Identification of chemosensory genes from the antennal transcriptome of Indian meal moth Plodia interpunctella

PLOS ONE, Jan 2018

Olfaction plays an indispensable role in mediating insect behavior, such as locating host plants, mating partners, and avoidance of toxins and predators. Olfactory-related proteins are required for olfactory perception of insects. However, very few olfactory-related genes have been reported in Plodia interpunctella up to now. In the present study, we sequenced the antennae transcriptome of P. interpunctella using the next-generation sequencing technology, and identified 117 candidate olfactory-related genes, including 29 odorant-binding proteins (OBPs), 15 chemosensory proteins (CSPs), three sensory neuron membrane proteins (SNMPs), 47 odorant receptors (ORs), 14 ionotropic receptors (IRs) and nine gustatory receptors (GRs). Further analysis of qRT-PCR revealed that nine OBPs, three CSPs, two SNMPs, nine ORs and two GRs were specifically expressed in the male antennae, whereas eight OBPs, six CSPs, one SNMP, 16 ORs, two GRs and seven IRs significantly expressed in the female antennae. Taken together, our results provided useful information for further functional studies on insect genes related to recognition of pheromone and odorant, which might be meaningful targets for pest management.

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Identification of chemosensory genes from the antennal transcriptome of Indian meal moth Plodia interpunctella

January Identification of chemosensory genes from the antennal transcriptome of Indian meal moth Plodia interpunctella Xiaojian Jia 0 1 Xiaofang Zhang 0 1 Hongmin Liu 1 Rongyan Wang 0 1 Tao Zhang 0 1 ☯ These authors contributed equally to this work. 1 cauzht@ 1 0 Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences/Integrated Pest Management Center of Hebei Province/Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture , Baoding , P. R. China , 2 College of Agronomy, Xinyang Agriculture and Forestry University , Xinyang , P. R. China 1 Editor: Yulin Gao, Chinese Academy of Agricultural Sciences Institute of Plant Protection , CHINA Olfaction plays an indispensable role in mediating insect behavior, such as locating host plants, mating partners, and avoidance of toxins and predators. Olfactory-related proteins are required for olfactory perception of insects. However, very few olfactory-related genes have been reported in Plodia interpunctella up to now. In the present study, we sequenced the antennae transcriptome of P. interpunctella using the next-generation sequencing technology, and identified 117 candidate olfactory-related genes, including 29 odorant-binding proteins (OBPs), 15 chemosensory proteins (CSPs), three sensory neuron membrane proteins (SNMPs), 47 odorant receptors (ORs), 14 ionotropic receptors (IRs) and nine gustatory receptors (GRs). Further analysis of qRT-PCR revealed that nine OBPs, three CSPs, two SNMPs, nine ORs and two GRs were specifically expressed in the male antennae, whereas eight OBPs, six CSPs, one SNMP, 16 ORs, two GRs and seven IRs significantly expressed in the female antennae. Taken together, our results provided useful information for further functional studies on insect genes related to recognition of pheromone and odorant, which might be meaningful targets for pest management. - Data Availability Statement: All relevant data are in the paper, its supporting information files, and at the following databases: The raw reads of P. interpunctella transcriptome have been deposited into the NCBI SRA database (accession number: SRR6002827 and SRR6002828), and the Transcriptome Shotgun Assembly (TSA) project has been deposited at DDBJ/ENA/GenBank under the accession GFWQ00000000. The version described in this paper is the first version, GFWQ01000000. The detailed TSA sequences could be obtained from Genbank (https://www. Introduction Indian meal moth, Plodia interpunctella (HuÈbener) (Lepidoptera: Pyraloidea, Pyralidae), is a notorious stored-product pest worldwide [ 1 ]. The larvae infest a variety of processed foods, including fruits, nuts, cereals, powdered milk, chocolate, birdseed, and pet food [ 2 ], causing extensive damage by impairing dry weight, germination, nutritional value, and quality grade. It is difficult to control P. interpunctella by conventional insecticides, because it often inhabits our kitchen, closet and warehouse, and its larvae are mixed with our processed foods. Accordingly, several novel strategies have been developed to monitor and control P. interpunctella. Among these novel methods, sex pheromone is widely acceptable due to its safety and efficiency. Meanwhile, host volatiles have been thought to affect the oviposition behavior of P. ncbi.nlm.nih.gov/Traces/wgs/?val= GFWQ01&display=contigs&page=1). Funding: This work was supported by the National Natural Science Foundation of China (3130914) to Tao Zhang; Special Finance of Hebei Province (F17C10007 and F17C10008) to Tao Zhang. