58(1):373–91. Epub 2012/10/02. pmid:23020622.
- 2. Syed Z, Leal WS. Maxillary palps are broad spectrum odorant detectors in Culex quinquefasciatus. Chem Senses. 2007;32(8):727–38. pmid:17569743.
- 3. Keil TA. Reconstruction and morphometry of silkmoth olfactory hairs: A comparative study of sensilla trichodea on the antennae of male Antheraea polyphemus and Antheraea pernyi (Insecta, Lepidoptera). Zoomorphology. 1984;104(3):147–56.
- 4. Vogt RG. Biochemical diversity of odor detection:OBPs, ODEs and SNMPs. Insect pheromone biochemistry and molecular biology. 2003;Edited by:Blomquist GJ, Vogt RG. Elsevier Academic Press; 2003:397–451.
- 5. Zhou JJ. Odorant-binding proteins in insects. Vitam Horm. 2010;83:241–72. Epub 2010/09/14. S0083-6729(10)83010-9 [pii] pmid:20831949.
- 6. Xu YL, He P, Zhang L, Fang SQ, Dong SL, Zhang YJ, et al. Large-scale identification of odorant-binding proteins and chemosensory proteins from expressed sequence tags in insects. BMC Genomics. 2009;10:632. pmid:20034407; PubMed Central PMCID: PMC2808328.
- 7. Pelosi P, Zhou JJ, Ban LP, Calvello M. Soluble proteins in insect chemical communication. Cell Mol Life Sci. 2006;63(14):1658–76. Epub 2006/06/21. pmid:16786224.
- 8. Pelosi P, Calvello M, Ban L. Diversity of odorant-binding proteins and chemosensory proteins in insects. Chem Senses. 2005;30 Suppl 1:i291–2. Epub 2005/03/02. 30/suppl_1/i291 [pii] pmid:15738163.
- 9. Rogers ME, Sun M, Lerner MR, Vogt RG. Snmp-1, a novel membrane protein of olfactory neurons of the silk moth Antheraea polyphemus with homology to the CD36 family of membrane proteins. J Biol Chem. 1997;272(23):14792–9. Epub 1997/06/06. pmid:9169446.
- 10. Vogt RG, Miller NE, Litvack R, Fandino RA, Sparks J, Staples J, et al. The insect SNMP gene family. Insect Biochem Mol Biol. 2009;39(7):448–56. Epub 2009/04/15. S0965-1748(09)00062-9 [pii] pmid:19364529.
- 11. Zhang J, Walker WB, Wang G. Pheromone reception in moths: from molecules to behaviors. Prog Mol Biol Transl Sci. 2015;130:109–28. Epub 2015/01/28. S1877-1173(14)00020-9 [pii]. pmid:25623339.
- 12. Crasto CJ. Olfactory Receptors. Methods in molecular biology™. 2013;Edited by Walker JM. Humana Press.
- 13. Clyne PJ, Warr CG, Carlson JR. Candidate taste receptors in Drosophila. Science. 2000;287(5459):1830–4. pmid:10710312.
- 14. Zhang HJ, Anderson AR, Trowell SC, Luo AR, Xiang ZH, Xia QY. Topological and functional characterization of an insect gustatory receptor. PLoS One. 2011;6(8):e24111. Epub 2011/09/14. PONE-D-11-09026 [pii]. pmid:21912618; PubMed Central PMCID: PMC3163651.
- 15. Briscoe AD, Macias-Munoz A, Kozak KM, Walters JR, Yuan F, Jamie GA, et al. Female behaviour drives expression and evolution of gustatory receptors in butterflies. PLoS Genet. 2013;9(7):e1003620. pmid:23950722; PubMed Central PMCID: PMC3732137.
- 16. Tunstall NE, Warr CG. Chemical communication in insects: the peripheral odour coding system of Drosophila melanogaster. Adv Exp Med Biol. 2012;739:59–77. Epub 2012/03/09. pmid:22399395.
- 17. Benton R, Vannice KS, Gomez-Diaz C, Vosshall LB. Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell. 2009;136(1):149–62. Epub 2009/01/13. S0092-8674(08)01561-4 [pii] pmid:19135896; PubMed Central PMCID: PMC2709536.
