Figures
Abstract
Olfaction plays vital roles in the survival and reproduction of insects. The completion of olfactory recognition requires the participation of various complex protein families. However, little is known about the olfactory-related proteins in Semiothisa cinerearia Bremer et Grey, an important pest of Chinese scholar tree. In this study, we sequenced the antennal transcriptome of S. cinerearia and identified 125 olfactory-related genes, including 25 odorant-binding proteins (OBPs), 15 chemosensory proteins (CSPs), two sensory neuron membrane proteins (SNMPs), 52 odorant receptors (ORs), eight gustatory receptors (GRs) and 23 ionotropic receptors (IRs). BLASTX best hit results and phylogenetic analyses indicated that these genes were most identical to their respective orthologs from Ectropis obliqua. Further quantitative real-time PCR (qRT-PCR) analysis revealed that three ScinOBPs and three ScinORs were highly expressed in male antennae, while seven ScinOBPs and twelve ScinORs were female-specifically expressed. Our study will be useful for the elucidation of olfactory mechanisms in S. cinerearia.
Citation: Liu P, Zhang X, Meng R, Liu C, Li M, Zhang T (2020) Identification of chemosensory genes from the antennal transcriptome of Semiothisa cinerearia. PLoS ONE 15(8): e0237134. https://doi.org/10.1371/journal.pone.0237134
Editor: J Joe Hull, USDA Agricultural Research Service, UNITED STATES
Received: April 30, 2020; Accepted: July 20, 2020; Published: August 7, 2020
Copyright: © 2020 Liu 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 sequences are available from the NCBI database (accession numbers from MT380331 to MT380456).
Funding: This study was supported by: TZ: HAAFS Agriculture Science and Technology Innovation Project (2019-1-2-2) and HAAFS Research and Development Program (2018120304). This study was also supported by Hebei Province Research and Development Program 20326518D.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Chemoreception, perceived through the olfactory system, plays a critical role in insect behavior (e.g., seeking hosts, selecting mates, locating oviposition sites, and avoiding adverse environments) [1–3]. The antennae, as the main olfactory organs of insects, comprise considerable numbers of olfactory sensilla, where chemical signals are recognized through olfactory receptor neurons (ORNs) [4]. When the external odorants enter the sensillum lymph, the binding proteins specifically bind to the odorants and transport them to the olfactory receptors on the neuron membrane, in which odorant stimulation is transformed into electric signals. Subsequently, these odorants are degraded by odorant degrading enzymes (ODEs) in the lymphatic cavity or in the sensor cells [5–7]. This complex process involves several important families, including odorant-binding proteins (OBPs), chemosensory proteins (CSPs), sensory neuron membrane proteins (SNMPs), olfactory receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs) and ODEs [8].
OBPs and CSPs, abundant in the antennae and mouthparts, are involved in binding and transporting various chemical signals [9]. Insect OBPs are soluble proteins, that are typically defined based on expression in the antennae [10, 11]. Recent studies, however, have revealed that OBPs also existed in non-olfactory organs (e.g. legs, wings and mouthparts) [9]. Generally, the classic OBP has a conserved pattern of six cysteines that form three disulfide bridges, while Minus-C OBPs and Plus-C OBPs have two and more than three disulfide bridges, respectively [8, 12]. Insect OBPs can specifically recognize and transmit a variety of signal and environmental molecules. Based on ligand binding assays, OBPs can function as pheromone-binding proteins (PBPs) and general odorant-binding proteins (GOBPs) that recognize various odorants [12, 13]. In Ectropis obliqua, EoblOBP6, abundant in antennae and legs, has been shown to bind plant volatiles (e.g. benzaldehyde, nerolidol, α-farnesene) as well as the aversive bitter alkaloid berberine [14]. Recent studies showed that silencing the OBP gene will lead to a decrease in the sensitivity of ORs to specific odors [15, 16]. CSPs, which are also small, soluble proteins, have four highly-conserved cysteines that form two disulfide bridges. CSPs are expressed in chemosensory organs [17, 18], as well as non-olfactory tissues [19, 20]. SNMPs, a homologue of the CD36 protein family, are signaling components crucial for odorant sensitivity [21]. The SNMP family is small, and usually have 2–3 subgroups in Diptera, Lepidoptera and other insect orders [22]. SNMP1 orthologs are mainly expressed in pheromone-sensitive ORNs, and participate in the perception of sex pheromones [21]. Bombyx mori SNMP1, a functional orthologue of Drosophila SNMP1, functions in pheromone detection by heteromirization with the BmOR1 pheromone receptor and the BmOroco co-receptor [23]. SNMP2s are expressed in supporting cells but their functions are still unclear [21]. SNMP3, a novel SNMP gene subfamily, has been identified in non-olfactory organs of moths and participates in the immunity response [23].
ORs and GRs are seven transmembrane domain membrane proteins located on the dendrite membrane of neurons. Their N-terminus and C-terminus are oriented to the cytoplasmic side and the extracellular sides of the plasma membrane, respectively [24]. Enormous OR diversity has been discovered in various insects. A special OR, called OR co-receptor (Orco), is necessary and highly conserved in many insects [25]. Each OR can bind with a species-specific Orco to form an Orco-ORx complex, that functions as a ligand-gated ion channel that determines the sensitivity and specificity of the ORN where it is expressed [26]. ORs and GRs are mainly responsible for detecting odorants and tastants, respectively. Recent studies also suggested that several GRs recognize carbon dioxide and pheromones [27]. For example, GR21a and GR63a of Drosophila are required for carbon dioxide detection, and GR39a is involved in sensing a female pheromone [28]. In addition, GRs are reported to be expressed not only in gustatory organs but also in antennae [27, 29]. Ionotropic receptors (IRs), a new kind of olfactory receptor, are related to ionotropic glutamate receptors (iGluRs) that respond to both environmental and cellular signals and function similar to chemosensory and gustatory receptors [30].
Semiothisa cinerearia Bremer et Grey (Lepidoptera: Geometridae) is one of the most important pests of the Chinese scholar tree (Sophora japonica L.), which is widely cultivated in the urban greenbelt in China for its significant medicinal and ornamental value. [31]. In recent years, the leaves of Chinese scholar tree have been severely damaged by S. cinerearia larvae in northern China. Currently, the control of S. cinerearia heavily relies on pesticides. Excessive usage of pesticides, however, can result in various negative effects on the environment [32]. Pheromone-based pest management strategies have been proven to be efficient and environmentally friendly methods of pest control [33, 34]. To date, sex pheromones have been identified from more than 570 Lepidopteran species. Sex pheromone components of moths can be classified into three types: Type-I (75%), Type-II (15%), and miscellaneous type (10%) [35, 36]. Type-I pheromones are comprised of saturated or unsaturated alcohols, aldehydes and esters. Type-II pheromones commonly are polyenic hydrocarbons and their corresponding epoxide derivatives [35, 36]. The main components of the sex pheromone of S. cinerearia are cis-3,4-epoxy-(Z.Z)-6,9-heptadecadiene, (3Z, 6Z, 9Z)-3,6,9-heptadecatriene and (3Z, 6Z, 9Z)-3,6,9-octadecatriene [37], which are typical Type-II sex pheromones. Although the sex pheromone has been identified, the mechanisms of pheromone and host plant volatile recognition have not yet been clarified.
