Antennal transcriptome and differential expression of olfactory genes in the yellow peach moth, Conogethes punctiferalis (Lepidoptera: Crambidae)

Abstract

The yellow peach moth (YPM), Conogethes punctiferalis (Guenée), is a multivoltine insect pest of crops and fruits. Antennal-expressed receptors are important for insects to detect olfactory cues for host finding, mate attraction and oviposition site selection. However, few olfactory related genes were reported in YPM until now. In the present study, we sequenced and characterized the antennal transcriptomes of male and female YPM. In total, 15 putative odorant binding proteins (OBPs), 46 putative odorant receptors (ORs) and 7 putative ionotropic receptors (IRs) were annotated and identified as olfactory-related genes of C. punctiferalis. Further analysis of RT-qPCR revealed that all these olfactory genes are primarily or uniquely expressed in male and female antennae. Among which, 3 OBPs (OBP4, OBP8 and PBP2) and 4 ORs (OR22, OR26, OR44 and OR46) were specially expressed in male antennae, whereas 4 ORs (OR5, OR16, OR25 and OR42) were primarily expressed in female antennae. The predicted protein sequences were compared with homologs in other lepidopteran species and model insects, which showed high sequence homologies between C. punctiferalis and O. furnacalis. Our work allows for further functional studies of pheromone and general odorant detection genes, which might be meaningful targets for pest management.

Antennal transcriptome and moth, Conogethes punctiferalis (Lepidoptera: Crambidae)

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Xiao-Jian Jia, Hai-XiangWang, Zeng-GuangYan, Min-Zhao Zhang, Chun-HuaWei, Xiao-Chun Qin, Wei-Rong Ji, Patrizia Falabella & Yan-Li Du

Conogethes punctiferali C. punctiferalis , and , , and , , and C. punctiferalis and O. furnacalis

The yellow peach moth Conogethes punctiferalis (Guene) is a kind of multivoltine and polyphagous insect pest, distributed in the south eastern Asia and Australia1,2. The adult female feeds, oviposits and develops primarily in buds and fruits of peach, plum, chestnut, maize and sunowers2. Aer hatching, larvae remain within the reproductive structures of the host plant and use them as food sources and a protected habitat to complete their life cycle. The endophytic behavior of larvae makes this insect difficult to control with conventional insecticides and other cultural practices. Thus, new methods to monitor C. punctiferalis population outbreaks and to achieve pest control have been initiated35. For example, sex pheromone composites of C. punctiferalis have been analyzed, synthetized and made into lure to attract male moths and disrupt their mating in elds1,57. At the same time, attention has been given to host plant volatiles usable to synergize response to sex attractant pheromone in the yellow peach moth4,8.

In insects, chemosensation serves to detect and react to environmental chemical cues, in virtually every aspect of their life cycle9,10. Olfaction, as a kind of chemosensation, is critical to food source identication, predator avoidance, oviposition site selection, kin recognition, mate choice, and toxic compound avoidance. It is, thus, an attractive target for pest control, for example, several olfactory-based strategies including mass trapping and mating disruption have been developed to control moth populations11. Better knowledge on the molecular

Beijing Key Laboratory for Agricultural Application and New Technique, College of Plant Science and Technology, College of Forestry, Shanxi Agricultural University, Taigu, State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Dipartimento di Scienze, Universit della Basilicata,

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Figure 1. The size distribution of the assembled unigenes from Conogethes punctiferalis male and female antennal transcriptome. A total of 47,109 unigenes were generated. Among which, 19,765 (41.96%) were longer than 500bp and 12,129 (25.75%) were longer than 1kb. The x-axis represents the unigene length (bp), and the y-axis represents the number of unigenes.

mechanisms by which an odor generates a neuronal signal could lead to the identication of targets for the development of new control strategies.