- 18. McKenna MP, Hekmat-Scafe DS, Gaines P, Carlson JR. Putative Drosophila pheromone-binding proteins expressed in a subregion of the olfactory system. J Biol Chem. 1994;269(23):16340–7. Epub 1994/06/10. pmid:8206941.
- 19. Picimbon JF, Gadenne C. Evolution of noctuid pheromone binding proteins: identification of PBP in the black cutworm moth, Agrotis ipsilon. Insect Biochem Mol Biol. 2002;32(8):839–46. Epub 2002/07/12. S0965174801001722 [pii]. pmid:12110291.
- 20. Xiu WM, Zhou YZ, Dong SL. Molecular characterization and expression pattern of two pheromone-binding proteins from Spodoptera litura (Fabricius). J Chem Ecol. 2008;34(4):487–98. Epub 2008/03/19. pmid:18347871.
- 21. Calvello M, Brandazza A, Navarrini A, Dani FR, Turillazzi S, Felicioli A, et al. Expression of odorant-binding proteins and chemosensory proteins in some Hymenoptera. Insect Biochem Mol Biol. 2005;35(4):297–307. Epub 2005/03/15. S0965-1748(05)00005-6 [pii] pmid:15763466.
- 22. Legeai F, Malpel S, Montagne N, Monsempes C, Cousserans F, Merlin C, et al. An expressed sequence tag collection from the male antennae of the Noctuid moth Spodoptera littoralis: a resource for olfactory and pheromone detection research. BMC Genomics. 2011;12:86. Epub 2011/02/01. 1471-2164-12-86 [pii] pmid:21276261; PubMed Central PMCID: PMC3045336.
- 23. Leitch O, Papanicolaou A, Lennard C, Kirkbride KP, Anderson A. Chemosensory genes identified in the antennal transcriptome of the blowfly Calliphora stygia. BMC Genomics. 2015;16:255. Epub 2015/04/17. 10.1186/s12864-015-1466-8 [pii]. pmid:25880816; PubMed Central PMCID: PMC4392625.
- 24. Wu Z, Zhang H, Wang Z, Bin S, He H, Lin J. Discovery of chemosensory genes in the oriental fruit fly, Bactrocera dorsalis. PLoS One. 2015;10(6):e0129794. Epub 2015/06/13. PONE-D-15-07024 [pii]. pmid:26070069; PubMed Central PMCID: PMC4466378.
- 25. Andersson MN, Videvall E, Walden KK, Harris MO, Robertson HM, Lofstedt C. Sex- and tissue-specific profiles of chemosensory gene expression in a herbivorous gall-inducing fly (Diptera: Cecidomyiidae). BMC Genomics. 2014;15(1):501. Epub 2014/06/21. 1471-2164-15-501 [pii] pmid:24948464.
- 26. Wang SN, Peng Y, Lu ZY, Dhiloo KH, Gu SH, Li RJ, et al. Identification and expression analysis of putative chemosensory receptor genes in Microplitis mediator by antennal transcriptome screening. Int J Biol Sci. 2015;11(7):737–51. Epub 2015/06/17. ijbsv11p0737 [pii]. pmid:26078716; PubMed Central PMCID: PMC4466455.
- 27. Robertson HM, Wanner KW. The chemoreceptor superfamily in the honey bee, Apis mellifera: expansion of the odorant, but not gustatory, receptor family. Genome Res. 2006;16(11):1395–403. Epub 2006/10/27. gr.5057506 [pii] pmid:17065611; PubMed Central PMCID: PMC1626641.
- 28. Olivier V, Monsempes C, Francois MC, Poivet E, Jacquin-Joly E. Candidate chemosensory ionotropic receptors in a Lepidoptera. Insect Mol Biol. 2011;20(2):189–99. Epub 2010/11/26. pmid:21091811.
- 29. Krieger J, Grosse-Wilde E, Gohl T, Dewer YM, Raming K, Breer H. Genes encoding candidate pheromone receptors in a moth (Heliothis virescens). Proc Natl Acad Sci U S A. 2004;101(32):11845–50. Epub 2004/08/04. [pii]. pmid:15289611; PubMed Central PMCID: PMC511062.