In the present study, using S. cinerearia antennal transcriptomes, we identified candidate olfactory-related proteins and used quantitative real-time PCR (qRT-PCR) to examine the expression profile of a subset of the transcripts. Our results provide useful information for further research on pheromone and host plant volatile recognition in insects.
Materials and methods
Insect rearing and total RNA extraction
Pupae of S. cinerearia were collected in July 2018 from soil under a Chinese scholar tree in a lot owned by a private seedling company located in Baoding, China (38.96°N, 115.46°E). The company had sought our assistance in controlling S. cinerearia because their scholar trees were under attack by the pest. Therefore, no specific permissions were required for the locations/activities; and our studies did not involve endangered or protected species. All pupae were separated by sex and maintained in a dark thermotank (26 ± 2°C and 70% RH) until emergence. The antennae of 3-day-old adults (100 of each sex) were cut off, frozen in liquid nitrogen immediately, and then ground with a mortar and pestle. Total RNA was extracted using TRIzol reagent (TransGen, China) following the manufacturer’s instructions. RNA quality was evaluated as previously described [38]. For transcriptome sequencing, two groups of 100 moths for each sex were used to collect antennae RNA. For qRT-PCR analysis, three biological repeats were conducted, and each RNA sample was extracted from antennae of 30 moths.
Preparation for transcriptome sequencing
One microgram of high-quality RNA per sample was sent to Novogene (Beijing, China) for constructing cDNA libraries. The NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) was used to generate sequencing libraries following the manufacturer’s recommendations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The double-stranded cDNA was synthesized using mRNA as a template and purified with the AMPure XP System (Beckman Coulter, Beverly, USA). Then, cDNA fragments 250~300 bp in length were preferentially selected after end repair and adaptor ligation. Three microliters of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system.
Clustering and sequencing
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq platform, and 150bp paired-end reads were generated.
Transcriptome assembly and gene functional annotation
Transcriptome assembly performed using high quality trimmed data and Trinity (r20140413p1) [39]. Corset was used to compare the number of reads and expression patterns of the above transcripts and to cluster the transcripts [40]. Finally, the longest transcript was selected as the unigene. BUSCO v3 was used to evaluate the completeness of the assembled unigenes using the metazoa_odb9 dataset [41].
All unigenes obtained from S. cinerearia were annotated against the NCBI nonredundant (Nr) protein database using BLASTn and BLASTx with a significant cut-off E-value of < 10−5. Then, the blast results were further imported into the Blast2GO pipeline for Gene Ontology (GO) annotation [42]. The functional annotation followed the latest databases (Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO and GO).
Analysis of differential gene expression
The abundance of transcripts was calculated by the FPKM (fragments per kilobase per million mapped reads) method [43]. Differential expression analysis of two samples was performed using the DEGseq R package (1.12.0). The P value was adjusted using q value [44] which was defined as q value < 0.005 & |log2(foldchange)| > 1 and was set as the threshold for significantly differential expression.
Identification and bioinformatic of putative olfactory genes
The putative OBPs, CSPs, SNMPs, ORs, GRs and IRs genes were retrieved from the S. cinerearia contigs as functional annotation based on our reference antennal transcriptome assembly. All candidate genes were manually checked using the BLASTx program with an E-value of < 10−5. All screened olfactory-related genes were given a four-letter code (uppercase first letter of the genus name + lowercase first three letters of the species name) followed by the abbreviation of the gene name and serial numbers [38]. The complete coding region was determined using ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).
The signal peptides of the putative OBPs and CSPs were predicted using the SignalP 5.0 server (http://www.cbs.dtu.dk/services/SignalP/). The transmembrane domains (TMDs) of ORs, GRs, IRs and SNMPs were predicted using TMHMM Server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Sequence alignments of OBPs, CSPs, and SNMPs were performed using ClustalX 1.83, and the results were formatted using GeneDoc software (http://nrbsc.org/gfx/genedoc). Secondary structures of full-length candidates were predicted using the online platform psipred (http://bioinf.cs.ucl.ac.uk/psipred/). The protein structure homology-model was constructed using SWISS-MODEL (https://swissmodel.expasy.org/).
Phylogenetic analysis
A total of 115 OBPs, 153 CSPs, 18 SNMPs, 181 ORs, 154 GRs and 138 IRs sequences were used for phylogenetic tree reconstruction. All sequences are listed in the supplementary information. Evolutionary analyses were conducted in MEGA7 using the neighbor-joining method with a bootstrap test (1000 replicates) [45]. The evolutionary distances were computed using the Poisson correction method [46]. Finally, phylogenetic trees were viewed and edited using FigTree v. 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).
qRT-PCR analysis
First-strand cDNA was synthesized with 1 μg of total RNA by using All-in-One First-Strand cDNA Synthesis SuperMix (TransGen, China) following the manufacturer’s instructions. qRT-PCR was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, USA) with specific primers (S1 Table) designed by Premier 6.0. The amplification efficiency of the primers was 92–95% according to the pre-experiment. Then, the expression data analyses were performed using the 2-ΔΔCT method [47]. One-way ANOVA was used to analyze gene expression in SPSS 22.0 software. Finally, graphs were made with Origin8 software. In addition, 17 genes were randomly selected to estimate the consistency between RNA-seq and qRT-PCR data.
Results
Transcriptome analysis and assembly
The antennal cDNA library was constructed from female and male S. cinerearia using the Illumina HiSeq™ platform, and the sequences were assembled by the TRINITY de novo program. A total of 25,769,348 (97.67%) and 22,137,323 (97.52%) clean reads were obtained from the transcriptomes of female and male antennae, respectively. After hierarchical clustering, 65,476 unigenes were generated with an N50 length of 1,702 bp. Among those unigenes, 64.01% (41,913) were longer than 500 bp, and 37.02% (24,239) were longer than 1,000 bp (S1 Fig). Furthermore, the evaluation of unigene completeness showed 869 unigenes are complete (88.9%), indicating the high quality of our assembly (S2 Table).