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. interpunctella [ 3 ]. However, the underlying molecular mechanisms of olfactory recognition of P. interpunctella remain largely unexplored. An accurate olfactory system plays crucial roles in survival, reproduction, and chemical communication for most insects [ 4 ]. Using the olfactory system in antennae, when peripheral odorants are detected, insects will activate olfactory sensory neurons (ORNs) and translate the signals into nerve impulses to the brain [ 5 ]. At least six gene families are involved in the olfactory sensory procedure, including three sensory protein families: odorant-binding proteins (OBPs), chemosensory proteins (CSPs), and sensory neuron membrane proteins (SNMPs); and three major chemosensory receptor families: odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs). Additionally, odorant degrading enzymes (ODEs) are also classified in olfactory system, due to their integral roles in the rapid inactivation of semiochemicals [6±7, 4]. Sensory proteins, functioning as molecular actors, are considered to play crucial roles in detection of semiochemicals. They participate in the initial transduction of olfactory signals. When the odorants are detected, binding proteins (OBPs, CSPs and SNMPs) will specifically bind the hydrophobic odorants, and transport them to cross the aqueous sensillum lymph that embeds olfactory neuron dendrites. Subsequently, the odorants interact with membranebound chemosensory receptors (ORs IRs, and GRs) in the receptor neuron membrane, in which the odorant signals are transformed into electric signals. Finally, signal termination is inactivated by ODEs, which prevent the continuous accumulation of stimulants and subsequent sensory adaptation, and allow insects to rapidly respond to changes in environmental odorants [8±9]. During the past decade, the emergence of next generation sequencing (NGS) technology has dramatically improved the efficiency of gene screening. Meanwhile, the entomological research has also benefited from the development of NGS technology [ 10 ]. With the improvement of high-throughput sequencing methods, olfactory-related genes have been identified from antennal transcriptomes in numerous Lepidoptera species, including several notorious agricultural pests [11±22]. Such technology has been widely used to identify genes involved in olfaction of insects. However, little information is available about the function of olfactoryralated genes of P. interpunctella due to the deficiency of the genomic data for this species. Although several transcriptomic studies related to P. interpunctella have been performed [23±25], antennal transcriptome analysis of olfactory system has not been conducted in previous studies. To identify the olfactory-related genes, we described the antennal transcriptome analysis of P. interpunctella in the present study. The expression levels of olfactory-related genes were investigated using quantitative real-time PCR. Taken together, our study successfully identified olfactory-related genes of P. interpunctella and provided useful information for further studies on pheromone and host volatile recognition. Materials and methods Insects material and RNA extraction Plodia interpunctella was the laboratorial population which was reared for more than 20 generations in our laboratory. The larvae were reared on crushed grains of wheat under constant conditions (28±1ÊC, 60±5% RH and 14:10 L:D photoperiod). Mature larvae were sorted by sex according to the black spot in the middle of male back. Antennae were excised from 3-day-old unmated moths, immediately frozen in liquid nitrogen and ground with a pestle. Total RNA was extracted from 100 antennae for each sex. The evaluation of RNA purity, RNA concentration and RNA quality were conducted following our previous method [ 13 ]. 