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. 2016 Sep 23;6:32636. doi: 10.1038/srep32636

Identification of putative chemosensory receptor genes from yellow peach moth Conogethes punctiferalis (Guenée) antennae transcriptome

Xing Ge 1, Tiantao Zhang 1,a, Zhenying Wang 1,b, Kanglai He 1, Shuxiong Bai 1
PMCID: PMC5034240  PMID: 27659493

Abstract

The yellow peach moth, Conogethes punctiferalis, is an extremely important polyphagous insect in Asia. The chemosensory systems of moth play an important role in detecting food, oviposition sites and mate attraction. Several antennal chemosensory receptors are involved in odor detection. Our study aims to identify chemosensory receptor genes for potential applications in behavioral responses of yellow peach moth. By transcriptomic analysis of male and female antennae, 83 candidate chemosensory receptors, including 62 odorant receptors, 11 ionotropic receptors and 10 gustatory receptors were identified. Through Blast and sequence alignment, the highly conserved co-receptor Orco was annotated, eight unigenes clustered into pheromone receptors, and two clustered as sugar receptor. Among the IRs, one unigenes was similar with co-receptors IR25a. Expression levels of 50 odorant receptors were further evaluated by quantitative real-time PCR in antennae. All the ORs tested were detected in antennae and some of which were associated with sex-biased expression. The chemosensory receptors identified in C. punctiferalis provide a foundational resource for further analysis on olfaction for behavior. The expression profiles of ORs in antennae indicated variant functions in olfactory recognition, and our results provided the possibility for the potential application of semiochemical to control this pest moth.

Insect olfactory system enables insects to detect and discriminate different odor molecules from their environment. The odorant and pheromone perception in insects are a complex series of process that transfer external stimulus from the environment to behavioral response. Diverse proteins consisting in this signal transduction pathway include odorant binding proteins (OBPs), chemosensory proteins (CSPs), chemosensory receptor (CRs), odorant degrading enzymes (ODEs) and sensory neuron membrane proteins (SNMPs)1,2,3,4,5,6,7.

The gene family encoding CRs in insects mainly divides into three groups: olfactory receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs)8,9,10,11,12. The insect ORs and the GRs were firstly identified in the genomic analysis of Drosophila melanogaster13. Previous studies suggested that the OR and GR families belong to a superfamily based on the transmembrane domain13,14. Both ORs and GRs are seven transmembrane-domain receptors with an inverted transmembrane topology in contrasts to the OR of mammalian14,15,16. ORs are housed targeted in the dendritic membrane of olfactory sensory neurons (OSNs). Most OSNs express a single OR gene, along with odorant receptor co-receptor (Orco)17. Insect OR genes are highly diverse, and their numbers among species are various13,18,19,20,21,22. ORs have been shown to form a voltage-gated ion channel following heterodimerization with Orco, which is an ortholog of the D. melanogaster OR, DmOr83b23,24. ORs in Lepidopteran pheromone system are classified into two major groups, pheromone receptors (PRs) and general odorant receptors according to perception of chemicals25. PRs are thought to be associated with olfactory signal transduction of pheromone compounds in peripheral olfactory reception, and general odorant receptors are considered to detect plant volatiles. PRs were reported in several species20,26,27,28. Also pheromone-pheromone receptor relationships also have been identified. In Bombyx mori, general odorant receptor BmOR19 responds to linalool, furthermore, BmOR45 and BmOR47 respond to benzoic acid, 2-phenylethanol, and benzaldehyde respectively29. While PRs (BmOR1 and BmOR3) respond specifically to bombykol (E10Z12-16: OH) and bombykal (E10Z12-16: Ald) respectively, which are major and minor sex pheromone components in B. mori.30,31. Although we classified receptors on the basis of their responses to chemicals, their functions are still unknown. PRs are more conserved compared with other ORs32,33. Orco is the only highly conserved OR protein among divergent insect species, which suggests the crucial role in chemical recognition34,35. The GRs are generally expressed in gustatory receptor neurons (GRNs) within gustatory organs. Differently, several GRs are usually expressed in one neuron. Most GRs sense non-volatile compounds, including sugar and bitter compounds36. Interestingly, Some GRs expressed in antennae cannot identify volatile odorants, but respond to carbon dioxide37. IRs belongs to a variant subfamily of ionotropic glutamate receptors (iGluRs), involving in detection of chemical signals38,39. Based on the expression location, two subfamily of IRs are identified: conserved antennal IRs and divergent IRs, indicating the function of IRs in olfactory and taste39. In antennal IRs, IR8a and IR25a appear to act as co-receptors and form heteromeric complexes with odor- specific receptors40,41. In contrast with ORs, many IR-expressing neurons expressed two or three IR genes17.