Functional annotation of unigenes
To obtain comprehensive information on gene function, we annotated the genes using seven databases. A total of 30,805 unigenes (47.04%), 24,197 unigenes (36.95%) and 21,887 unigenes (33.42%) had significantly hits in the Nr, Pfam and Swiss-Prot databases, respectively (S3 Table). Furthermore, there were 6,077 unigenes with matches to all the Nr, Nt, Pfam, KOG, and GO databases (S2A Fig). Among the Nr-hit unigenes, 13,317 genes (43.2%) matched to B. mori, and 5,439 genes (17.7%) matched to Danaus plexippus (S2B Fig).
Gene ontology and KEGG pathway analysis
To better understand the function of the unigenes, Gene Ontology analysis was carried out on 24,346 unigenes, which were divided into 3 distinct subsets: biological process, cellular component and molecular function (S3 Fig). In the category of biological process, cellular process (13,848) was the largest of 26 groups, followed by metabolic process (11,785). In the cellular component category, the unigenes were mainly enriched in “cell”, “cell part” and “organelle”. In the molecular function category, the annotations were mostly enriched in “binding” (13,546) and “catalytic activity” (9,902) (S3 Fig).
Furthermore, KEGG pathway classification was carried out to enrich the function of unigenes. A total of 12,722 unigenes were divided into five branches covering 36 subgroups: cellular processes, environmental information processing, genetic information processing, metabolism and organismal systems. Among them, “signal transduction” is the most significant pathway, which involved 1606 genes (S4 Fig).
Candidate OBPs in antennae of S. cinerearia
A total of 25 transcripts encoding putative OBPs were identified in the antennae of S. cinerearia, including 20 OBPs, 3 PBPs and 2 GOBPs (Table 1). Of which, 15 OBPs had full-length complete open reading frames (ORFs). The BLASTx results indicated that 12 OBPs (ScinOBP3-4, ScinOBP6, ScinOBP8, ScinOBP10-14, and ScinOBP17-19) and two PBPs (ScinPBP1 and ScinPBP3) had highest amino acid identities (>50%) with those of E. obliqua (Table 1), a closely related species of S. cinerearia.
According to the number of cysteine residues, OBPs can be divided into three subclasses: Classic OBPs (six cysteine residues at conserved positions), Plus-C OBPs (4–6 additional cysteines) and Minus-C OBPs (loss of cysteine residues, generally C2 and C5) [8, 12]. A total of 115 OBPs from moths were divided into several different branches in the reconstructed phylogenetic trees: the PBP subfamily, GOBP subfamily, Plus-C OBP subfamily and Minus-C OBP subfamily (Fig 1). The neighbor-joining tree showed that ScinOBP10, which was clustered with EoblOBP10 with a high bootstrap value, belonged to the Plus-C OBP subfamily. Additionally, ScinOBP8, which clustered with EoblOBP8, was in the Minus-C OBP subfamily (Fig 1). Notably, ScinPBP1 was clustered together with EbolPBP1, the geometrid PBP for binding Type-II sex pheromones [48]. The results of amino acid sequence alignment showed that 10 OBPs (ScinOBP1, ScinOBP3, ScinOBP6, ScinOBP11, ScinOBP18, ScinPBP1-3, and ScinGOBP1-2) exhibited a Cys spacing profile of “C1-X25-30-C2-X3-C3-X37-42-C4-X8-14-C5-X8-C6”, which is typical of Classic OBPs. These OBPs were highly conserved with the EoblOBPs of E. obliqua, a closely related geometrid moth (Fig 2).
The sequences used in this analysis are listed in S4 Table.
Based on the FPKM values, ScinOBP6, ScinOBP12, ScinPBP1, ScinGOBP1 and ScinGOBP2 were highly expressed in the antennae of both female and male S. cinerearia. Further qRT-PCR tests using 17 OBPs with complete sequences showed that ScinOBP8, ScinOBP10, ScinOBP14, ScinOBP18, ScinOBP19, ScinPBP2 and ScinGOBP2 were significantly expressed in female antennae, whereas ScinOBP11, ScinOBP17 and ScinPBP1 were male antenna-biased (Fig 3A). Additionally, the correlation between RNA-seq and qRT-PCR data (R2 = 0.9207) confirmed the accuracy of the results (S5 Fig).
* indicates significant difference between female and male at p < 0.05, and ** indicates significant difference at p < 0.01.
Candidate CSPs and SNMPs in antennae of S. cinerearia
Fifteen transcripts were annotated as CSPs from the antennal transcriptome of S. cinerearia. All identified ScinCSPs had sequence lengths ranging from 71 to 303 aa and contained at least one signal peptide. There were 13 ScinCSPs with complete sequence, exceptions were ScinCSP5 and ScinCSP15. Based on BlastX, the majority of ScinCSPs were most similar to OBPs from E. obliqua with most exhibiting more than 60% identity (Table 2). In addition, ScinCSP2 was highly expressed and matched HarmCSP5 with an identity of 55.12%, which was reported to interact with 60 odorants (Table 2) [49].
The phylogenetic analysis of 153 sequences showed that ScinCSPs were distributed to different branches and had closest evolutionary relationships with corresponding proteins in E. obliqua (Fig 4). ScinCSP11 clustered 5-helix CSP subfamily. Secondary structure prediction result showed that ScinCSP11 lost the last helix (S6 Fig). All ScinCSPs had a relatively conserved structure, and ScinCSP1-14 conformed to a Cys spacing profile of “C1-X6-C2-X18-C3-X2-C4” (S7 Fig).
Sequences used were from Hymenoptera (Solenopsis invicta), Coleoptera (Tribolium castaneum), Diptera (Drosophila melanogaster), and Lepidoptera (Bombyx mori, Helicoverpa armigera, Heliothis virescens, Mamestra brassicae, Manduca sexta, Plutella xylostella, Ectropis obliqua, Spodoptera exigua, Ostrinia furnacalis). The sequences listed in S5 Table.
The FPKM analysis showed that ScinCSP5 displayed the highest expression levels among all CSPs, followed by ScinCSP7 (Table 2). Furthermore, the relative expression of 12 ScinCSPs (ScinCSP1-9 and ScinCSP13-15) in the qRT-PCR showed that ScinCSP3 and ScinCSP14 were significantly more expressed in the female antennae, whereas ScinCSP4 and ScinCSP7 had male antenna-biased expression (Fig 3B).
We also identified two full-length transcripts encoding putative SNMPs. ScinSNMP1 and ScinSNMP2 showed 73.86% and 80.39% identity with their orthologs in E. obliqua (Table 3). Sequence alignment results showed that SNMPs had five conserved cysteine residues and were highly conserved among insects (S8 Fig). The evolutionary tree showed that ScinSNMP1 and ScinSNMP2 were clustered with EbolSNMP1 and EbolSNMP2, respectively (Fig 5). The qRT-PCR results showed that both ScinSNMP1 and ScinSNMP2 were highly expressed in male antennae (Fig 3B).