Antennae are the primary olfactory sensor of insects and their cuticular surface is covered with several different types of small sensory structures, named sensilla, in which olfactory receptor neurons extend dendrites into the antennal lymph where peripheral olfactory signal transduction events occur. Previous studies reported diverse olfactory proteins, including odorant-binding proteins (OBPs), odorant receptors (ORs), chemosensory proteins (CSPs), sensory neuron membrane proteins (SNMPs), ionotropic receptors (IRs) and odorant degrading enzymes (ODEs) involved in dierent odor perception steps in signal transduction pathway1214. OBPs are widely engaged in the initial biochemical recognition steps in insect odorant perception and play a key role in transporting hydrophobic odorants across the sensillum lymph to the ORs15,16. Recently, OBPs have attracted the attention of many researchers1719. OBP family notably includes two sub-families: the pheromone-binding proteins (PBPs), transporting pheromone molecules, and the general odorant-binding proteins (GOBPs), transporting general odorants such as plant volatiles20,21. As to the procedures of olfaction transmission, the volatile hydrophobic molecules are rstly bound by the sensilla-enriched binding proteins (OBPs and CSPs) to cross the aqueous sensillum lymph that embeds the olfactory neuron dendrites, thus interacting with the membrane-bound chemosensory receptors (ORs and IRs) located in the dendritic membrane of receptor neurons22. The chemical signal is then transformed into an electric signal that is transmitted to the brain. Sensory neuron membrane proteins (SNMPs), located in the dendritic membrane of pheromone sensitive neurons, are thought to trigger ligand delivery to the receptor. Subsequently, signal termination may then be ensured by ODEs14,21,2325.

Lepidopteran species have been widely used as models of insect olfaction because of their highly specic and sensitive olfactory senses and complex olfactory behaviors. The emergence of next-generation sequencing (high-throughput deep sequencing) technology has dramatically improved the efficiency and quantity of gene annotation26. Similarly, application of the high-throughput sequencing technology in the eld of entomological research has greatly promoted its progress2729. Recently, studies on antennal transcriptomes have led to the identication of olfactory-related genes in several moth species18,19,28,3032, which demonstrated the power of transgenomic strategies for olfactory gene identication. However, in C. punctiferalis, only two olfactory-related genes (CpunOrco and CpunPBP1) with their expression proling were reported to date33,34. Hence, little is known about the function of olfactory genes of C. punctiferalis, due to the deciency of the genomic data for this species.

In this study, we used next generation sequencing (NGS) to gain insights into the complexity of the antennal transcriptome and to identify genes related to chemosensation of C. punctiferalis. We also report the results from gene ontology (GO) annotation as well as sets of putative OBPs, ORs and IRs in C. punctiferalis. Moreover, using real-time quantitative-PCR (RT-qPCR), we screened all the annotated olfactory genes from C. punctiferalis antennal transcriptomes. The results will be the basis for further studies of the olfactory mechanisms of C. punctiferalis and to select some of the olfactory genes that may be used as targets in management programs of this destructive insect pest.

Results and Discussion

Two non-normalized cDNA libraries (SRR2976624 and SRR2976631) of the male and female C. punctiferalis antennae were constructed. Aer a trimming of adaptor sequences, contaminating or low quality sequences, 70.3 and 74.2 million clean-reads comprised of 8.88 and 9.34 gigabases were generated from male and female antennae respectively, and remained for the following assembly.

All clean reads from male and female antennae were assembled and a total of 47,109 unigenes were generated. The transcript dataset was 41.82 mega bases in size and with a mean length of 887.83 bp and N50 of 1,808 bp. Among these unigenes, 19,765 (41.96%) were longer than 500 bp and 12,129 (25.75%) were longer than 1 kb (Fig.1 and Table1). Compared with the published Lepidoptera antennal transcriptomes, especially

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Length (bp) Contig Transcript Unigene

201300 10,879,481 (99.69%) 24,147 (23.82%) 17,266 (36.65%)

301500 12,435 (0.11%) 18,792 (18.54%) 10,078 (21.39%)

5011000 9,011 (0.08%) 20.661 (20.38%) 7,636 (16.21%)

10012000 6,688 (0.06%) 20.364 (20.09%) 6,467 (13.73%)

2000+ 5,710 (0.05%) 17,402 (17.17%) 5,662 (12.02%)

Total Number 10,913,325 101,366 47,109

Total Length 467,980,625 116,001,455 41,824,959

N50 Length 45 2,031 1,808

Mean Length 42.88 1144.38 887.83

Table 1. An overview of the sequencing and assembly process.