The yellow peach moth Conogethes punctiferalis (Guenée) (Lepidoptera: Crambidae), is a serious polyphagous insect of crop (sunflower, maize, sorghum, cotton, etc.) and fruit (peach, plum, durian, etc.). It’s hard to control by traditional method, due to larvae bored the inside fruit or stem42,43. Few olfactory receptor genes have been reported in this moth44, however more receptor genes investigated allows a better understanding of the molecular basis of olfaction. Identification of the chemosensory genes can lead to comprehensive understanding of how to recognize and locate host for this moth. In the present study, the antennal transcriptome of C. punctiferalis were sequenced on the Illumina HiSeq 4000 platform. In total 83 chemoreceptor genes including 62 ORs, 11 IRs and 10 GRs were identified. The expression levels of ORs, which may play important roles in chemoreception of C. punctiferalis were analyzed by using of quantitative real-time PCR (qRT-PCR). The results assist with the identification of genes involved in C. punctiferalis olfactory, help better understand the gene expression in sex, and provide the molecular basis for further study of pheromone and host volatile recognition.

Results

Illumina sequencing

To identify the chemosensory protein genes from C. punctiferalis, the cDNA from male and female antennae were sequenced using the Illumina HiSeqTM 4000 sequencing platform. A total of 59,375,582 and 57,353,054 raw reads were obtained from female and male antennae, respectively. After removing adaptor sequences, low quality sequences and containing N sequences, 58,053,354 and 55,592280 clean reads were generated from the antennae of female and male raw data, respectively. After assembled, the two datasets result in 112,933 contigs with a mean length of 902 bp and an N50 of 1750 bp. From these datasets, 76,486 unigenes were obtained with a mean length of 727 bp and an N50 of 1499 bp (Table 1). The raw reads of the C. punctiferalis are available from the SRA database (accession number: (SRX 1604701 and SRX 1604705).

Table 1. Summary of assembled contigs and unigenes.

  Contigs Unigenes
Total length (bp) 101,865,459 55,582,691
Minimum length (bp) 201 201
Mean length (bp) 902 727
Median length (bp) 411 320
Maximum length (bp) 29,150 29,150
N50 (bp) 1,782 1,499
N90 (bp) 306 254
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Functional annotation of assembled unigenes

The proportion of unigenes annotated in at least one database of Nr, Nt, KO, SwissProt, PFAM, GO, KOG reach to 40%. Only 4.75% unigenes were annotated in all databases (Table 2). Using BlastX database, 26,114 (34.14%) unigenes were matched to known proteins. For species distribution, the results showed 33.43%, 24.85%, and 1.91% of protein similar with the sequences from B. mori, Danaus plexippus and Papilio xuthus, respectively (Fig. 1). GO annotation was used to classify the function of transcripts according to the GO terms (Fig. 2A). In the biological process terms, cellular, metabolic and single-organism were the highest classified. In the cellular component terms, cell, cell part and organelle were the most abundant. In the molecular function category, the genes expressed in the antennae were mostly related to binding, catalytic activity and transporter activity. KOG annotation was based on the relationship between orthologous genes from different species (Fig. 2B). Using KOG functional annotation, “General function prediction only”, “Signal transduction”, and “Posttranslational modification, protein turnover, chaperones” were three largest groups assigned to. For KEGG annotation, 10,298 unigenes were classified into five groups, Cellular processes, environmental information processing, genetic information processing, metabolism and organismal systems (Fig. 2C). Transport and catabolism, signal transduction, translation, carbohydrate metabolism, endocrine systems were the main pathways in each group, respectively.

Table 2. Summary of annotations of unigenes.

  Number of Unigenes Percentage (%)
Annotated in NR 26,114 34.14
Annotated in NT 11,354 14.84
Annotated in KO 10,298 13.46
Annotated in SwissProt 18,229 23.83
Annotated in PFAM 18,142 23.71
Annotated in GO 18,434 24.1
Annotated in KOG 11,475 15
Annotated in all Databases 3,640 4.75
Annotated in at least one Database 30,596 40
Total Unigenes 76,486 100
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Figure 1. Best hits of the BLASTx results.

Figure 1

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Figure 2.

Figure 2

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(A) Gene Ontology (GO) analysis for the transcriptomic sequences. (B) euKaryotic Ortholog Groups (KOG) annotation for the unigenes.(C) KEGG pathway annotation of the transcriptome. (A) Cellular processes. (B) Environmental information processing. (C) Genetic information processing. (D) Metabolism. (E) Organismal systems.

Putative chemoreceptor genes

All the unigenes were searched by BLAST to identified chemoreceptor genes. Total 62 OR genes, 11 IR genes and 10 GR genes were identified through bioinformatics analysis. All the chemosensory receptor genes of C. punctiferalis were submitted to the GenBank (accession numbers: KX084452-KX084521 and KX096203-KX096215). Of these genes, 45 unigenes (39 ORs and 6 IRs) showed sequence identity to the previously identified in C. punctiferalis which are already submitted in GenBank. Among the identified ORs, 38 were found to represent full-length open reading frames (ORF) with 4–7 transmembrane domains (Table 3). Half of those sequences were annotated against homologs of Ostrinia furnacalis. As expected, Orco/OR2 was identified with high identity compared with the conserved insect OR2/Orco. The IRs and GRs sequences in C. punctiferalis antennal transcriptome were identified according to the similarity with known insect IRs and GRs. Seven of eleven putative IRs were similar with IRs of O. furnacalis. Six unigenes with full-length ORF, contained 3~4 transmembrane domains. Of GR genes, 3 putative GR genes contained full-length ORF with 6 transmembrane regions, while 7 sequences were partial (Table 4).