2 / 25 cDNA library preparation for transcriptome sequencing cDNA library were constructed following previous method [ 17 ]. Briefly, 3 μg RNA per sample was used as input material for the RNA sample preparation. Sequencing libraries were generated using NEBNext1Ultra™ RNA Library Prep Kit for Illumina1 (NEB, USA) following manufacturer's instructions. Newly isolated mRNA was further purified using with Oligo (dT) magnetic beads and sheared into 200±700 nucleotides sections using fragmentation buffer. The fragmented mRNA was used as templates for first-strand cDNA synthesis using random hexamer primers. Subsequently, second-strand cDNA was synthesized using DNA polymerase I and RNaseH. All remaining overhangs were passivated via polymerase. After adenylation of 30 ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated for hybridization. In order to select cDNA fragments of preferentially 150~200 bp, the library fragments were purified using an AMPure XP system. Then 3 μL USER Enzyme (NEB, USA) was incubated with size-selected, adaptor-ligated cDNA at 37ÊC for 15 min followed by incubation at 95ÊC for 5 min before PCR reaction. Subsequently, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Amplicons were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The cDNA library of P. interpunctella was sequenced on Illumina Hiseq™ 2500 using paired-end technology in a single run by Beijing Biomake Company (Beijing, China). Clustering and sequencing Following a previous report [ 17 ], clustering and sequencing were performed on a cBot Cluster Generation System and an Illumina Hiseq 2500 platform, respectively. Sequence analysis and assembly Raw reads of fastq format were firstly processed through in-house perl scripts. In this step, clean reads were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads. At the same time, Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. Cleaned reads shorter than 60 bases were removed because the short reads might represent sequencing artifacts [ 26 ]. The qualified reads were assembled into unigenes using short reads assembling program-Trinity [ 10 ]. The obtained contigs were annotated against the NCBI non-redundant protein (NR) database using BLASTn (E-value<10−5) and BLASTx (E-value<10−5) programs [ 11 ]. To annotate the assembled sequences with Gene Ontology (GO) terms, the Swiss-Prot BLAST results were imported into BLAST2GO, a software package that retrieves GO terms, allowing determination and comparison of gene functions [ 27 ]. The unigene sequences were also aligned to the Clusters of Orthologous Groups of proteins (COG) database to predict and classify the unigene sequences [ 28 ]. Pathway annotations for unigenes were determined using Kyoto Encyclopedia of Genes and Genomes (KEGG) ontology [ 29 ]. Finally, the best matches were used to identify coding regions and determine the sequence direction [ 30 ]. Olfactory gene identification and phylogenetic analysis The annotations of OBP, CSP, SNMP, OR, IR and GR genes in P. interpunctella were verified by BLASTx and BLASTn programs NCBI. The complete coding region was predicted using the open reading frame (ORF) finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) based on the results given by BLASTx. After completing the alignments of the candidate chemosensory genes using ClustalX (2.1), phylogenetic reconstruction for the analysis of OBPs, CSPs, ORs, IRs and GRs was performed by MEGA5.0 software using the neighbor-joining method with 3 / 25 1000 Bootstrap iterations [ 31 ]. In addition, the evolutionary distances were assumed by using the Poisson correction method [ 11 ]. Analysis of differentially expressed genes and qRT-PCR verification To compare the differential expression of chemosensory genes between the male and female antennal transcriptomes of P. interpunctella, the read number of each olfactory-related gene was converted to FPKM (fragments per kilobase of exon model per million mapped reads) [ 32 ]. qRT-PCR was performed to quantify the expression levels of olfactory-related genes in male and female antennae. Total RNA was extracted from 100 antennae as above description. cDNA from antennae of both sexes was synthesized using the SMARTTMPCR cDNA synthesis kit (Clontech, Mountain View, CA, USA). The β-actin gene (SRP05157) was used as an internal control in each sample, and it was selected as a housekeeping gene in our qRT-PCR test. Real-time PCR was performed on an ABI 7500 using SYBR green dye binding to doublestranded DNA at the end of each elongation cycle. Primer sequences were designed using the Primer Premier 5.0 program (S1 Table). Real-time PCR was conducted with our previous method [ 13 ]. Briefly, 10.0 μL of 2×SYBR Green PCR Master Mix, 0.4 μL of primer, 2.0 μL of sample cDNA (100 ng μL-1) and 7.2 μL of sterilized ultrapure water were mixed to form a 20 μL reaction system.After an initial denaturation step at 95ÊC for 3 min, amplifications were carried out with 40 cycles at a melting temperature of 95ÊC for 10 s and an annealing temperature of 60ÊC for 30 s. To check reproducibility, qRT-PCR test for each sample was performed with three technical replicates and three biological replicates. qRT-PCR analysis Relative quantification was determined using the comparative 2-ΔΔCt method [ 33 ]. All data