- 30. Kang N, Koo J. Olfactory receptors in non-chemosensory tissues. BMB reports. 2012;45(11):612–22. pmid:23186999.
- 31. Huang CM, Cheng XY. Insecta Vol. 50: Diptera Syrphidae. Fauna Sinica. 2012;Edited by: Huang CM, Cheng XY. Science Press; pp 825
- 32. Xiong HZ, Dong HF. The oviposition behavior of Scaeva pyrastri (HYM.: Surphidae) and its control effct on aphids in greenhouses. CHINESE J BIOLOGICAL CONTROL. 1991;7(2):49–52.
- 33. Xiong HZ, Dong HF. The oviposition behavior of Scaeva pyrastri (HYM.:Surphidae) and its control effect on aphids in greenhouses. CHINESE J BIOLOGICAL CONTROL. 1991;7(2):49–52.
- 34. Güncan A, Yoldaș Z, Madanlar N. Studies on the aphids (Hemiptera: Aphididae) and their natural enemies on peach orchards in Izmir. Turk Entomol. 2010;34(3):399–408.
- 35. Jones VP, Horton DR, Mills NJ, Unruh TR, Baker CC, Melton TD, et al. Evaluating plant volatiles for monitoring natural enemies in apple, pear and walnut orchards. Biol Control. 2015.
- 36. Zhang YN, Zhu XY, Fang LP, He P, Wang ZQ, Chen G, et al. Identification and expression profiles of sex pheromone biosynthesis and transport related genes in Spodoptera litura. PLoS One. 2015;10(10):e0140019. pmid:26445454.
- 37. Zhang YN, Jin JY, Jin R, Xia YH, Zhou JJ, Deng JY, et al. Differential expression patterns in chemosensory and non-chemosensory tissues of putative chemosensory genes identified by transcriptome analysis of insect pest the purple stem borer Sesamia inferens (Walker). PLoS One. 2013;8(7):e69715. pmid:23894529; PubMed Central PMCID: PMC3722147.
- 38. Xia YH, Zhang YN, Hou XQ, Li F, Dong SL. Large number of putative chemoreception and pheromone biosynthesis genes revealed by analyzing transcriptome from ovipositor-pheromone glands of Chilo suppressalis. Scientific reports. 2015;5:7888. pmid:25601555
- 39. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29(7):644–52. pmid:21572440; PubMed Central PMCID: PMC3571712.
- 40. Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, et al. De novo assembly of human genomes with massively parallel short read sequencing. Genome Res. 2010;20(2):265–72. Epub 2009/12/19. gr.097261.109 [pii] pmid:20019144; PubMed Central PMCID: PMC2813482.
- 41. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics. 2005;21(18):3674–6. Epub 2005/08/06. bti610 [pii] pmid:16081474.
- 42. Robertson HM, Warr CG, Carlson JR. Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2003;100 Suppl 2:14537–42. Epub 2003/11/11. pmid:14608037; PubMed Central PMCID: PMCPmc304115.
- 43. Smadja C, Shi P, Butlin RK, Robertson HM. Large Gene Family Expansions and Adaptive Evolution for Odorant and Gustatory Receptors in the Pea Aphid, Acyrthosiphon pisum. Mol Biol Evol. 2009;26(9):2073–86. pmid:19542205
- 44. Smadja CM, Canbäck B, Vitalis R, Gautier M, Ferrari J, Zhou J-J, et al. Large-scale candidate gene scan reveals the role of chemoreceptor genes in host plant specialization and speciation in the pea aphid. Evolution. 2012;66(9):2723–38. pmid:22946799
- 45. Cao D, Liu Y, Walker WB, Li J, Wang G. Molecular characterization of the Aphis gossypii olfactory receptor gene families. PLoS One. 2014;9(6):e101187. Epub 2014/06/28. PONE-D-14-16877 [pii]. pmid:24971460; PubMed Central PMCID: PMC4074156.
- 46. Hill CA, Fox AN, Pitts RJ, Kent LB, Tan PL, Chrystal MA, et al. G Protein-Coupled Receptors in Anopheles gambiae. Science. 2002;298(5591):176–8.