Table 3. Odorant receptors in Conogethes punctiferalis antennae.

contigs Gene name Accession number residues Full length Top blastx hit Score E-value % ID TM FPKM
Female Male
c45607_g1 OR1 KX084452 449 Yes BAG71417.1 | olfactory receptor-1 [Diaphania indica] 739 0.00E + 00 81% 4 0.68 9.61
c50176_g1 OR2 /Orco KX084453 474 Yes AGF29886.1 | odorant co-receptor [Conogethes punctiferalis] 951 0.00E + 00 99% 7 534.09 523.08
c45303_g1 OR3 KX084454 329 No AFK30395.1 | odorant receptor 3 [Ostrinia furnacalis] 280 3.00E−87 42% 4 2.33 283.65
c35425_g1 OR4 KX084455 418 No BAJ22891.1 | odorant receptor [Ostrinia furnacalis] 283 5.00E−87 39% 3 11.5 0
c43578_g2 OR5 KX084456 421 Yes NP_001296037.1 | odorant receptor 13a-like [Plutella xylostella] 318 1.00E−100 45% 4 3.86 91.95
c43248_g1 OR6 KX084457 419 No AFK30403.1 | odorant receptor 6 [Ostrinia furnacalis] 316 1.00E−99 43% 4 0.17 170.9
c42117_g1 OR7 KX084458 378 Yes AIZ94614.1 | putative pheromone receptor OR6 [Ostrinia nubilalis] 175 2.00E−47 41% 7 0.08 24.79
c40235_g2 OR8 KX084459 428 Yes BAG71424.1 | olfactory receptor [Diaphania indica] 449 1.00E−151 50% 6 43.43 9.77
c39383_g1 OR9 KX084460 426 Yes AFC91723.1 | putative odorant receptor OR15 [Cydia pomonella] 211 3.00E−61 45% 6 5.81 1.27
c50460_g1 OR10 KX084461 394 Yes BAR43452.1 | putative olfactory receptor 10 [Ostrinia furnacalis] 475 1.00E−162 59% 6 5.04 2.8
c40543_g1 OR11 KX084462 389 Yes BAR43453.1 | putative olfactory receptor 11 [Ostrinia furnacalis] 493 4.00E−170 68% 6 5.17 4.22
c44553_g2 OR12 KX084463 353 No BAR43454.1 | putative olfactory receptor 12 [Ostrinia furnacalis] 505 3.00E−175 65% 4 31.85 12.76
c46066_g1 OR13 KX084464 402 Yes BAR43455.1 | putative olfactory receptor 13 [Ostrinia furnacalis] 645 0.00E + 00 81% 5 32.09 14.17
c46413_g1 OR14 KX084465 416 Yes BAR43456.1 | putative olfactory receptor 14 [Ostrinia furnacalis] 546 0.00E + 00 64% 6 19.59 8.43
c50375_g1 OR15 KX084466 388 Yes NP_001166603.1 | olfactory receptor 13 [Bombyx mori] 442 3.00E−150 61% 6 22.17 9.13
c46668_g4 OR16 KX084467 420 Yes BAR43458.1 | putative olfactory receptor 16 [Ostrinia furnacalis] 523 1.00E−180 60% 5 10.49 4.84
c47537_g1 OR17 KX084468 418 No EHJ78030.1 | olfactory receptor 29 [Danaus plexippus] 574 0.00E + 00 73% 0 2.79 2.99
c45438_g1 OR18 KX084469 424 No BAR43460.1 | putative olfactory receptor 18 [Ostrinia furnacalis] 593 0.00E + 00 74% 6 5.9 3.44
c45263_g1 OR19 KX084470 415 Yes BAR43461.1 | putative olfactory receptor 19 [Ostrinia furnacalis] 290 6.00E−90 40% 7 13.51 7.2
c42557_g3 OR20 KX084471 399 Yes BAR43462.1 | putative olfactory receptor 20 [Ostrinia furnacalis] 437 1.00E−147 59% 5 24.84 8.24
c42436_g1 OR21 KX084472 395 Yes AGK90020.1 | olfactory receptor 17 [Helicoverpa assulta] 461 2.00E−157 64% 5 25.06 19.27
c14092_g1 OR22 KX096208 150 No XP_013191807.1 | PREDICTED: gustatory and odorant receptor 22 [Amyelois transitella] 305 2.00E−99 94% 3 0.91 0.22
c45775_g1 OR23 KX084473 401 Yes BAR43467.1 | putative olfactory receptor 25 [Ostrinia furnacalis] 423 5.00E−142 52% 6 14.52 7.73
c46864_g1 OR24 KX084474 450 Yes BAR43466.1 | putative olfactory receptor 24 [Ostrinia furnacalis] 627 0.00E + 00 80% 6 16.34 14.37
c49436_g1 OR25 KX084475 432 Yes BAR43467.1 | putative olfactory receptor 25 [Ostrinia furnacalis] 671 0.00E + 00 78% 6 10.79 10.78
c44068_g1 OR26 KX084476 396 Yes NP_001166611.1 | olfactory receptor 59 [Bombyx mori] 363 1.00E−118 47% 5 50.52 26.05
c42831_g1 OR27 KX084477 401 Yes BAR43469.1 | putative olfactory receptor 27 [Ostrinia furnacalis] 647 0.00E + 00 86% 6 16.3 6.65
c43774_g1 OR28 KX084478 326 No BAR43470.1 | putative olfactory receptor 28 [Ostrinia furnacalis] 309 2.00E−98 47% 6 22.87 14.79
c38000_g1 OR29 KX084479 413 Yes AJF23799.1 | olfactory receptor OR32 [Planotortrix octo] 365 8.00E−119 48% 5 7.71 2.3
c47502_g1 OR30 KX084480 458 Yes AIG51850.1 | odorant receptor [Helicoverpa armigera] 177 5.00E−50 72% 6 21.24 0.9
c45926_g1 OR31 KX084481 388 Yes CUQ99414.1 | Olfactory receptor 34 [Manduca sexta] 399 4.00E−133 55% 7 12.74 7.83