were normalized to endogenous β-actin levels from the same individual samples. The relative fold change was assessed by comparing the expression level in male moths to that in females [ 34 ]. The results were presented as the means of the fold change in three biological duplicates. The comparative analyses of chemosensory genes between sexes were determined by one-way analysis of variance (ANOVA) using SPSS 19.0, with p-value of 0.05 considered significant. Results Sequence analysis and assembly cDNA library of Plodia interpunctella was constructed using the TRINITY de novo assembly program, and short-read sequences were assembled into 150,633 transcripts with a mean length of 1,491 bp and an N50 of 3,567 bp. A total of 20,261 scaffolds (13.45%) were longer than 1,000 bp, and 36,148 scaffolds (24.00%) were longer than 2,000 bp. The scaffolds were subjected to cluster and assembly analyses. Subsequently, 87,300 unigenes were obtained with a mean length of 699 bp and an N50 of 1,282 bp (Fig 1, Table 1). The length distribution of unigenes revealed that 26,054 unigenes (29.84%) were longer than 500 bp and 12,485 unigenes (14.30%) were longer than 1,000 bp (Table 1). The raw reads of P. interpunctella transcriptome have been deposited into the NCBI SRA database (accession number: SRR6002827 and SRR6002828), and the Transcriptome Shotgun Assembly (TSA) project has been deposited at DDBJ/ENA/GenBank under the accession GFWQ00000000. The version described in this paper is the first version, GFWQ01000000. The detailed TSA sequences could be obtained from Genbank (https://www.ncbi.nlm.nih.gov/Traces/wgs/?val=GFWQ01&display= contigs&page=1). 4 / 25 Fig 1. Distribution of Unigene length of Plodia interpunctella. Sequence annotation The unigene annotation showed that 27,920 unigenes (31.98%) significantly matched in the NR database and 15,815 unigenes (18.12%) had significant matches in the Swiss-Prot database. A total of 31,921 unigenes (36.56%) were successfully annotated in the NR, Swiss-Prot, KEGG, GO and COG databases (Table 2), whereas 55,379 unigenes (63.44%) were unmapped in those databases. NR database queries revealed that a high percentage of P. interpunctella sequences had closely matched sequences in Bombyx mori (6,087, 21.84%), followed by Danaus plexippus (4,612, 16.54%), Acyrthosiphon pisum (4,329, 15.53%) and Bactrocera dorsalis (3,839, 13.77%) (Fig 2). For GO analysis, 15,893 unigenes (18.21%) could be assigned to three GO terms as follows: cellular components, molecular functions and biological process (Fig 3). The ªcellular componentsº and ªmolecular functionsº were most represented by 18.79% and 21.04% transcripts, respectively. In the ªcellular componentsº ontology, the terms were mainly distributed in cell Transcript 39,574(26.27%) (26.27%) 31,475(20.90%) (20.90%) 23,172(15.38%)) (15.38%) 20,261(13.45%) 36,148(24.00%) 150,633 224,546,425 3,567 1490.69 Unigene 35,516(40.68%) 25,729(29.47%)(29.74%) 13,569(15.54%) 6,176(7.07%) 6,309(7.23%) 87,300 61,027,187 1,282 699.05 5 / 25 (20.71%) and cell part (20.71%). In the ªmolecular functionsº ontology, the terms of binding function and catalytic activity were the most represented (39.91% and 39.80%, respectively) (Fig 3). To predict and classify the functional genes, all unigenes were searched against the COG database. A total of 10,106 unigenes could be assigned to 25 specific categories according to the COG classification results. ªGeneral function predictionº (2,494, 24.68%) was the largest group, and the categories of ªcell motilityº (20, 0.20%) and ªnuclear structureº (11, 0.11%) were the smallest groups (Fig 4). In addition, 290 pathways were predicted in the KEGG database, representing 15,016 unigenes. Identification of olfactory-related genes In the present study, we identified 117 olfactory-related genes from antennal transcriptome of P. interpunctella, including 29 OBPs, 15 CSPs, three SNMPs, 47 ORs, nine GRs and 14 IRs. All genes were named according to a four-letter code (first letter of the genus name followed by the first three letters of the species name) + OR + number according to the ORF lengths. Analysis of differential expression of unigenes indicated that 1,031 genes showed differences between the antennal transcriptomes of male and female P. interpunctella, including 93 upregulated and 938 down-regulated genes using female result as the reference standard. Fig 2. Characteristics of homology search for Plodia interpunctella unigenes. The number of unigenes matching the top ten species using BlastX in the Nr database is indicated in square brackets. 