- 47. Wanner KW, Robertson HM. The gustatory receptor family in the silkworm moth Bombyx mori is characterized by a large expansion of a single lineage of putative bitter receptors. Insect Mol Biol. 2008;17(6):621–9. pmid:19133074.
- 48. Croset V, Rytz R, Cummins SF, Budd A, Brawand D, Kaessmann H, et al. Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction. PLoS Genetics. 2010;6(8):e1001064. Epub 2010/09/03. e1001064 [pii] pmid:20808886; PubMed Central PMCID: PMC2924276.
- 49. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23(21):2947–8. Epub 2007/09/12. btm404 [pii] pmid:17846036.
- 50. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59(3):307–21. pmid:20525638.
- 51. Le SQ, Gascuel O. An improved general amino acid replacement matrix. Mol Biol Evol. 2008;25(7):1307–20. pmid:18367465.
- 52. Liu Y, Gu S, Zhang Y, Guo Y, Wang G. Candidate olfaction genes identified within the Helicoverpa armigera antennal transcriptome. PLoS One. 2012;7(10):e48260. Epub 2012/10/31. PONE-D-12-23655 [pii]. pmid:23110222; PubMed Central PMCID: PMC3482190.
- 53. Li SW, Yang H, Liu YF, Liao QR, Du J, Jin DC. Transcriptome and gene expression analysis of the rice leaf folder, Cnaphalocrosis medinalis. PLoS One. 2012;7(11):e47401. Epub 2012/11/28. PONE-D-11-23279 [pii]. pmid:23185238; PubMed Central PMCID: PMC3501527.
- 54. Bengtsson JM, Trona F, Montagne N, Anfora G, Ignell R, Witzgall P, et al. Putative chemosensory receptors of the codling moth, Cydia pomonella, identified by antennal transcriptome analysis. PLoS One. 2012;7(2):e31620. Epub 2012/03/01. PONE-D-11-20430 [pii]. pmid:22363688; PubMed Central PMCID: PMC3282773.
- 55. Liu NY, Xu W, Papanicolaou A, Dong SL, Anderson A. Identification and characterization of three chemosensory receptor families in the cotton bollworm Helicoverpa armigera. BMC Genomics. 2014;15:597. Epub 2014/07/17. [pii]. pmid:25027790; PubMed Central PMCID: PMC4112213.
- 56. Krieger J, Grosse-Wilde E, Gohl T, Breer H. Candidate pheromone receptors of the silkmoth Bombyx mori. Eur J Neurosci. 2005;21(8):2167–76. Epub 2005/05/05. EJN4058 [pii] pmid:15869513.
- 57. Wang G, Carey AF, Carlson JR, Zwiebel LJ. Molecular basis of odor coding in the malaria vector mosquito Anopheles gambiae. Proc Natl Acad Sci U S A. 2010;107(9):4418–23. pmid:20160092; PubMed Central PMCID: PMC2840125.
- 58. Croset V, Rytz R, Cummins SF, Budd A, Brawand D, Kaessmann H, et al. Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction. PLoS Genet. 2010;6(8):e1001064. Epub 2010/09/03. e1001064 [pii] pmid:20808886; PubMed Central PMCID: PMC2924276.
- 59. Patch HM, Velarde RA, Walden KK, Robertson HM. A candidate pheromone receptor and two odorant receptors of the hawkmoth Manduca sexta. Chem Senses. 2009;34(4):305–16. Epub 2009/02/04. [pii]. pmid:19188280.
- 60. Xu H, Guo M, Yang Y, You Y, Zhang L. Differential expression of two novel odorant receptors in the locust (Locusta migratoria). BMC Neurosci. 2013;14(1):50. Epub 2013/04/24. 1471-2202-14-50 [pii] pmid:23607307.
- 61. Tanaka K, Uda Y, Ono Y, Nakagawa T, Suwa M, Yamaoka R, et al. Highly selective tuning of a silkworm olfactory receptor to a key mulberry leaf volatile. Curr Biol. 2009;19(11):881–90. Epub 2009/05/12. S0960-9822(09)01034-3 [pii] pmid:19427209.