The sequences used in this analysis are listed in S6 Table.
Candidate chemoreceptors in S. cinerearia
In this study, we identified 52 candidate ORs from the antennal transcriptome of S. cinerearia. Among them, 23 ORs were full-length ORs with 4–7 transmembrane domains. The incomplete ORs also contained 1–6 transmembrane domains (Table 3). ScinOrco had high identity (89.18%) with EoblOrco. The neighbor-joining tree reconstructed from 181 ORs from S. cinerearia, E. obliqua, B. mori, H. armigera and Chilo. suppressalis revealed that four ORs (ScinOR6, ScinOR10, ScinOR29 and ScinOR33) were clustered into the traditional pheromone receptor (PR) subfamily, that is thought to recognize Type-I pheromones. As expected, ScinOrco belonged to the Orco subfamily, with high homology to EoblOrco (Fig 6). The FPKM analysis revealed that ScinOrco had the highest FPKM value. qRT-PCR expression showed that 12 ORs (ScinOrco, ScinOR3-4, ScinOR7, ScinOR11-12, ScinOR15, ScinOR17, ScinOR20-21 and ScinOR 26–27) were highly expressed in female antennae. Only three ORs (ScinOR5, ScinOR6 and ScinOR8) were significantly more expressed in males (Fig 9A).
The sequences used in this analysis are listed in S7 Table.
Eight putative GRs were obtained by bioinformatic analysis. Although only one GR was intact (ScinGR1), all ScinGRs had 1–6 transmembrane domains (Table 4). The phylogenetic analysis showed that ScinGR1 and ScinGR6 were clustered with fructose receptors EoblGR2 and BmorGr10. ScinGR4 and ScinGR5 were members of the “sugar” receptor subfamily (Fig 7). qRT-PCR results showed that two GRs (ScinGR4 and ScinGR7) were highly expressed in female antennae, and three (ScinGR3, ScinGR6 and ScinGR8) exhibited high expression in males (Fig 9B).
The sequences used in this analysis are listed in S8 Table.
A total of 23 putative ScinIRs were also identified from transcriptomic analyses. Among them, seven candidate IRs (ScinIR2, ScinIR6, ScinIR8, ScinIR10, ScinIR11, ScinIR12 and ScinIR14) had full-length ORFs. The others were incomplete at the 5' or 3' end (Table 5). The evolutionary tree showed that five ScinIRs (ScinIR4, ScinIR6, ScinIR13, ScinIR15 and ScinIR16) were clustered with the IR75 subfamily and ScinIR17 was clustered with the IR93 subfamily. In addition, ScinIR10 was close to OfurIR41a, and ScinIR12 belonged to the IR76 subfamily (Fig 8). The qRT-PCR results revealed that four IRs (ScinIR2, ScinIR8, ScinIR9 and ScinIR12) were highly expressed in female antennae, and ScinIR13 had male-specific expression (Fig 9B).
The sequences used in this analysis are listed in S9 Table.
* indicates significant difference between female and male at p <0.05, and ** indicates significant difference at p < 0.01.
Discussion
During the past decade, RNA-Seq-based transcriptome analysis has been widely used for screening the olfactory-related genes of insects [38, 50, 51]. In the present study, a total of 125 candidate olfactory-related genes were identified from the transcriptome of S. cinerearia, including 25 OBPs, 15 CSPs, 2 SNMPs, 52 ORs, 8 GRs and 23 IRs. Compared to that in the antennal transcriptome in Lepidoptera from E. obliqua (24 OBPs, 21 CSPs, 2 SNMPs, 4 ORs and 3 GRs) and H. armigera (26 OBPs, 21 CSPs, 2 SNMPs, 60 ORs, 19 IRs and 9 GRs), the number of olfactory-related genes in S. cinerearia was comparable [52, 53]. The number of olfactory transcripts in S. cinerearia was less than those in Ectropis grisescens (40 OBPs, 30 CSPs, 59 ORs, and 24 IRs) and B. mori (44 OBPs, 24 CSPs, 66 ORs, 17 IRs and 65 GRs) [53–55]. The difference among species might result from complex environmental changes or the diversity of gene functions. In addition, antennal cDNA libraries are notorious for under representation of transcripts, especially those with low expression [13, 56, 57].
Odorant binding is considered the first critical step in olfactory signal transduction pathways [58, 59]. Due to the various expression patterns of most OBPs, PBPs and GOBPs, their functions are more complex than previously thought [60–62]. For example, in Drosophila melanogaster, OBP49a is mainly expressed in the lip of taste organs and interacts with bitter chemicals [63]. In E. obliqua, EoblOBP6, which is highly abundant in the legs, can integrate with the benzaldehyde emitted from tea plants. EoblOBP6 also shows high binding abilities to nerolidol and α-farnese, herbivore-induced volatiles [14]. In S. cinerearia, ScinOBP6, abundantly expressed in antennae, was homologous to EoblOBP6 with >50% sequence identity (Fig 2; Table 1). Therefore, ScinOBP6 is predicted to bind similar volatiles, but this prediction requires further experimental verification. ScinGOBP2 might also bind volatiles from S. japonica leaves, similar to its homolog EoblGOBP2 (Fig 1), which has strong binding abilities with seven tea volatiles [64]. In addition, many insect PBPs are reported to bind pheromones [65]. ScinPBP1 and ScinPBP2 had high expression in antennae. In the phylogenetic tree, ScinPBP1 clustered together with EbolPBP1, the geometrid PBP for detecting Type-II sex pheromones [48, 66]. Combining the qRT-PCR results that ScinPBP1 was significantly expressed in male antennae, we speculate that ScinPBP1 likewise interacts with Type-II sex pheromones.
In insects, most CSPs are composed of 6 α-helices containing four highly-conserved cysteine residues over the protein structure [18]. However, some CSPs have only five α-helix domains [67]. The 5-helical CSP5 is likely involved in an essential ubiquitous function rather than chemosensation [68]. In our study, the modeled ScinCSP11 has completely lost helix 6, similar to the conserved 5-helical CSP5 (S6 Fig), indicating its potential multifunction. Insect SNMPs probably have similar functions in the binding and membrane translocation of fatty acids [69]. In the present study, two ScinSNMPs had typical features of the SNMP family, with two transmembrane domains and highly conserved sequences with other Lepidopteran insects (Fig 5; S8 Fig). Previous studies have shown that SNMP1 and SNMP2 are mostly abundant in the antennae, especially in males [21, 70]. The expression of the two ScinSNMPs was consistent with the above regularity (Fig 6), and the ScinSNMPs may have functions similar to those in other insects.