6 / 25 Fig 3. Functional annotation of assembled sequences based on gene ontology (GO) categorization. Candidate OBPs in antennae of Plodia interpunctella In antennal transcriptomes of P. interpunctella, 29 OBP genes were annotated based on the tBLASTn results, including four pheromone-binding proteins (PBPs) and one general odorant-binding protein (GOBP) (Table 3). Among the 29 OBP genes, 17 had intact ORFs with lengths ranging from 291 bp to 1,014 bp. The BLASTx results indicated that 24 identified PintOBPs shared relatively higher amino acid identities (>50%) with Lepidoptera OBPs in NCBI. A neighbor-joining tree of 123 OBP sequences was constructed using OBPs of Lepidoptera species, including four species in Pyraloidea family (P. interpunctella, Conogethes punctiferalis, Ostrinia furnacalis and Chilo suppressalis), and Bombyx mori. Due to the lack of antennal transcriptome information of genus Plodia, we selected three closer relatives of P. interpunctella to Fig 4. Cluster of orthologous groups (COG) classification. 7 / 25 Y Y N N N Y N Y Y N Y N Y Y Y Y Y N Y Y N Y N N N N 8 / 25 56 69 67 71 47 81 76 44 61 36 49 83 68 51 77 83 78 90 61 63 61 47 94 62 69 100 compare the OBPs. B. mori was chosen to study the patterns and functions of OBPs, because BmorOBPs were widely recognized and verified. Most PintOBPs had a high similarity to known Pyralidae OBPs, which could possibly be attributed to that both P. interpunctella and Pyralidae belong to Pyraloidea family. Phylogenetic tree showed that the PintPBP2-4 was clustered into the PBP family, and the PintGOBP1 was clustered into the GOBP family. In the PBP family, PintPBP2, PintPBP3 and PintPBP4 were stretched in the same branch with the bootstrap values as high as 62 (Fig 5). Based on the number of conserved cysteines, OBPs can be divided into three subclasses: classic OBPs, Plus-C OBPs and Minus-C OBPs [ 35 ]. As for P. interpunctella, PintOBP7, PintOBP10, PintOBP15 and PintOBP19 were clustered into the Minus-C OBP family. Meanwhile PintOBP5 belonged to the Plus-C OBP family. According to multiple amino acid sequence alignments, 16 OBPs (PintOBP1-4, PintOBP6, PintOBP8-9, PintOBP11-17, PintOBP20, PintPBP1-3 and PintGOBP1) totally matched with C1-X25-30-C2X3-C3-X36-42-C4-X8-14-C5-X8-C6 (X stands for any amino acid), and they were identified as classic OBPs (Fig 6) [ 36 ]. Base on FPKM measure, the OBPs with an FPKM value greater than 1,000 were defined as high-expression genes [ 30 ]. The FPKM analysis revealed that 10 OBP genes (PintOBP8, PintOBP10, PintOBP11, PintOBP15, PintOBP17, PintPBP1-4 and PintGOBP1) were highly abundant in antennae of P. interpunctella (FPKM>1,000) (Table 3). Furthermore, the qRT-PCR expression levels of 29 PintOBP genes indicated that nine OBP genes (PintOBP4, PintOBP6, PintOBP9, PintOBP13, PintOBP17 PintOBP20, PintOBP22 and PintPBP2-3) were significantly expressed in the male antennae (1.8 to 33.5 times compared with females). Eight OBPs (PintOBP5, PintOBP7, PintOBP12, PintOBP15-16, PintOBP18, PintPBP1 and PintGOBP1) were significantly expressed in the female antennae (1.7 to 3.8 times compared with males). The other eight OBP genes (PintOBP1-3, PintOBP8, Pint10-11, PintOBP14 and PintOBP21) showed similar expression levels in the male and female antennae (Fig 7). Candidate CSPs in antennae of Plodia interpunctella In the antennal transcriptomes of P. interpunctella, 15 putative CSPs were identified with lengths ranging from 291 bp to 492 bp. All identified PintCSPs were verified according to the four-cysteines pattern C1-X6-8-C2-X18-19-C3-X2-C4 (Fig 8) [ 36 ]. Among the 15 PintCSP genes, eight had intact ORFs with lengths ranging from 318 bp to 492 bp. The BLASTx results indicated that 13 identified PintCSPs shared relatively higher amino acid identities (>50%) with Lepidoptera CSPs in NCBI (Table 4). A neighbor-joining tree of 78 CSP sequences was constructed based on Lepidoptera species from C. punctiferalis, O. furnacalis, C. suppressalis and B. mori. PintCSPs were distributed on various branches throughout the cladogram (Fig 9). The phylogenetic tree showed that PintCSP14, PintCSP2, PintCSP5 and PintCSP1 were clustered together with OfurCSPs, with relatively higher bootstrapping values. 9 / 25 Fig 5. Neighbor-joining tree of candidate OBPs from Plodia interpunctella, Conogethes punctiferalis, Ostrinia furnacalis, Chilo suppressalis and Bombyx mori. The protein names and sequences of OBPs that were used in this analysis are listed in S2 Table. The FPKM analysis revealed that only PintCSP4 was highly abundant in antennal transcriptomes of P. interpunctella (FPKM>1,000) (Table 4). The qRT-PCR results indicated that three PintCSP genes (PintCSP11, PintCSP14 and PintCSP15) were significantly expressed in the male antennae (1.5 to 3.5 times compared with females). Seven PintCSPs (PintCSP1-2, PintCSP5, PintCSP9-10 and PintCSP12-13) were specifically expressed in the female antennae (1.7 to 3.2 times compared with males) (Fig 10). Fig 6. Sequences alignment of classic PintOBPs. 