- 62. Latorre-Estivalis JM, de Oliveira ES, Esteves BB, Guimaraes LS, Ramos MN, Lorenzo MG. Patterns of expression of odorant receptor genes in a Chagas disease vector. Insect Biochem Mol Biol. 2015;In press. Epub 2015/05/25. S0965-1748(15)00083-1 [pii] pmid:26003917.
- 63. Gu XC, Zhang YN, Kang K, Dong SL, Zhang LW. Antennal transcriptome analysis of odorant reception genes in the Red Turpentine Beetle (RTB), Dendroctonus valens. PLoS One. 2015;10(5):e0125159. Epub 2015/05/06. PONE-D-14-52146 [pii]. pmid:25938508.
- 64. Li X, Ju Q, Jie W, Li F, Jiang X, Hu J, et al. Chemosensory gene families in adult antennae of Anomala corpulenta Motschulsky (Coleoptera: Scarabaeidae: Rutelinae). PLoS One. 2015;10(4):e0121504. Epub 2015/04/10. PONE-D-14-20592 [pii]. pmid:25856077.
- 65. Andersson MN, Grosse-Wilde E, Keeling CI, Bengtsson JM, Yuen MM, Li M, et al. Antennal transcriptome analysis of the chemosensory gene families in the tree killing bark beetles, Ips typographus and Dendroctonus ponderosae (Coleoptera: Curculionidae: Scolytinae). BMC Genomics. 2013;14:198. Epub 2013/03/23. [pii]. pmid:23517120; PubMed Central PMCID: PMC3610139.
- 66. Zhou JJ, Field LM, He XL. Insect odorant-binding proteins: Do they offer an alternative pest control strategy?. Outlooks Pest Manag. 2010;21(1):31–4.
- 67. V LEM and, Dicke M. Ecology of infochemical use by natural enemies in a tritrophic context. Annu Rev Entomol. 1992;37(1):141–72.
- 68. Ma L, Gu SH, Liu ZW, Wang SN, Guo YY, Zhou JJ, et al. Molecular characterization and expression profiles of olfactory receptor genes in the parasitic wasp, Microplitis mediator (Hymenoptera: Braconidae). J Insect Physiol. 2014;60:118–26. Epub 2013/12/03. S0022-1910(13)00242-4 [pii]. pmid:24291166.
- 69. Sanchez-Gracia A, Vieira FG, Rozas J. Molecular evolution of the major chemosensory gene families in insects. Heredity. 2009;103(3):208–16. Epub 2009/05/14. hdy200955 [pii] pmid:19436326.
- 70. Menuz K, Larter NK, Park J, Carlson JR. An RNA-seq screen of the Drosophila antenna identifies a transporter necessary for ammonia detection. PLoS Genet. 2014;10(11):e1004810. pmid:25412082; PubMed Central PMCID: PMC4238959.
- 71. Mitchell RF, Hughes DT, Luetje CW, Millar JG, Soriano-Agaton F, Hanks LM, et al. Sequencing and characterizing odorant receptors of the cerambycid beetle Megacyllene caryae. Insect Biochem Mol Biol. 2012;42(7):499–505. pmid:22504490; PubMed Central PMCID: PMC3361640.
- 72. Vogt RG. Molecular Basis of Pheromone Detection in Insects. In Comprehensive Insect Physiology, Biochemistry, Pharmacology and Molecular BiologyVolume 3 Endocrinology (LI Gilbert, K Iatro, SS Gill eds). 2005:pp. 753–804. Elsevier, London. ISBN: 044451516X.
- 73. Krieger J, Klink O, Mohl C, Raming K, Breer H. A candidate olfactory receptor subtype highly conserved across different insect orders. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2003;189(7):519–26. Epub 2003/06/27. pmid:12827420.
- 74. Xu P, Leal WS. Probing insect odorant receptors with their cognate ligands: Insights into structural features. Biochem Biophys Res Commun. 2013;435(3):477–82. Epub 2013/05/16. S0006-291X(13)00785-7 [pii]. pmid:23673297.