Olfactory receptors play crucial roles in odorant detection and recognition [71]. A total of 52 ORs, 8 GRs and 23 IRs were identified of S. cinerearia. For most insects, a coreceptor (Orco) is required for membrane targeting of canonical ORs on ORN membranes [71, 72]. ScinOrco showed the highest expression of all ScinORs, suggesting that it might have the potential to receive chemical signals [73]. In the present study, ScinOrco showed higher expression in female antennae (Fig 9A), which is opposite to the results of most species in Lepidoptera [74]. This difference may be attributable to sample size (i.e. three biological duplication) which potentially resulted more mistakes and consequently contributed to the significant differences. However, Orco showed higher expression in other moths, e.g. Manduca sexta and E. grisescens [55, 75], indicating the possibility that Orco is highly expressed in females. PRs, which function to detect sex pheromones, are reported to be male-specifically expressed in moths [76, 77]. In S. cinerearia, four ORs (ScinOR6, ScinOR10, ScinOR29 and ScinOR33) phylogenetically sorted with PR subfamily for Type-I sex pheromones components (Fig 6). In particular, ScinOR6 was highly expressed in male antennae (Figs 6 and 9A), suggesting that it might contribute to detecting sex pheromone. In Geometridae moths, including E. grisescens and S. cinerearia, Type-II pheromones dominate the chemical communication. The homology comparison with EgriORs related Type-II pheromone from E. grisescens revealed that ScinOR5 had high homology with EgriOR31 and EgriOR24, which function as Type-II pheromone receptors (unpublished data). Moreover, ScinOR5 was highly expressed in male antennae, further indicating that it may function as a PRs for Type-II sex pheromones [55]. The coexpression of PRs and PBPs could greatly enhance the sensitivity to pheromones [15]. Consequently, linking research on the function of ScinPBPs and ScinOR6 will be more interesting and meaningful.
GRs, mainly expressed in the gustatory organs, detect different sugars, bitter compounds, and contact pheromones [78]. Eight GRs were annotated from S. cinerearia, including two putative sugar receptors (ScinGR4 and ScinGR5). The expression of ScinGR4 and ScinGR5 was female-specific. Sugars and sugar alcohols are reported to affect host plant selection and egg-laying behavior of codling moth females [79]. However, most of the identified GRs were incomplete, and no CO2 receptor was found in S. cinerearia, which might be due to the low expression of GRs in antennae [24, 80]. Furthermore, we found that two coreceptors from IRs, ScinIR2 and ScinIR7, belong to the IR8a/IR25a subfamily, which are thought to act as coreceptors such as Orco [81, 82].
Transcriptome sequencing is ideal for obtaining target genes of interest [38]. GO analysis showed that the majority of ScinORs were mainly clustered in “signaling” and “molecular transducer activity”. Twenty ScinIRs (except ScinIR1, ScinIR8 and ScinIR20) matched with “molecular transducer activity”, “signaling” and “transporter activity”. Four ScinGRs (ScinGR1, ScinGR 2, ScinGR 4, and ScinGR 7) were enriched in “molecular transducer activity” (S3 Fig). This information confirmed the accuracy of gene identification from transcriptome data and increased the possibility of detecting odorant receptor function.
Conclusion
In summary, our study provided the first report on antennal transcriptome analysis in S. cinerearia. A total of 65,476 unigenes were generated, and 30,805 unigenes were successfully annotated by the Nr database. A total of 125 unigenes were identified as olfactory-related genes, including 25 OBPs, 15 CSPs, two SNMPs, 52 ORs, 8 GRs and 23 IRs. Most olfactory-related genes showed female- or male-biased expression in the antennae. This will facilitate functional research on olfactory mechanisms in S. cinerearia.
Supporting information
S1 Fig. Distribution of length of Semiothisa cinerearia.
https://doi.org/10.1371/journal.pone.0237134.s001
(TIF)
S2 Fig. Summary for the annotation of Semiothisa cinerearia antennal unigenes.
(A) The number of unigenes matching in five databases. (B) The species distribution of the best Blastx hits in Nr database.
https://doi.org/10.1371/journal.pone.0237134.s002
(TIF)
S3 Fig. Gene Ontology (GO) functional classification of unigenes.
The purple boxes indicate the process of accumulation of olfactory receptors.
https://doi.org/10.1371/journal.pone.0237134.s003
(TIF)
S4 Fig. KEGG pathway functional classification of unigenes.
A: Cellular Processes. B: Environmental Information Processing. C: Genetic Information Processing. D: Metabolism. E: Organismal Systems.
https://doi.org/10.1371/journal.pone.0237134.s004
(TIF)
S5 Fig. The correlation analysis between the results of qRT-PCR and RNA-seq.
The data was based on the average value of more than three repetitions, and students’ t-test was used to determine the statistical significance.
https://doi.org/10.1371/journal.pone.0237134.s005
(TIF)
S6 Fig. Secondary and 3D structures of ScinCSP11.
(A) Sequence alignment of ScinCSP11 with CSP1 from Bombyx mori (template library identity: 2jnt.1A). α-helices are displayed as squiggles. The signal peptides are removed. (B) The predicted 3D structure of ScinCSP11.
https://doi.org/10.1371/journal.pone.0237134.s006
(TIF)
S7 Fig. Amino acid sequence alignment of CSPs in Semiothisa cinerearia and Ectropis obliqua.
https://doi.org/10.1371/journal.pone.0237134.s007
(TIF)
S8 Fig. Amino acid sequence alignment of SNMPs in Semiothisa cinerearia and Ectropis obliqua.
The red letters indicate conservative cysteine residues. The red box represents the transmembrane domains.
https://doi.org/10.1371/journal.pone.0237134.s008
(TIF)
S2 Table. BUSCO assessment of Semiothisa cinerearia.
https://doi.org/10.1371/journal.pone.0237134.s010
(DOCX)
S3 Table. Summary for the annotation of Semiothisa cinerearia unigenes.
https://doi.org/10.1371/journal.pone.0237134.s011
(DOCX)
S4 Table. Amino acid sequences of OBPs used in phylogenetic analyses.
https://doi.org/10.1371/journal.pone.0237134.s012
(DOCX)
S5 Table. Amino acid sequences of CSPs used in phylogenetic analyses.
https://doi.org/10.1371/journal.pone.0237134.s013
(DOCX)
S6 Table. Amino acid sequences of SNMPs used in phylogenetic analyses.
https://doi.org/10.1371/journal.pone.0237134.s014
(DOCX)
S7 Table. Amino acid sequences of ORs used in phylogenetic analyses.
https://doi.org/10.1371/journal.pone.0237134.s015
(DOCX)
S8 Table. Amino acid sequences of GRs used in phylogenetic analyses.
https://doi.org/10.1371/journal.pone.0237134.s016
(DOCX)
S9 Table. Amino acid sequences of IRs used in phylogenetic analyses.