10 / 25 Fig 7. P. interpunctella OBP transcript levels in different antennae measured by qRT-PCR. MA: male antennae; FA: female antennae. The β-actin was used as internal control to normalize transcript levels in each sample. The standard error represented by the error bar, and the asterisk above each bar denote significant differences (p<0.05). Candidate ORs We identified 47 OR genes in the antennal transcriptomes of P. interpunctella, in which 36 PintORs had intact ORFs with lengths ranging from 219 bp to 1,422 bp with four to seven transmembrane domains (Table 5). In the neighbor-joining tree of ORs (Fig 11), the PintOR1 was clustered into the ORco family and four PintORs (PintOR5, PintOR7, PintOR22 and PintOR30) were clustered into the pheromone receptor (PR) family. Two groups of ORs (PintOR14 and PintOR35, PintOR29 and PintOR26) were clustered into the same branch with bootstrapping values of 98 and 87, respectively. All of the other PintORs were distributed on various branches throughout the phylogenetic tree. PintOR1 (ORco) showed the highest qRT-PCR expression level among the 47 PintORs, with FPKM values of 576.23 and 430.52 in the male and female antennae, respectively. However, the other 46 typical ORs showed a relatively lower expression level (FPKM ranged from 0 to 214). The qRT-PCR results indicated that nine OR genes (PintOR1, PintOR5, PintOR15, PintOR18, PintOR22, PintOR38, PintOR41-42 and PintOR47) were highly expressed in the male antennae. Meanwhile, 16 OR genes (PintOR3, PintOR7, PintOR9-11, PintOR23-25, Fig 8. Sequences alignment of candidate PintCSPs. 11 / 25 PintOR28, PintOR30-31, PintOR35, PintOR37, PintOR40 and PintOR45-46) exhibited female antenna-specific expressions (Fig 12). Candidate GRs In the present study, we identified nine candidate PintGR encoding transcripts from antennal transcriptome of P. interpunctella. Five PintGR genes had intact ORFs with lengths ranging from 198 bp to 1,461 bp. The BLASTx results indicated that seven identified PintGRs shared relatively higher amino acid identities (>50%) with Lepidoptera GRs in NCBI (Table 6). In the neighbor-joining tree of GRs (Fig 13), PintGRs were present on various branches throughout the cladogram. PintGR1 and PintGR3 were clustered into the same branch, with a bootstrapping value of 65. The FPKM analysis showed that all PintGRs had a relatively low expression level (FPKM ranged from 0.27 to 33.37). The qRT-PCR results indicated that PintGR1 and PintGR8 were highly 12 / 25 Fig 9. Neighbor-joining tree of candidate CSPs from Plodia interpunctella, Conogethes punctiferalis, Ostrinia furnacalis, Chilo suppressalis and Bombyx mori. The protein names and sequences of CSPs that were used in this analysis are listed in S3 Table. expressed in the male antennae (1.9 and 3.7 times compared with females, respectively). Moreover, five GRs (PintGR3, PintGR5-7 and PintGR9) displayed female antenna-specific expressions (Fig 14.). Fig 10. P. interpunctella CSP transcript levels in different antennae measured by qRT-PCR. MA: male antennae; FA: female antennae. The internal control β-actin was used to normalize transcript levels in each sample. The standard error represented by the error bar, and the asterisk above each bar denote significant differences (p<0.05). 13 / 25 Candidate IRs In the present study, we identified 14 candidate PintIR genes encoding transcripts from antennal transcriptome of P. interpunctella (Table 6). Nine PintIRs had intact ORFs with lengths ranging from 384 bp to 2,706 bp. In the neighbor-joining tree of IRs (Fig 15), PintIR1 and PintIR2 were phylogenetically clustered into the highly conserved IR8a and IR21a sub-families, respectively. The FPKM analysis revealed that all PintIRs showed a low expression level (FPKM value ranged from 0.36 to 113.52). The qRT-PCR results indicated that PintIR1, PintIR3-5, PintIR10, and PintIR13-14 were highly expressed in the female antennae (1.2 to 5.3 times compared with males) (Fig 14). 15 / 25 Fig 11. Neighbor-joining tree of candidate OR proteins from Plodia interpunctella, Ostrinia furnacalis, Chilo suppressalis and Bombyx mori. The protein names and sequences of ORs that were used in this analysis are listed in S4 Table. Discussion In recent years, RNA-Seq transcriptome sequencing technology has been widely used due to the development of high-throughput sequencing technology, resulting in great progress in non-model organisms [11, 37±39]. In the present study, we used NGS technology to analyze Fig 12. P. interpunctella OR transcript levels in different antennae measured by qRT-PCR. MA: male antennae; FA: female antennae. The internal control β-actin was used to normalize transcript levels in each sample. The standard error represented by the error bar, and the asterisk above each bar denote significant differences (p<0.05). 