- 75. Nakagawa T, Sakurai T, Nishioka T, Touhara K. Insect sex-pheromone signals mediated by specific combinations of olfactory receptors. Science. 2005;307(5715):1638–42. Epub 2005/02/05. 1106267 [pii] pmid:15692016.
- 76. Jones WD, Nguyen TA, Kloss B, Lee KJ, Vosshall LB. Functional conservation of an insect odorant receptor gene across 250 million years of evolution. Curr Biol. 2005;15(4):R119–21. Epub 2005/02/23. S0960982205001466 [pii] pmid:15723778.
- 77. Jiang XJ, Guo H, Di C, Yu S, Zhu L, Huang LQ, et al. Sequence similarity and functional comparisons of pheromone receptor orthologs in two closely related Helicoverpa species. Insect Biochem Mol Biol. 2014;48:63–74. Epub 2014/03/19. S0965-1748(14)00036-8 [pii]. pmid:24632377.
- 78. Mitsuno H, Sakurai T, Murai M, Yasuda T, Kugimiya S, Ozawa R, et al. Identification of receptors of main sex-pheromone components of three Lepidopteran species. Eur J Neurosci. 2008;28(5):893–902. Epub 2008/08/12. EJN6429 [pii] pmid:18691330.
- 79. Zhang J, Yan S, Liu Y, Jacquin-Joly E, Dong S, Wang G. Identification and functional characterization of sex pheromone receptors in the common cutworm (Spodoptera litura). Chem Senses. 2015;40(1):7–16. Epub 2014/10/27. [pii]. pmid:25344681.
- 80. Zhang YN, Zhang J, Yan SW, Chang HT, Liu Y, Wang GR, et al. Functional characterization of sex pheromone receptors in the purple stem borer, Sesamia inferens (Walker). Insect Mol Biol. 2014;23(5):611–20. Epub 2014/07/22. pmid:25039606.
- 81. Ha TS, Smith DP. A pheromone receptor mediates 11-cis-vaccenyl acetate-induced responses in Drosophila. J Neurosci. 2006;26(34):8727–33. pmid:16928861.
- 82. Kurtovic A, Widmer A, Dickson BJ. A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature. 2007;446(7135):542–6. pmid:17392786.
- 83. Abuin L, Bargeton B, Ulbrich MH, Isacoff EY, Kellenberger S, Benton R. Functional architecture of olfactory ionotropic glutamate receptors. Neuron. 2011;69(1):44–60. Epub 2011/01/12. S0896-6273(10)00984-0 [pii] pmid:21220098; PubMed Central PMCID: PMC3050028.
- 84. Rytz R, Croset V, Benton R. Ionotropic receptors (IRs): chemosensory ionotropic glutamate receptors in Drosophila and beyond. Insect Biochem Mol Biol. 2013;43(9):888–97. Epub 2013/03/06. S0965-1748(13)00031-3 [pii]. pmid:23459169.
- 85. Koh TW, He Z, Gorur-Shandilya S, Menuz K, Larter NK, Stewart S, et al. The Drosophila IR20a clade of ionotropic receptors are candidate taste and pheromone receptors. Neuron. 2014;83(4):850–65. pmid:25123314
- 86. Xu W, Papanicolaou A, Liu NY, Dong SL, Anderson A. Chemosensory receptor genes in the Oriental tobacco budworm Helicoverpa assulta. Insect Mol Biol. 2015;24(2):253–63. pmid:25430896.
- 87. Sparks JT, Vinyard BT, Dickens JC. Gustatory receptor expression in the labella and tarsi of Aedes aegypti. Insect Biochem Mol Biol. 2013;43(12):1161–71. pmid:24157615.
- 88. Jones WD, Cayirlioglu P, Kadow IG, Vosshall LB. Two chemosensory receptors together mediate carbon dioxide detection in Drosophila. Nature. 2007;445(7123):86–90. pmid:17167414.
- 89. Kwon JY, Dahanukar A, Weiss LA, Carlson JR. The molecular basis of CO2 reception in Drosophila. Proc Natl Acad Sci U S A. 2007;104(9):3574–8. pmid:17360684; PubMed Central PMCID: PMC1805529.