https://doi.org/10.1371/journal.pone.0237134.s017
(DOCX)
References
- 1. Carey AF, Wang G, Su CY, Zwiebel LJ, Carlson JR. Odorant reception in the malaria mosquito Anopheles gambiae. Nature. 2010; 464(7285): 66–71. pmid:20130575
- 2. Sun L, Mao TF, Zhang YX, Wu JJ, Bai JH, Zhang YN et al. Characterization of candidate odorant-binding proteins and chemosensory proteins in the tea geometrid Ectropis obliqua Prout (Lepidoptera: Geometridae). Archives of Insect Biochemistry and Physiology. 2017 Apr; 94(4): e21383. pmid:28321909
- 3. Brito NF, Moreira MF, Melo AC. A look inside odorant-binding proteins in insect chemoreception. Journal of Insect Physiology. 2016 Dec; 95: 51–65. pmid:27639942
- 4. 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 May; 10(5): e0125159. pmid:25938508
- 5. Vogt RG, Riddiford LM. Pheromone binding and inactivation by moth antennae. Nature. 1981; 293: 161–163. pmid:18074618
- 6. Leal WS. Pheromone reception. Topics in Current Chemistry. 2005; 240:1–36.
- 7. He P, Zhang YN, Li ZQ, Yang K, Zhu JY, Liu SJ et al. An antennae-enriched carboxylesterase from Spodoptera exigua displays degradation activity in both plant volatiles and female sex pheromones. Insect Molecular Biology. 2017; 23(4): 475–486. pmid:24628907
- 8. Leal WS. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annual of Review of Entomology. 2013; 58: 373–391. pmid:23020622
- 9. Xu P, Wang Y, Akami M, Niu CY. Identification of olfactory genes and functional analysis of BminCSP and BminOBP21 in Bactrocera minax. PLoS one. 2019 Sep; 14(9): e0222193. pmid:31509572
- 10. Pelosi P, Zhu J, Knoll W. Odorant-binding proteins as sensing elements for odour monitoring. Sensors. 2018 Sep; 18(10): 3248. pmid:30262737
- 11. Xiao S, Sun JS, Carlson JR. Robust olfactory responses in the absence of odorant binding proteins. Elife. 2019 Oct; 8. pii: e51040. pmid:31651397
- 12.
Ozaki M. Odorant-Binding Proteins in Taste System: Putative Roles in Taste Sensation and Behavior. In: Picimbon JF, editors. Olfactory Concepts of Insect Control-Alternative to Insecticides. 2019. pp. 187–204. https://doi.org/10.1007/978-3-030-05165-5_8
- 13. Zhou JJ, Vieira FG, He XL, Smadja C, Liu R, Rozas J, et al. Genome annotation and comparative analyses of the odorant-binding proteins and chemosensory proteins in the pea aphid Acyrthosiphon pisum. Insect Molecular Biology. 2010 Mar; 19 (Suppl. 2): 113–122. pmid:20482644
- 14. Ma L, Li Z, Zhang W, Cai X, Luo Z, Zhang Y, et al. The odorant binding protein 6 expressed in sensilla chaetica displays preferential binding affinity to host plants volatiles in Ectropis obliqua. Frontiers in Physiology. 2018 May; 9: 534. pmid:29867573
- 15. Chang H, Liu Y, Yang T, Pelosi P, Dong S, Wang G. Pheromone binding proteins enhance the sensitivity of olfactory receptors to sex pheromones in Chilo suppressalis. Scientific Reports. 2015; 5:13093. pmid:26310773
- 16. Sun M, Liu Y, Walker WB, Liu C, Lin K, Gu S, et al. Identification and characterization of pheromone receptors and interplay between receptors and pheromone binding proteins in the diamondback moth, Plutella xyllostella. PloS one. 2013; 8(4): e62098. pmid:23626773
- 17. Briand L, Swasdipan N, Nespoulous C, Bézirard V, Blon F, Huet JC, et al. Characterization of a chemosensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier. European Journal of Biochemistry. 2002 Sep; 269(18):4586–96. pmid:12230571
- 18.
Picimbon JF. Evolution of protein physical structures in insect chemosensory systems. In: Picimbon JF, editors. Olfactory concepts of insect control. Nature Springer Switzerland AG. 2019. pp. 234–238. https://doi.org/10.1007/978-3-030-05165-5_10
- 19. He P, Li ZQ, Zhang YF, Chen L, Wang J, Xu L, et al. Identification of odorant-binding and chemosensory protein genes and the ligand affinity of two of the encoded proteins suggest a complex olfactory perception system in Periplaneta americana. Insect Molecular Biology. 2017; 26(6), 687–701. pmid:28719023
- 20. Maleszka J, Forêt S, Saint R, Maleszka R. RNAi-induced phenotypes suggest anovel role for a chemosensory protein CSP5 in the development ofembryonic integument in the honeybee (Apis mellifera). Development Genes and Evolution. 2007 Mar; 217(3): 189–96. pmid:17216269
- 21. Gu SH, Yang RN, Guo MB, Wang GR, Zhang YJ. Molecular identification and differential expression of sensory neuron membrane proteins in the antennae of the black cutworm moth Agrotis ipsilon. Journal of Insect Physiology. 2013 Apr; 59(4): 430–443. pmid:23454276
- 22. Zhao YJ, Li GC, Zhu JY, Liu NY. Genome-based analysis reveals a novel SNMP group of the Coleoptera and chemosensory receptors in Rhaphuma horsfieldi. Genomics. 2020; 112(4): 2713–2728. pmid:32145380
- 23. Zhang HJ, Xu W, Chen QM, Sun LN, Anderson A, Xia QY, et al. A phylogenomics approach to characterizing sensory neuron membrane proteins (SNMPs) in Lepidoptera. Insect Biochemistry and Molecular Biology. 2020; 118: 103313. pmid:31911087
- 24. He P, Engsontia P, Chen GL, Yin Q, Wang J, Lu X, et al. Molecular characterization and evolution of a chemosensory receptor gene family in three notorious rice planthoppers, Nilaparvata lugens, Sogatella furcifera and Laodelphax striatellus, based on genome and transcriptome analyses. Pest Management Science. 2018; 74(9): 2156–2167. pmid:29542232
- 25.
Breer H, Fleischer J, Pregitzer P, Krieger J. Molecular mechanism of insect olfaction: olfactory receptors. In: Picimbon JF, editors. Olfactory Concepts of Insect Control-Alternative to insecticides. 2019. pp. 93–114.