16 / 25 the antennal transcriptome of P. interpunctella. Sequence analysis and assembly results demonstrated that Illumina sequencing technology could effectively and rapidly captured a large portion of the transcriptome, providing molecular foundations for rapid characterization of functional genes and better reference of target genes [ 40 ]. 17 / 25 Fig 13. Neighbor-joining tree of candidate GR proteins from Plodia interpunctella, Conogethes punctiferalis, Ostrinia furnacalis, Chilo suppressalis and Bombyx mori. The protein names and sequences of GR that were used in this analysis are listed in S5Table. The unigene annotation showed that 55,379 unigenes (63.44%) were unmapped in those databases, which could be attributed to the short sequence reads generated by the sequencing technology. It also suggested that the unmapped sequences could represent unannotated or new genes. In fact, fewer than 5% of unmapped unigenes are likely to represent new genes. Generally, the 5' ends of sequences show less conservation than the body. Therefore, partial transcripts (unigenes representing the 5' CDS, but not the body) may not be found matches in the databases. For GO analysis, the antennal unigenes were annotated into different functional groups [ 16 ], which were similar to those in the antennal transcriptomes of Conogethes punctiferalis [ 13 ], Spodoptera littoralis [ 41 ] and Helicoverpa armigera [ 11 ]. Therefore, we inferred that the success rates of functional annotation of genes highly depended on the sequence length of the splicing unigene: the shorter the length of the sequence, the less possibility of the annotation. Others reasons might also result in partial information failure, such as the 18 / 25 Fig 14. P. interpunctella GR and IR transcript levels in antennae measured by qRT-PCR. MA: male antennae; FA: female antennae. The internal control β-actin was used to normalize transcript levels in each sample. The standard error represented by the error bar, and the asterisk above each bar denote significant differences (p<0.05). incompleteness of P. interpunctella gene transcription group information, and/or the insufficiency of the sequence of partial RNA-Seq sequencing data in public database. Olfactory-related genes might be used as potential targets for management programs of P. interpunctella. As the first step of odor detection [ 6 ], OBPs have attracted wide interests of researchers [ 13, 17, 42 ]. In the present study, we identified 29 PintOBP genes from antennal transcriptome of P. interpunctella. The number of identified PintOBPs was equivalent to that from H. armigera (26) [ 11 ], Dendrolimus kikuchii (27) [ 17 ] and Agrotis ipsilon (33) [ 21 ], and it was significantly greater than that from Cnaphalocrocis medinalis (12) [ 12 ], C. punctiferalis (14) [ 13 ], Manduca sexta (18) [ 43 ] and S. exigua (11) [ 44 ]. The small number of OBPs in above species could be attributed to that the actual number of OBPs was less in P. interpunctella, or there should be more OBPs that were not caught by the sequencing. Therefore, we speculated that the transcriptomic sequencing might not be strong enough to detect all the OBPs, especially for some OBPs with low expression levels in the antennae [45]. The OBP trees from five Lepidopteran species indicated that after a long history evolution, the Lepidopteran OBPs differentiated into several branches (Fig 5), which was consistent with previous reports [46]. In the evolutionary tree for GOBPs and PBPs, these two sub-families were clustered respectively, indicating that these genes might have the same ancestor gene and differentiate along sex isolation and speciation. The qRT-PCR results indicated that nine PintOBP genes (PintOBP4, PintOBP6, PintOBP9, PintOBP13, PintOBP17 PintOBP20, PintOBP22 and PintPBP2-3) were significantly expressed in the male antennae, suggesting that these OBPs played essential roles in the detection of sex pheromones. Eight PintOBPs (PintOBP5, PintOBP7, PintOBP12, PintOBP15-16, PintOBP18, PintPBP1 and PintGOBP1) were significantly expressed in the female antennae, revealing that these OBPs played important roles in the detection of general odorants, such as host plant volatiles [ 21 ]. CSPs represent a newly-discovered class of soluble carrier proteins with similar functions to OBPs in insect chemoreception [47]. CSPs have been found in insect chemosensory tissues and non-chemosensory organs, such as antennae [ 11 ], legs [ 48 ], labial palps [ 49 ], tarsi [ 50 ], brain [ 51 ], proboscis [ 52 ], pheromone gland [53±54] and wings [ 55 ]. We identified 15 putative CSP encoding transcripts, and found that six PintCSP genes were significantly expressed in the female antennae. These PintCSPs might play important roles in the detection of general odorants, such as host plant volatiles. 