Annotated databases unigene 300bp 1000bp

COG_annotation 5,076 4,676 3,444

GO_annotation 10,411 8,900 5,826

KEGG_annotation 4,931 4,495 3,119

SwissProt_annotation 11,489 10,399 7,245

nr_annotation 18,924 15,781 9,224

Total 18,990 15,803 9,228

Table 2. Functional annotation of the Conogethes punctiferalis. COG= Cluster of Orthologous Groups of proteins; GO= Gene Ontology; KEGG= Kyoto Encyclopedia of Genes and Genomes; nr=non-redundant protein.

the two Crambidae species Chilo suppressalis (66,560 unigenes, mean length 761bp, N50 1,271bp)35 and Ostrinia furnacalis (37,687 unigenes, mean length 818bp, N50 1,022bp)18, the assembly quality of our transcriptome was qualied and even better than most of these transcriptomes. These results further demonstrated the eectiveness of Illumina sequencing technology in rapidly capturing a large portion of the transcriptome, and provided a sequence basis for future studies, such as rapid characterization of a large portion of the transcriptome and better reference of the genes of interest36. The assembled sequences have been deposited in the NCBI Transcriptome Shotgun Assembly (TSA) Database with the title as BioProject: PRJNA304355 and accession numbers GEDO01000001 to GEDO01000068.

C. punctiferalis The unigenes were annotated by aligning with the deposited ones in diverse protein databases including the National Center for Biotechnology Information (NCBI) non-redundant protein (nr) database, the Kyoto Encyclopedia of Genes and Genomes (KEGG), the UniProt/Swiss-Prot, Gene Ontology (GO), Cluster of Orthologous Groups of proteins (COG) and the UniProt/TrEMBL databases, using BLASTx with a cut o E-value of 105 (Table2). The analyses showed that a total of 18990 unigenes (40.31%) were successfully annotated in all above-mentioned databases. Of which, 18924 unigenes (40.17%) had signicant matches in the nr database, followed by 11489 unigenes (24.39%) in the Swiss-Prot database. However, 28119 unigenes (59.69%) were unmapped in these databases. The higher percentage of sequences without annotation information could be attributable to the insufficient sequences in public databases for phylogenetically closely related species to date37. For example, in the two published Crambidae antennal transcriptomes, the ratio of the unigenes annotated in nr database in C. suppressalis35 and O. furnacalis18 was 45.4% and 41.2% respectively, similar to the results in present study. On the other hand, short reads obtained from sequencing would rarely be matched to known species because the signicance of the BLAST comparison depends in part on the length of the query sequence37. In the present study, more than one third (36.65%) unigenes were shorter than 300bp, which might be too short to allow for statistically meaningfully matches. As to sequences longer than 1 kb, the annotation rate was 76.08%, whereas for sequences longer than 300 bp, the percentage decreased to 52.95% (Tables1 and 2). In addition, the low annotated percentage might be due to non-conserved areas of proteins where homology is not detected38,39. For example, the 5 ends of genes generally show less sequence conservation than the body40. Therefore, partial transcripts, especially unigenes representing the 5 CDS, may not nd matches in the various databases.

For GO analysis, a total of 10411 unigenes (22.10%) could be assigned to three ontologies, including biological process ontology, cellular components ontology and molecular function (Fig.2). In biological process ontology, the metabolic process and cellular process were most represented, with 5853 (22.64%) and 5651 (21.85%) uni-genes, respectively. In the cellular component ontology, the terms were mainly distributed in cell (3337 unigenes, 19.59%) and cell part (3364 unigenes, 19.74%). In the molecular function ontology, the terms binding functions (5546 unigenes, 41.27%) and catalytic activity (5120 unigenes, 38.10%) were the most represented. These results were also similar to those found in the antennal transcriptomes of Manduca sexta30, Spodoptera littoralis41, and Agrotis ipsilon42.

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Figure 2. Functional annotation of assembled sequences based on gene ontology (GO) categorization. GO analysis was performed at the level two for three main categories (cellular component, molecular function, and biological process).