- 26. Sato K, Pellegrino M, Nakagawa T, Nakagawa T, Vosshall LB, Touhara K. Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature. 2008; 452:1002–1006. pmid:18408712
- 27. Nei M, Niimura Y, Nozawa M. The evolution of animal chemosensory receptor gene repertoires: roles of chance and necessity. Nature Reviews Genetics. 2008 Dec; 9(12): 951–963. pmid:19002141
- 28. Watanabe K, Toba G, Koganezawa M, Yamamoto D. Gr39a, a highly diversified gustatory receptor in Drosophila, has a role in sexual behavior. Behavior Genetics. 2011; 41:746–753. pmid:21416142
- 29. Liu H, Zhang X, Liu C, Liu Y, Mei X, Zhang T. Identification and expression of candidate chemosensory receptors in the white-spotted flower chafer, Protaetia brevitarsis. Scientific Reports. 2019 Mar; 9(1): 3339. pmid:30833589
- 30. 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: e1001064. pmid:20808886
- 31. Fan XL, Liang YM, Ma R, Tian CM. Morphological and phylogenetic studies of Cytospora (Valsaceae, Diaporthales) isolates from Chinese scholar tree, with description of a new species. Mycoscience. 2014; 55(4): 252–259.
- 32. Zhuo Z, Li PY, Chan Z, Guang CL. Potential Distribution Prediction of Semiothisa cinerearia in China Based on GARP Ecological Niche Model. Advanced Materials Research. 2013; 726–731:4678–4681.
- 33. Caparros MR, Haubruge E, Verheggen F. Pheromone-based management strategies to control the tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). Biotechnologie Agronomie Société Et Environnement. 2013; 17(3): 475–482.
- 34. Cui GZ, Zhu JJ. Pheromone-based pest management in China: past, present, and future prospects. Journal of Chemical Ecology. 2016 Jul; 42(7): 557–70. pmid:27481347
- 35. Watanabe H, Tabunoki H, Miura N, Sato R, Ando T. Analysis of odorant-binding proteins in antennae of a geometrid species, Ascotis selenaria cretacea, which produces lepidopteran Type II sex pheromone components. Invertebrate Neuroence. 2007; 7(2):109–118. pmid:17516105
- 36.
Ando T, Inomata S, Yamamoto M. Lepidopteran sex pheromones. In: Schulz S, editors. The chemistry of pheromones and other Semiochemicals I. Springer. 2004. pp. 51–96. https://doi.org/10.1007/b95449 pmid:22160231
- 37. Li ZM, Yao EY, Liu TL, Liu ZP, Wang SH, Zhu HQ, et al. Structural elucidation of sex pheromone components of the Geometridae Semiothisa cinerearia (Bremer et Grey) in China. Chinese Journal of Chemistry, 1993; 11(3): 251–256.
- 38. Jia X, Zhang X, Liu H, Wang R, Zhang T. Identification of chemosensory genes from the antennal transcriptome of Indian meal moth Plodia interpunctella. PLoS one. 2018 Jan; 13(1): e0189889. pmid:29304134
- 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. Nature Biotechnology. 2011 May; 29(7): 644–652. pmid:21572440
- 40. Davidson NM, Oshlack A. Corset: enabling differential gene expression analysis for de novo assembled transcriptomes. Genome Biology. 2014 Jul; 15(7): 410. pmid:25063469
- 41.
Seppey M, Manni M, Zdobnov EM. BUSCO: Assessing Genome Assembly and Annotation Completeness. In: Kollmar M, editors. Gene Prediction. Methods in Molecular Biology. 2019. pp. 227–245. https://doi.org/10.1007/978-1-4939-9173-0_14 pmid:31020564
- 42. Götz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, et al. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Research. 2008 Jun; 36(10): 3420–35. pmid:18445632
- 43. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, Van B, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology. 2010; 28(5): 511–515. pmid:20436464
- 44. Storey J D. The positive false discovery rate: a Bayesian interpretation and the q-value. The Annals of Statistics. 2003; 31(6): 2013–2035.
- 45. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular biology and evolution. 2016 Mar; 33(7), 1870–1874. pmid:27004904
- 46. Zuckerkandl E, Pauling L. Evolutionary divergence and convergence in proteins. Evolving Genes and Proteins. 1965. pp. 97–166.
- 47. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001; 25(4): 402–408.
- 48. Sun L, Wang Q, Zhang Y, Tu XH, Yan YT, Wang Q, et al. The sensilla trichodea-biased EoblPBP1 binds sex pheromones and green leaf volatiles in Ectropis obliqua Prout, a geometrid moth pest that uses Type-II sex pheromones. Journal of Insect Physiology. 2019; 116: 17–24. pmid:31009623
- 49. Zhang TT, Wang WX, Zhang ZD, Zhang YJ, Guo YY. Functional characteristics of a novel chemosensory protein in the cotton bollworm Helicoverpa armigera (Hübner). Journal of Integrative Agriculture. 2013; 12(5): 853–861.
- 50. Pitts RJ, Rinker DC, Jones PL, Rokas A, Zwiebel LJ. Transcriptome profiling of chemosensory appendages in the malaria vector Anopheles gambiae reveals tissue-and sex-specific signatures of odor coding. BMC Genomics. 2011 May; 12: 271. pmid:21619637
- 51. Yuan H, Chang H, Zhao L, Yang C, Huang Y. Sex-and tissue-specific transcriptome analyses and expression profiling of olfactory-related genes in Ceracris nigricornis Walker (Orthoptera: Acrididae). BMC Genomics. 2019 Nov; 20(1): 808. pmid:31694535
- 52. Ma L, Li ZQ, Bian L, Cai XM, Luo ZX, Zhang YJ, et al. Identification and comparative study of chemosensory genes related to host selection by legs transcriptome analysis in the tea geometrid Ectropis obliqua. PLoS one. 2016 Mar; 11(3): e0149591. pmid:26930056
- 53. Chang H, Ai D, Zhang J, Dong S, Wang G. Candidate odorant binding proteins and chemosensory proteins in the larval chemosensory tissues of two closely related noctuidae moths, Helicoverpa armigera and H. assulta. PLoS one. 2017 Jun; 12(6). pmid:28594956
- 54. 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. Current Biology. 2009; 19: 881–890. pmid:19427209
- 55. Li ZQ, Luo ZX, Cai XM, Bian L, Xin ZJ, Liu Y, et al. Chemosensory gene families in Ectropis grisescens and candidates for detection of type-II sex pheromones. Frontiers in Physiology. 2017 Nov; 8: 953. pmid:29209233
- 56. Vogt RG, Callahan FE, Rogers ME, Dickens JC. Odorant binding protein diversity and distribution among the insect orders, as indicated by LAP, an OBP-related protein of the true bug Lygus lineolaris (Hemiptera, Heteroptera). Chemical Senses. 1999 Oct; 24: 481–495. pmid:10576256
- 57. Wanner KW, Isman MB, Feng Q, Plettner E, Theilmann DA. Developmental expression patterns of four chemosensory protein genes from the Eastern spruce budworm, Chroistoneura fumiferana. Insect Molecular Biology. 2005 Jun; 14(3): 289–300. pmid:15926898
- 58. Pelosi P, Zhou JJ, Ban LP, Calvello M. Soluble proteins in insect chemical communication. Celluar and Molecular Life Sciences. 2006 Jul; 63(14): 1658–1676. pmid:16786224
- 59. Wu JD, Shen ZC, Hua HQ, Zhang F, Li YX. Identification and sex expression profiling of odorant-binding protein genes in Trichogramma japonicum (Hymenoptera: Trichogrammatidae) using RNA-Seq. Applied Entomology and Zoology. 2017; 52(4): 623–633.