c45783_g1 OR32 KX084482 390 Yes AII01045.1 | odorant receptor [Dendrolimus houi] 354 2.00E−115 44% 6 4.86 1.1
c46810_g1 OR33 KX084483 404 No BAR43475.1 | putative olfactory receptor 33 [Ostrinia furnacalis] 646 0.00E + 00 81% 7 4.97 1.95
c47522_g3 OR34 KX084484 306 No BAR43476.1 | putative olfactory receptor 34 [Ostrinia furnacalis] 320 6.00E−103 56% 4 0.69 14.2
c41738_g1 OR35 KX084485 430 Yes BAR43477.1 | putative olfactory receptor 35 [Ostrinia furnacalis] 809 0.00E + 00 89% 6 3.52 1.89
c42979_g1 OR36 KX084486 328 No BAR43478.1 | putative olfactory receptor 36 [Ostrinia furnacalis] 370 1.00E−122 60% 6 13.92 5.12
c49487_g1 OR37 KX084487 389 Yes BAR43479.1 | putative olfactory receptor 37 [Ostrinia furnacalis] 579 0.00E + 00 77% 6 11.94 5.43
c50585_g1 OR38 KX084488 398 Yes BAR43480.1 | putative olfactory receptor 38 [Ostrinia furnacalis] 411 2.00E−137 49% 6 10.58 7.68
c42903_g1 OR39 KX084489 405 Yes BAR43481.1 | putative olfactory receptor 39 [Ostrinia furnacalis] 249 2.00E−74 35% 5 13.98 0
c46193_g1 OR40 KX084490 414 No BAR43461.1 | putative olfactory receptor 19 [Ostrinia furnacalis] 296 6.00E−92 44% 5 20.14 6.25
c45009_g1 OR41 KX084491 356 Yes BAR43483.1 | putative olfactory receptor 41 [Ostrinia furnacalis] 429 5.00E−145 61% 6 4.75 1.93
c40483_g1 OR42 KX084492 370 No BAR43484.1 | putative olfactory receptor 42 [Ostrinia furnacalis] 452 6.00E−155 61% 6 4.26 1.55
c44512_g1 OR43 KX084493 326 No NP_001091818.1 | olfactory receptor 42 [Bombyx mori] 305 4.00E−97 54% 6 10.81 6.51
c45800_g1 OR44 KX084494 433 Yes BAR43486.1 | putative olfactory receptor 44 [Ostrinia furnacalis] 555 0.00E + 00 69% 5 28.41 14.73
c44781_g1 OR45 KX084495 401 Yes NP_001157210.1 | olfactory receptor 17 [Bombyx mori] 333 5.00E−107 41% 7 13.46 8.28
c45498_g3 OR46 KX084496 215 No BAR43488.1 | putative olfactory receptor 46 [Ostrinia furnacalis] 377 6.00E−127 83% 2 9.53 5.48
c39174_g1 OR47 KX084497 203 No XP_013195066.1 | PREDICTED: putative odorant receptor 92a [Amyelois transitella] 272 2.00E−86 63% 2 1.94 1.09
c28776_g1 OR48 KX084498 277 No BAR43481.1 | putative olfactory receptor 39 [Ostrinia furnacalis] 199 3.00E−57 39% 4 1.45 0
c42096_g2 OR49 KX084499 431 Yes BAR43491.1 | putative olfactory receptor 49 [Ostrinia furnacalis] 523 0.00E + 00 66% 6 14.61 7.74
c43755_g1 OR50 KX084500 410 Yes KOB74670.1 | Odorant receptor 50 [Operophtera brumata] 477 6.00E−163 53% 7 6.07 1.65
c47178_g1 OR51 KX084501 330 No AII01110.1 | odorant receptor [Dendrolimus kikuchii] 393 2.00E−131 53% 4 9.2 4.48
c30858_g1 OR52 KX084502 408 Yes BAR43494.1 | putative olfactory receptor 52 [Ostrinia furnacalis] 491 1.00E−168 56% 6 7.22 1.26
c40944_g1 OR53 KX084503 402 Yes BAR43495.1 | putative olfactory receptor 53 [Ostrinia furnacalis] 496 1.00E−170 66% 5 15.15 0
c48055_g1 OR54 KX084504 410 Yes AFC91736.1 | putative odorant receptor OR28 [Cydia pomonella] 457 4.00E−155 52% 5 14.89 5.16
c49083_g1 OR55 KX084505 415 Yes BAR43458.1 | putative olfactory receptor 16 [Ostrinia furnacalis] 538 0.00E + 00 63% 5 32.58 30.96
c48693_g1 OR56 KX084506 393 Yes BAR43452.1 | putative olfactory receptor 10 [Ostrinia furnacalis] 425 4.00E−143 56% 6 20.86 8.35
c45498_g2 OR57 KX084507 215 No BAR43488.1|putative olfactory receptor 46 [Ostrinia furnacalis] 340 2.00E−112 75% 3 10.91 4.52
c16587_g1 OR58 KX096203 195 No BAR43458.1 | putative olfactory receptor 16 [Ostrinia furnacalis] 234 3.00E−71 61% 2 0.69 0.78
c38597_g3 OR59 KX096204 125 No ACF32962.1 | olfactory receptor 4 [Helicoverpa armigera] 229 1.00E−70 87% 1 1.62 1.12
c10513_g1 OR60 KX096205 107 No BAR43458.1 | putative olfactory receptor 16 [Ostrinia furnacalis] 156 4.00E−43 70% 2 0.4 1.46
c84526_g1 OR61 KX096206 107 No AIT69908.1 | olfactory receptor 66 [Ctenopseustis herana] 122 1.00E−30 51% 1 1.65 0.98
c47522_g1 OR62 KX096207 135 No XP_013165286.1 | PREDICTED: odorant receptor 85b-like [Papilio xuthus] 102 7.00E−23 35% 0 0.69 14.2
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Table 4. Ionotropic and gustatory receptors in Conogethes punctiferalis antennae.