19 / 25 Fig 15. Neighbor-joining tree of candidate IR proteins from Plodia interpunctella, Conogethes punctiferalis, Ostrinia furnacalis, Chilo suppressalis and Bombyx mori. The protein names and sequences of IR that were used in this analysis are listed in S6 Table. OR proteins are key players in insect olfaction [ 56 ]. We identified 47 PintOR genes in antennal transcriptome of P. interpunctella. The number of PintORs identified in this study was less than that identified from the antennal transcriptomes of Bombyx mori (72) [ 57 ], C. punctiferalis (62) [ 58 ] and Ostrinia furnacalis (56) [ 59 ]. However, the difference in identified OR gene numbers might be caused by sequencing methods and depth, or sample preparation. In the neighbor-joining tree of ORs, four PintORs (PintOR5, PintOR7, PintOR22 and PintOR30) were clustered into the PR family, indicating that parts or all of them contributed to sex pheromone detection. The qRT-PCR results indicated that PintOR5 and PintOR22 were highly expressed in the male antennae, suggesting they are highly related to sex pheromone. PintOR7 and PintOR30 specifically expressed in the female antennae. The expression profiles of these sequences showed that not all of them were male-specific [ 60 ]. Recent studies also showed that some PR genes are expressed in both sexes [54]. The OR tree showed that the PintORco (PintOR1) was highly conserved. 20 / 25 In recent years, 12 HarmIRs in H. armigera [ 11 ], 17 SlitIRs in S. littoralis [ 61 ] and 15 CpomIRs in C. pomonella [ 22 ] have been identified. In this study, we identified 14 PintIRs, including highly conserved IR co-receptors PintIR1 and PintIR2 (IR8a and IR21a) from antennal transcriptome of P. interpunctella. Therefore, we speculated that IRs were relatively highly conserved sequences, implying that IRs had conservative features. Several recent reports simultaneously tested the qRT-PCR expression of olfactory-related genes in various tissues of insect, including bodies, heads, legs or abdomens [13±15, 20±21, 54]. In present study, we only focused on the qRT-PCR analysis of P. interpunctella antennae. To the best of our knowledge, P. interpunctella moths do not eat anything, suggesting they have no food demands, so location of mate partners and oviposition sites should be the main function of olfactory. While most olfactory genes related to recognition of pheromone and host volatiles distribute in insect antennae, therefore, we only compared the expression between female and male antennae of P. interpunctella, to verify the olfactory-related genes. Conclusion In this study, we identified a few olfactory gene families in antennal transcriptome of P. interpunctella, including 29 PintOBPs, 15 PintCSPs, three PintSNMPs, 47 PintORs, nine PintGRs and 14 PintIRs. The identification of antennal olfactory-related proteins in P. interpunctella reinforced our knowledge on the molecular and cellular basis of insect chemoreception. More importantly, our data suggested that new methods could be developed to control this pest by interfering their olfactory perception. Supporting information S1 Table. Primers used for RT-qPCR. (DOCX) S2 Table. Amino acid sequences of PintOBPs used in phylogenetic analyses. (DOC) S3 Table. Amino acid sequences of PintCSPs used in phylogenetic analyses. (DOC) S4 Table. Amino acid sequences of PintORs used in phylogenetic analyses. (DOC) S5 Table. Amino acid sequences of PintGRs used in phylogenetic analyses. (DOC) S6 Table. Amino acid sequences of PintIRs used in phylogenetic analyses. (DOC) Acknowledgments This work was funded by the National Natural Science Foundation of China (3130914), Special Finance of Hebei Province (F17C10007 and F17C10008). Author Contributions Data curation: Xiaojian Jia, Hongmin Liu. Formal analysis: Hongmin Liu, Rongyan Wang. Funding acquisition: Tao Zhang. 21 / 25 Investigation: Hongmin Liu. Methodology: Xiaojian Jia. Resources: Xiaofang Zhang, Hongmin Liu, Rongyan Wang. Software: Xiaojian Jia. Supervision: Tao Zhang. Validation: Xiaofang Zhang, Rongyan Wang. 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Xiaojian Jia, Xiaofang Zhang, Hongmin Liu, Rongyan Wang, Tao Zhang. Identification of chemosensory genes from the antennal transcriptome of Indian meal moth Plodia interpunctella, PLOS ONE, 2018, DOI: 10.1371/journal.pone.0189889