- 60. Zeng FF, Sun X, Dong HB, Wang MQ. Analysis of a cDNA library from the antenna of Cnaphalocrocis medinalis and the expression pattern of olfactory genes. Biochemical and Biophysical Research Communications. 2013 Apr; 433(4): 463–469. pmid:23523786
- 61. 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. 2013b Jul; 8(7): e69715. pmid:23894529
- 62. Zhang S, Zhang Z, Wang H, Kong X. Molecular characterization, expression pattern, and ligand-binding property of three odorant binding protein genes from Dendrolimus tabulaeformis. Journal of Chemical Ecology. 2014 Apr; 40(4): 396–406. pmid:24728949
- 63. Jeong YT, Shim J, Oh SR, Yoon HI, Kim CH, Moon SJ, et al. An odorant-binding protein required for suppression of sweet taste by bitter chemicals. Neuron. 2013 Aug; 79(4): 725–37. pmid:23972598
- 64. Zhang YL, Fu XB, Cui HC, Zhao L, Yu JZ, Li HL. Functional characteristics, electrophysiological and antennal immunolocalization of general odorant-binding protein 2 in tea geometrid, Ectropis obliqua. International journal of molecular sciences. 2018; 19(3): 875. pmid:29543772
- 65. Honson NS, Gong Y, Plettner E. Structure and function of insect odorant and pheromone-binding proteins (OBPs and PBPs) and chemosensory-specific proteins (CSPs). Chemical Ecoiogy and Phytochemistry Forest Ecosystems. 2005; pp. 227–268.
- 66. Guo F, Yu J, Yang YQ, Wan XC. Response to enantiomers of (Z3Z9)-6, 7-epoxy-octadecadiene, sex pheromone component of Ectropis obliqua Prout (Lepidoptera: Geometridae): electroantennagram test, field trapping, and in silico study. Florida Entomologist. 2019; 102(3): 549–554.
- 67. Kulmuni , Havukainen H. Insights into the evolution of the CSP gene family through the integration of evolutionary analysis and comparative protein modeling. PLoS ONE. 2013; 8: e63688. pmid:23723994
- 68. Missbach C, Vogel H, Hansson BS, Groβe-Wilde Ewald. Identification of odorant binding proteins and chemosensory proteins in antennal transcriptomes of the jumping bristletail Lepismachilis y-signata and the firebrat Thermobia domestica: evidence for an independent OBP–OR origin. Chemical senses. 2015; 40(9): 615–626.
- 69. Liu C, Zhang J, Liu Y, Wang G, Dong S. Expression of SNMP1 and SNMP2 genes in antennal sensilla of Spodoptera exigua (Hübner). Archives of Insect Biochemistry and Physiology. 2014 Feb; 85(2): 114–26.
- 70. Liu S, Qiao F, Liang QM, Huang YJ, Zhou WW, Gong ZJ, et al. Molecular characterization of two sensory neuron membrane proteins from Chilo suppressalis (Lepidoptera: Pyralidae). Annals of Entomological Society of America. 2013; 106(3): 378–384.
- 71. Fleischer J, Pregitzer P, Breer H, Krieger J. Access to the odor world: olfactory receptors and their role for signal transduction in insects. Cellular and Molecular Life Sciences. 2018 Feb; 75(3): 485–508. pmid:28828501
- 72. DeGennaro M, McBride CS, Seeholzer L, Nakagawa T, Dennis EJ, Goldman C, et al. Orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET. Nature. 2013 Jun; 498(7455): 487–91. pmid:23719379
- 73. Dong X, Zhong G, Hu M, Yi X, Zhao H, Wang H. Molecular cloning and functional identification of an insect odorant receptor gene in Spodoptera litura (F.) for the botanical insecticide rhodojaponin III. Journal of Insect Physiology. 2013 Jan; 59(1): 26–32. pmid:23195879
- 74. Liu S, Huang Y, Qiao F, Zhou WW, Gong ZJ, Cheng JA, et al. Cloning, tissue distribution, and transmembrane orientation of the olfactory co-receptor Orco from two important Lepidopteran rice pests, the leaffolder (Cnaphalocrocis medinalis) and the striped stem borer (Chilo suppressalis). Journal of Integrative Agriculture. 2013; 12(10): 1816–1825.
- 75. Howlett N, Dauber KL, Shukla A, Morton B, Glendinning JI, Brent E, et al. Identification of chemosensory receptor genes in Manduca sexta and knockdown by RNA interference. BMC Genomics. 2012; 13: 211. pmid:22646846
- 76. Sakurai T, Nakagawa T, Mitsuno H, Mori H, Endo Y, Tanoue S, et al. Identification and functional characterization of a sex pheromone receptor in the silkmoth Bombyx mori. Proceedings of the National Academy of Sciences. 2004 Nov; 101(47): 16653–8. pmid:15545611
- 77. 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. European Journal of Neuroscience. 2008 Sep; 28(5): 893–902. pmid:18691330
- 78. Vosshall LB, Stocker RF. Molecular architecture of smell and taste in Drosophila. Annual Review of Neuroscience. 2007; 30(1): 505–533. pmid:17506643
- 79. Lombarkia N, Derridj S. Resistance of apple trees to Cydia pomonella egg-laying due to leaf surface metabolites. Entomologia Experimentalis et Applicata. 2008; 128(1): 57–65.
- 80. Yang B, Ozaki K, Ishikawa Y, Matsuo T. Identification of candidate odorant receptors in asian corn borer Ostrinia furnacalis. PLoS One. 2015; 10(3): e0121261. pmid:25803580
- 81. Abuin L, Bargeton B, Ulbrich MH, Isacoff EY, Kellenberger S, Benton R. Functional architecture of olfactory ionotropic glutamate receptors. Neuron. 2011 Jan; 69(1): 44–60. pmid:21220098
- 82. Li XM, Zhu XY, Wang ZQ, Wang Y, He P, Chen G, et al. Candidate chemosensory genes identified in Colaphellus bowringi by antennal transcriptome analysis. BMC Genomics. 2015 Dec, 16(1): 1028. pmid:26626891