Contigs Gene name Accession number Residues Full length Top blastx hit Score E-value % ID TM
c45400_g1 IR25a KX084508 937 No BAR64798.1|ionotropic receptor [Ostrinia furnacalis] 1709 0.00E + 00 97% 3
c46878_g3 IR1 KX084509 652 Yes BAR64810.1|ionotropic receptor [Ostrinia furnacalis] 1144 0.00E + 00 87% 3
c47407_g1 IR2 KX084510 657 Yes KOB72397.1|Ionotropic receptor [Operophtera brumata] 662 0.00E + 00 53% 4
c43961_g1 IR3 KX084511 659 NO XP_013194002.1|PREDICTED: glutamate receptor ionotropic, NMDA 2A [Amyelois transitella] 1098 0.00E + 00 78% 4
c47528_g1 IR4 KX084512 548 Yes BAR64809.1|ionotropic receptor [Ostrinia furnacalis] 875 0.00E + 00 77% 3
c51268_g2 IR5 KX084513 488 No BAR64796.1|ionotropic receptor [Ostrinia furnacalis] 756 0.00E + 00 74% 0
c45899_g2 IR6 KX084514 499 Yes BAR64797.1|ionotropic receptor [Ostrinia furnacalis] 781 0.00E + 00 85% 4
c46846_g2 IR7 KX084515 578 Yes BAR64800.1|ionotropic receptor [Ostrinia furnacalis] 805 0.00E + 00 71% 3
c49721_g1 IR8 KX084516 371 Yes BAR64803.1|ionotropic receptor [Ostrinia furnacalis] 451 3.00E−150 60% 2
c12165_g1 IR9 KX096209 162 No KPJ10740.1|Glutamate [NMDA] receptor subunit 1 [Papilio machaon] 258 6.00E−82 98% 1
c43148_g1 IR10 KX096210 133 No ADR64688.1|putative chemosensory ionotropic receptor IR1 [Spodoptera littoralis] 153 8.00E−41 62% 1
c37515_g1 GR1 KX084517 372 No NP_001233217.1|gustatory receptor 68 [Bombyx mori] 194 6.00E−54 37% 8
c42471_g1 GR2 KX084518 333 Yes NP_001233217.1|gustatory receptor 68 [Bombyx mori] 148 4.00E−35 34% 6
c38597_g1 GR3 KX084519 221 No AGA04648.1|gustatory receptor [Helicoverpa armigera] 224 2.00E−66 74% 4
c40152_g1 GR4 KX084520 454 Yes AGK90023.1|gustatory receptor 1 [Helicoverpa assulta] 622 0.00E + 00 72% 6
c45098_g1 GR5 KX084521 427 Yes AGK90012.1|gustatory receptor 5 [Helicoverpa armigera] 444 2.00E−149 53% 6
c30207_g1 GR6 KX096211 156 No XP_013189983.1|PREDICTED: gustatory receptor for sugar taste 64f-like [Amyelois transitella] 214 8.00E−64 73% 3
c16511_g1 GR7 KX096212 170 No KOB74472.1|Gustatory receptor 53 [Operophtera brumata] 230 2.00E−70 71% 3
c127_g1 GR8 KX096213 147 No DAA06380.1|TPA: gustatory receptor 17 [Bombyx mori] 113 8.00E−27 38% 3
c21792_g1 GR9 KX096214 122 No XP_013189983.1|PREDICTED: gustatory receptor for sugar taste 64f-like [Amyelois transitella] 122 5.00E−30 54% 3
c15265_g1 GR10 KX096215 117 No XP_013189983.1|PREDICTED: gustatory receptor for sugar taste 64f-like [Amyelois transitella] 153 2.00E−41 48% 3
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Phylogenetic tree