Figure 3. Cluster of orthologous groups (COG) classication. In total, 5076 of the 47109 unigenes with non-redundant database hits were grouped into 25 COG classications.

In addition, all unigenes were subjected to a search against the COG database for functional prediction and classication (Fig.3). As result, a total of 5076 unigenes with hits in the nr database could be assigned to COG classication and divided into 25 specic categories. The category of general function prediction, similarly to that found in Dialeurodes citri36, was also the largest group (1517 unigenes, 29.89%), followed by the classication of replication, recombination and repair (785 unigenes, 15.46%). The categories of cell motility (11 unigenes, 0.22%) and nuclear structure (3 unigenes, 0.06%) were the smallest groups.

The unigenes metabolic pathway analysis was also conducted using the KEGG annotation system. This pro

cess predicted a total of 197 pathways, which represented a total of 4931 unigenes.

A total of 68 olfactory genes, including 15 OBPs, 46 ORs and 7 IRs, were identied from antennal transcriptome of

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Unigene reference

RPKM Value

male female

Gene name

ORF

(bp) Accession number BLASTx annotation Score E-value Identify

Unigene_32154 OBP1 456 GEDO01000008.1 gb|AGM38610.1|odorant binding protein

[Chilo suppressalis] 156 5e45 51% 2.78 3.96

Unigene_24192 OBP2 417 GEDO01000009.1 gb|AFG73000.1| odorant-binding protein 2

[Cnaphalocrocis medinalis] 251 3e84 84% 497.19 506.90

Unigene_26427 OBP3 384 GEDO010000010.1 gb|AFG72998.1| odorant-binding protein 1

[Cnaphalocrocis medinalis] 235 6e78 84% 0.15 0.14

Unigene_33249 OBP4* 435 GEDO010000011.1 gb|AGP03455.1| odorant-binding protein 9

[Spodoptera exigua] 111 3e29 50% 7.80 2.12

Unigene_32695 OBP5 522 GEDO010000012.1 gb|AER27567.1| odorant binding protein

[Chilo suppressalis] 191 1e59 62% 812.60 2059.91

Unigene_11213 OBP6 420 GEDO010000013.1 gb|AGI37362.1| general odorant-binding

protein 3 [Cnaphalocrocis medinalis] 228 4e75 80% 6061.33 6056.06

Unigene_34662 OBP7 837 GEDO010000014.1 gb|AER27567.1| odorant binding protein

[Chilo suppressalis] 290 8e44 49% 1273.48 687.08

Unigene_25150 OBP8* 417 GEDO010000015.1 gb|AGI37366.1| general odorant-binding

protein 2 [Cnaphalocrocis medinalis] 226 2e74 88% 15576.65 4538.34

Unigene_33044 PBP1 483 GEDO010000018.1 gb|AGS46557.1| pheromone binding

protein 1 [Maruca vitrata] 257 8e86 75% 5223.90 8946.71

Unigene_31490 PBP2* 510 GEDO010000019.1 gb|BAG71419.1|pheromone binding

protein [Diaphania indica] 249 1e82 74% 44872.29 2143.21

Unigene_29089 PBP3 570 GEDO010000020.1 gb|ACF48467.1| pheromone binding

protein female 1 [Loxostege sticticalis] 186 1e57 70% 5402.32 4224.34

Unigene_33607 PBP4 486 GEDO010000021.1 gb|AGI37368.1| pheromone binding

protein 4 [Cnaphalocrocis medinalis] 224 4e73 69% 2130.20 1742.68

Unigene_37211 GOBP1 522 GEDO010000016.1 gb|AFG72996.1| general odorant binding

protein 1 [Cnaphalocrocis medinalis] 243 7e80 83% 2121.17 7257.42

Unigene_33256 GOBP2 483 GEDO010000017.1 gb|AIN41151.1| general odorant-binding

protein 2 [Maruca vitrata] 311 5e107 91% 19358.14 33411.03

Unigene_34301 ABP 432 GEDO010000022.1 gb|AAL60415.1| antennal binding protein 4

[Manduca sexta] 206 2e66 67% 172.80 305.29

Table 3. Candidate OBP genes in Conogethes punctiferalis antennae.