Phylogenetic tree of ORs was constructed using the sequences of 162 ORs from B. mori, O. furnacalis and C. punctiferalis (Fig. 3). The hits with a length of less than 200 amino acids were ignored. The OR sequences were clustered into PRs, Orco and other divergent ORs. The 8 amino acid sequences, OR1 and OR3-9 clustered into a clade with pheromone receptors of B. mori and O. furnacalis. Orco/OR2 was highly conserved and clustered with Orco/OR2 family. In the neighbor-joining tree of IRs (Fig. 4), the IRs were clustered into ionotropic glutamate receptors (iGluRs), IR25/IR8a and other divergent IRs. Among the nine IRs in C. punctiferalis, CpunIR25a grouped with the highly conserved IR25a/IR8a. Alignment analysis revealed that the similarity of IR25a was higher than 76.3% in D. melanogaster, B. mori, Tribolium castaneum and C. punctiferalis (Fig. 5). CpunIR4, CpunIR6, CpunIR3, CpunIR7 shared identity with IR76b, IR21a, IR68a, IR41a from D. melanogaster, B. mori and T. castaneum, respectively. Unfortunately, we cannot obtain the sequences belong to iGluRs. In phylogenetic tree of the GRs, GRs from B. mori, Manduca sexta and C. punctiferalis were analyzed (Fig. 6), CpunGR5 and CpunGR4 were respectively classified into BmorGr64a and MexGR6 subgroup, which were known as sugar receptors. CpunGR3 was cluster into a clade of fructose receptors.

Figure 3. Phylogenetic relationship of putative olfactory receptors from Conogethes punctiferalis and other insects.

Figure 3

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The tree was constructed by MEGA 5.2 program using the neighbor-joining method with the Bootstrapping model by 1000 replication.

Figure 4. Phylogenetic relationship of putative ionotropic receptors from Conogethes punctiferalis and other insects.

Figure 4

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The tree was constructed by MEGA 5.2 program using the neighbor-joining method with the Bootstrapping model by 1000 replication.

Figure 5. Amino acid alignment of the putative IR25a with other insects.

Figure 5

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Blue shadings indicate the same sequence among insects. Pink shading indicated amino acids which show 75% identity between sequences. Green shading ink shading indicated amino acids which show 50% identity between sequences.

Figure 6. Phylogenetic relationship of putative gustatory receptors from Conogethes punctiferalis and other insect.

Figure 6

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The tree was constructed by MEGA 5.2 program using the neighbor-joining method with the Bootstrapping model by 1000 replication.

Expression level of OR genes in female and male antennae

Quantitative real-time PCR (qPCR) was used to analyze the expression levels of 50 OR genes in the antennae of male and female moths. The results indicated that all the tested genes were detected in antennae. Among the OR genes, OR8, OR39, OR48, OR4 and OR30 were highly enriched in female, with 182.6, 93.9, 84.4, 15.6, and 13.5 times to male, respectively. PR subfamily members, OR1, OR3, OR5 and OR7 were highly expressed in male antennae, as well as the odorant receptor OR62 (Fig. 7). Meanwhile, Orco/OR2, OR26, and OR8 had high expression level in female with an FPKM value of 534.09, 50.52, and 43.43, respectively. Orco/OR2, OR3 and OR6 had high expression level in male with an FPKM value of 523.08, 283.65, 170.90, respectively (Table 3). As co-receptor, Orco/OR2 was most abundant in female and male antennae. Because OR4, OR39, OR48 and OR53 were only detected in female antenna, so we cannot obtain the FPKM value (Table 3).