C. punctiferalis. Analysis of gene expression dierences at a single time indicated that the antennal transcriptomes of male and female C. punctiferalis were dierent, mainly distributed in the expression of 1308 genes. Using female antennae as the reference standard, we found 759 up-regulated genes and 549 down-regulated genes. Among which, 3 OBPs (OBP4, OBP8, PBP2) and 4 ORs (OR22, OR26, OR44, OR46) are male antennae-specic expression, whereas 4 ORs (OR5, OR16, OR25, OR42) are female antennae-enriched expression.

Candidate odorant binding proteins in the C. punctiferalis In the antennal transcriptome of C. punctiferalis, a total of 15 OBP genes, including four pheromone binding proteins (PBPs), two general odorant binding proteins (GOBPs) and one antennal binding protein (ABP) were identied (Table3). The BLASTx results indicated that all of these 15 identied CpunOBPs shared a typical structural feature of OBPs (i.e. having typical six conserved cysteins) with other insects43 and twelve of them shared relatively high amino acid identities (6291%) with Lepidoptera OBPs at NCBI. Thirteen of these presented intact ORFs with lengths ranging from 384bp to 837bp, and the other two genes, CpunPBP1 and CpunABP, were represented as partial ORFs with length 483bp and 432bp, respectively.

Among the 15 putative OBP genes in the C. punctiferalis antennal transcriptome data, the gene of CpunPBP1 has been reported in our previous study34, but the remaining 14 CpunOBPs are reported here for the rst time. The number of C. punctiferalis OBPs was less than those identied from the antennal transcriptome of Bombyx mori (44)17, Helicoverpa armigera (26)44, Dendrolimus houi (23)45, O. furnacalis (23)18 and Spodoptera litura (38)19, but comparable with those identied in M. sexta (18)30 and more than those identied in Spodoptera exigua (11)46. Since we used the same methods and technologies reported for previously cited papers we hypothesized the possible reasons of the small number of OBPs identied in C. punctiferalis in actually less number of OBPs than other caterpillar or that some OBPs may be larvae-biased ones, some species-specic ones and some ones that low expressed in antennae. For example, some of the genes might be expressed only in the larva47,48.

The RPKM value analysis revealed that 12 OBP genes (OBP2, OBP5, OBP6, OBP7, OBP8, PBP1, PBP2, PBP3, PBP4, GOBP1, GOBP2 and ABP) were highly expressed in both male and female antennal transcriptomes (RPKM value much higher than 100). The other 3 OBP genes (OBP1, OBP3 and OBP4), however, showed a relative low expression level (RPKM ranged from 0 to 8). Six OBPs (OBP4, OBP7, OBP8, PBP2, PBP3 and PBP4) showed a higher RPKM in the male antennae than in the female antennae (about 1 to 20 times) (Table3).

Furthermore, RT-qPCR analysis was performed to compare the accurate quantitative expression levels of these OBP genes among dierent tissues between sexes (Fig.4). The results indicated that three OBPs (OBP4, OBP8 and PBP2) were signicantly overexpressed in male antennae and have male antennae-specic expression, which suggests that these OBPs may play essential roles in the detection of sex pheromones. Comparatively, the expression of 2 GOBPs (GOBP1, GOBP2) in female antennae were almost twice to three times higher than those in male antennae)

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Figure 4. Conogethes punctiferalis OBP transcript levels in dierent tissues as measured by RT-qPCR.

MA: male antennae; FA: female antennae; MB: male body with antennae cut o; FB: female body with antennae cut o. The internal controls -actin was used to normalize transcript levels in each sample. The standard error is represented by the error bar, and the dierent letters (ac) above each bar denote signicant dierences (p<0.05).

(Table3, Fig.4), which suggests that these OBPs may play important roles in the detection of general odorants such as host plant volatiles. Especially, three OBPs (OBP5, PBP1 and ABP) showed somewhat higher RPKM in the female antennae than in the male antennae (Table3), lack concordance with the results of RT-qPCR (Fig.4), which maybe the sequencing depth of Hiseq2500 is not good enough, or may need more repetition to further test in the future study.

In addition, the RT-qPCR results showed that all of the 15 C. punctiferalis OBPs were signicantly overex-pressed in the antennae compared with the bodies (P< 0.05) (Fig.4). The result of high expression in antennae was not only concordant with that from RPKM values in present study, but also same as that in Anopheles gambiae10, H. armigera44, Ips typographus and Dendroctonus ponderosae49, Ag. Ipsilon42 and Sp. Litura19. For the body parts with antennae cut o, no signicant dierence appeared between male and female OBP gene expression levels, excepting OBP7 and PBP1 signicantly overexpressed in the male body, whereas ABP overexpressed in female body. Up regulation in antennae indicate their participation in moth olfaction during attraction to the host plants and may oer targets for disrupting this activity.

A neighbor-joining tree of 126 OBP sequences was built from six dierent Lepidoptera species, including C. punctiferlis, O. furnacalis, B. mori, H. armigera, Ag. ipsilon and Sp. exigua (Fig.5). The OBP trees indicated that the six Lepidoptera species were extremely divergent; however, the GOBPs (GOBP1 and GOBP2) were highly conserved among dierent species. All PBPs, GOBPs, and OBPs from C. punctiferlis were grouped into corresponding

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Figure 5. Neighbor-joining dendrogram based on protein sequences of candidate odorant binding proteins (OBPs). The protein names and sequences of OBPs used in this analysis are listed in Supplementary Table 3.

branches except CpunPBP3 clustered with OBP group. No evident specic expansion of OBP lineages was found except CpunOBP5 and CpunOBP7 were grouped together.

C. punctiferalis In the process of recognizing smells, insect ORs are the most important players in sex pheromone and general odorant detection. In this research, the OR candidates from the C. punctiferalis antennal transcriptomes were identied carefully, and a total of 46 ORs (including the full-length or almost full-length OR candidates) were submitted for further analysis. Of which, ten ORs (OR2, 10, 17, 19, 21, 22, 23, 25, 30 and 32) had intact ORF, whereas the other 36 ORs were represented as partial open reading frames. In addition, 45 of these submitted 46 ORs were rst report in C. punctiferalis and identied as typical ORs, whereas one OR (OR23) has been reported and was identied as atypical coreceptor33

(Table4). The number of C. punctiferalis ORs identied in this study was comparable with the numbers identied in M. sexta (47)30, H. armigera (47)44 and Ag. ipsilon (42)42, and more than those identied in Sesamia inferens(39)32, Dendrolimus houi (33) and Dendrolimus kikuchii (33)45, but less than those identied in B. mori (72)17 and O. furnacalis (56)18. Considering that those OR candidates with partial ORFs were discarded in the present study, we speculated that more ORs may be identied in the future.

The RPKM value analysis revealed that the ORco (OR23) had the highest expression level among the 46 ORs, with RPKM value of 320 and 531 in the male and female antennae, respectively. The other 45 typical ORs, however, showed a relative low expression level (RPKM ranged from 0 to 233) compared with the ORco (OR23) and OBP genes. In detail, ve ORs (OR17, OR22, OR26, OR44 and OR46) showed a higher RPKM in the male antennae than in the female antennae (more than 10 times), whereas OR16 and OR42 showed opposite results, with RPKM from the male antennae almost 20 times lower compared to female antennae (Table4). The RT-qPCR results indicated that ORco (OR23) had a signicant higher expression level in the antennae than in the bodies of C. punctiferalis, which was concordant with previous results33. Moreover, 4 ORs (OR22, OR26, OR44 and OR46)

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Gene name

ORF

(bp) Accession number BLASTx annotation Score E-value Identify

RPKM Value

Male Female

Unigene

Unigene_10429

OR1

555

GEDO010000024.1

gb|BAR43480.1| putative olfactory receptor 38 [Ostrinia furnacalis]

173

3e48

45%

1.36

1.11

Unigene_35486 OR2 1203 GEDO010000025.1 gb|NP001157210.1| olfactory

receptor 17 [Bombyx mori] 334 3e107 44% 10.88 17.06

Unigene_11235

OR3

420

GEDO010000026.1

gb|BAR43481.1| putative olfactory receptor 39 [Ostrinia furnacalis]