Figure 7. Relative expression levels of putative ORs in the female and male moth antennae.

Figure 7

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FA: female antennae; MA: female antennae. The expression levels were estimated using delta delta CT method. Standard error for each sample is represented by error bar.

Discussion

With the rapidly development of next generation sequencing technology, a large number of unigenes were identified. RNA-seq effectively increased the depth of sequencing compared to the previous sequence data from cDNA library46,47,48. From the transcriptome of C. punctiferalis, we identified 83 chemoreceptors including 62 ORs, 11 IRs and 10 GRs. In Crambidae family insect, totally 56 ORs were identified in O. furnacalis20, and in other moths, Cao et al. found 47 ORs and 20 IRs in Chilo suppressalis49, suggested that the olfactory related genes we identified were equivalent to other moths. Total number of identified OR genes varied in different orders or species due to sequencing methods and depth, and/or sample preparations. In Lepidopteran, B. mori, totally 66 ORs were reported in genomic data50. In Dipteran, there were 79 candidate OR genes from Anopheles gambiae and in Hemipteran, 170 OR genes from Apis mellifera were annotated18,21. Especially in Nasonia vitripennis, total number of ORs was up to 30151. However, identified OR genes were only obtained from the antennae, ORs expressed in other tissues might be difficult to identify and the physiological states perhaps influence the amount of ORs in the antennae22. It seemed that more OR genes in Hymenopteran insect, especially the parasitoid wasp, might be due to need more receptors were needed to identify and locate the host.

In Lepidopteran insects, the PRs are more conserved and clustered in a subgroup within the OR family9. In our study, OR1 and OR3-9 of C. punctiferalis were clustered with pheromone receptors of B. mori and O. furnacalis, and we inferred that some or all of them might be putative pheromone receptors. But the expression profiles of these sequences showed that not all of them were male-specific. Koenig considered that at least one of the putative PR should exhibit male-specific expression associated with trichoid sensilla9. Recent studies showed that some PR genes expressed in both sexes. The PR1, 2 and 4 identified from the antennae of C. suppressalis have been detected in both male and female antennae49. From the antennal transcriptome of Spodoptera littoralis, four unigenes were enriched in male antennae, only two of which were annotated as putiative PRs and found to be expressed in antennae of both sexes48. In B. mori, Bmor19, Bmor45 and Bmor47 were also reported as female-specific or highly female-biased receptors, which responded to linalool, benzoic acid and benzaldehyde, suggesting the potential roles in detecting oviposition site and male-produced sex pheromone29. In our study, five unigenes were enriched in the female antennae than in the male, which may have provide important function during the host plant seeking.

IRs are derived from ionotropic glutamate receptors and comprise a novel family of chemosensory receptors. In D. melanogaster, 15 of total 66 IR genes were specifically expressed in antennae38 that was similar with our study that only 11 IRs were identified from the antennae transcriptomes of C. punctiferalis. Phylogenetic analysis showed that IRs from C. punctiferalis were not closely related to the iGluRs. Animal iGluRs are highly conserved family of ligand-gated ion channels39, while IRs are extremely divergent exclusive of IR8a and IR25a38. IR25a and IR8a are most similar primary sequence to iGluRs, suggesting that they are the IR genes most similar to the ancestral IRs9,39,52. In our study, CpunIR25a clustered with the highly conserved IR25a from B. mori, D. melanogaster and T. castenum, while in N. vitripennis and Microplitis mediator, two putative IR25a orthologues were identified22,39. Therefore, the congruent function of IR25a orthologues is still not clear. In Lepidoptera insect, IR8a were identified from the antennal transcriptome of Helicoverpa armigera, H. assulta and C. suppressalis26,49. Surprisingly, IR8a was not found in our antennal transcriptome. We speculated that it maybe need more deeply sequencing and increasing the samples, suggesting that IR8a maybe not expressed in our present samples.

In the previous studies, 56 and 77 candidate GR genes were respectively identified from the Drosophila and Acyrthosiphon pisum genome14,53. In B. mori, 65 GR genes were annotated from the silkworm genome54. However, only 10 GRs were identified from the antennal transcriptome of C. punctiferalis. It might be that GRs have a low expression level in the antennae and mainly expressed in gustatory organs (proboscis, legs, wings and genitalis)55,56. GRs have been divided into four classes: non-fructose sugar, bitter, CO2, and fructose receptors9,54. In phylogenetic tree of GRs, the gustatory receptor genes, GR3-5 were clustered into sugar and fructose receptors, suggesting the potential sugar detection function in the antennae of this moth. However, the recent report showed a wide range of non-gustatory sensory functions of GRs57, indicating that GRs may have more divergent functions in insect antennae.

In conclusion, the main purpose of this study was to identify the chemosensory receptor genes involved in the chemoreception. Based on the transcriptomic analysis, 83 CRs were identified from the antennae of C. punctiferalis. Our method was successful in identifying CR genes with low-expressing levels and the results made it possible for further studies on the molecular level and behaviors, providing the possibility to applicate potential target genes for controlling this pest.

Materials and Methods

Insects rearing and antennae collection

C. punctiferalis larvae were collected from the infested sunflower at Langfang Experimental Station of Chinese Academy of Agricultural Sciences, Hebei Province, China. In laboratory, the larvae were raised on fresh corn ear under conditions 27 ± 1 °C, with 70–80% relative humidity (RH) and a photo period of 16:8 h light: dark (L:D). After eclosion, adults were fed with 10% honey solution. The antennae (70 pairs of each sex) from adults within three days after eclosion were separately collected and frozen immediately in liquid nitrogen.

RNA extraction

Total RNAs were separately extracted and purified using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) following the product manuals. The integrity of total RNA was assessed with Agilent 2100 (Agilent Technologies Inc., CA, USA) and the purity was detected on a NanoDrop 2000 spectrophotometer (NanoDrop products, Wilmington, DE, USA). The concentration was accurately measured by Qubit. Illumina sequencing was performed at Novogen Co., Ltd. Beijing, China, The cDNA libraries were sequenced using Illumina HiSeq 4000 system under effective concentration. In order to obtain the high-quality transcriptome sequence data, the raw reads were filtered to eliminate adaptor sequences and low quality reads. Reads with uncertain nucleotides larger than 10% of the fragment sequence were removed. Trinity de novo program with a default k-mer was used to assemble the clean reads45. Redundant sequences were removed to obtain unigenes sequences by means of selecting longest transcript contigs.

Unigenes annotation and classification

To obtain comprehensive information of gene functions, assembled unigenes were searched against Nr (NCBI non-redundant protein sequences), Nt (NCBI nucleotide), Pfam (Protein family), KOG (euKaryotic Ortholog Groups), SwissProt, KEGG (Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology) database using BlASTX with an E-value < 10−5. CDS (coding sequence) were predicted in two-step process, the unigenes were firstly aligned using NR and SwissProt protein database to obtain ORF. If the sequences mismatch with the two databases, estscan (version 3.0.3) software was used to predict the ORF to obtain the nucleic acid and amino acid sequence. FPKM (fragments per kilobase pairs per million mapped reads) was used for gene expression analysis.

Identification of chemosensory receptor genes

Homology searches of OR, GR and IR sequence were performed using BLAST (http://blast.ncbi.nlm.nih.gov/blast.cgi), and the ORFs were predicted using ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Transmembrane domain was predicted using TMHMM (http://www.cbs.dtu.dk/services/TMHMM/). Phylogenetic tree was constructed by MEGA 5.2 software using the neighbor-joining method with the Bootstrapping model by 1000 replication. The evolutionary distances were computed by using the Poisson correction method. The phylogenetic tree image was further created by Figtree software.

Quantitative real-time PCR

The expression levels of ORs were examined in male and female antennae using qRT-PCR. Total RNA was extracted as described above. First-strand cDNAs were synthetize followed One-Step gDNA removal and cDNA Synthesis kit (TransGen Biotech, China) with oligo dT-primer following the kit manual. The primers for qPCR were designed using Primer premier5.0 program (Table S1). qRT-PCR were performed on ABI 7500 fast real-time PCR system (Applied Biosysterm, USA) in a reaction volume of 20 μL using SYBR Premix Ex Taq II (Tli RNaseH Plus) master mix (Takara-Bio, Shiga, Japan), according to the manufacturers’ instructions. Two-step program were performed as follows, 95 °C for 30 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. For each gene, three biological replications were performed with each biological replication measured in three technique replications. Relative quantification was analyzed using the comparative 2−ΔΔCT method, with the housekeeping genes β-actin (accession number JX119014) as the reference gene.

Additional Information

How to cite this article: Ge, X. et al. Identification of putative chemosensory receptor genes from yellow peach moth Conogethes punctiferalis (Guenée) antennae transcriptome. Sci. Rep. 6, 32636; doi: 10.1038/srep32636 (2016).

Supplementary Material

Supplementary Information
srep32636-s1.pdf (173.2KB, pdf)

Acknowledgments

This work was supported by Special Fund for Agro-scientific Research in the Public Interest (201303026) and China Agriculture Research System (CARS-02).

Footnotes

Author Contributions X.G., T.Z. and Z.W. conceived and designed the experimental plan. X.G. preformed the experiments. X.G., T.Z., K.H., S.B. and Z.W. analyzed the sequence data. T.Z. and Z.W. revised the manuscript. All authors read and approved the final manuscript.

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Supplementary Materials

Supplementary Information
srep32636-s1.pdf (173.2KB, pdf)

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