99

1e21

39%

0

1.51

Unigene_38154

OR4

1170

GEDO010000027.1

gb|BAR43452.1| putative olfactory receptor 10 [Ostrinia furnacalis]

471

4e161

59%

13.85

26.63

Unigene_6365

OR5*

501

GEDO010000028.1

gb|NP001296037.1| odorant receptor 13a-like [Plutella xylostella]

172

2e48

54%

0.4

1.76

Unigene_47068

OR6

282

GEDO010000029.1

gb|ALM26253.1| gustatory receptor 3, partial [Athetis dissimilis]

155

7e45

77%

0.73

0.60

Unigene_31536

OR7

1185

GEDO010000030.1

gb|AGK90020.1| olfactory receptor 17 [Helicoverpa assulta]

462

1e157

64%

10.3

19.54

Unigene_37424

OR8

1164

GEDO010000031.1

gb|.XP0143628661| odorant receptor 46a, isoform A-like [Papilio machaon]

483

6e166

63%

4.37

13.62

Unigene_21797

OR9

978

GEDO010000032.1

gbXP013186820|.1| gustatory and odorant receptor 22-like [Amyelois transitella]

580

0.0

89%

0.84

1.33

Unigene_39046

OR10

1296

GEDO010000033.1

gb|BAR43467.1| putative olfactory receptor 25 [Ostrinia furnacalis]

672

0.0

79%

7.4

8.04

Unigene_39333 OR11 1272 GEDO010000034.1 gb|NP001103476.1| olfactory

receptor 35 [Bombyx mori] 388 7e128 52% 16.46 25.88

Unigene_34286

OR12

1161

GEDO010000035.1

gb|BAR43487.1| putative olfactory receptor 45 [Ostrinia furnacalis]

422

4e142

57%

6.53

10.24

Unigene_33960

OR13

1230

GEDO010000036.1

gb|ALM26238.1| odorant receptor 53 [Athetis dissimilis]

454

3e154

55%

1.40

5.45

Unigene_35288

OR14

1368

GEDO010000037.1

gb|BAR43460.1| putative olfactory receptor 18 [Ostrinia furnacalis]

626

0.0

73%

5.94

11.87

Unigene_41196

OR15

201

GEDO010000038.1

gb|BAR43490.1| putative olfactory receptor 48 [Ostrinia furnacalis]

142

9e40

97%

0

1.42

Unigene_30767

OR16*

1322

GEDO010000039.1

gb|BAR43476.1| putative olfactory receptor 34 [Ostrinia furnacalis]

412

7e137

51%

0.36

13.29

Unigene_36352

OR17

1245

GEDO010000040.1

gb|BAR43461.1| putative olfactory receptor 19 [Ostrinia furnacalis]

290

1e89

40%

16.96

1.18

Unigene_33377

OR18

1245

GEDO010000041.1

gb|BAR43468.1| putative olfactory receptor 19 [Ostrinia furnacalis]

525

0.0

65%

1.01

4.15

Unigene_36402

OR19

1269

GEDO010000042.1

gb|BAR43488.1| putative olfactory receptor 46 [Ostrinia furnacalis]

706

0.0

79%

5.08

9.87

Unigene_35705

OR20

807

GEDO010000043.1

gb|BAR43491.1| putative olfactory receptor 49 [Ostrinia furnacalis]

401

4e136

68%

3,71

6.37

Unigene_33043

OR21

1281

GEDO010000044.1

gb|ADB89183.1| olfactory receptor 6 [Ostrinia furnacalis]

315

2e99

44%

8.43

11.63

Unigene_32177

OR22*

1239

GEDO010000045.1

gb|BAR43471.1| putative olfactory receptor 29 [Ostrinia furnacalis]

474

2e161

62%

197.27

4.26

Unigene_39439

OR23

1422

GEDO010000046.1

gb|AFG29886.1| odorant co-receptor [Conogethes punctiferalis]

951

0.0

99%

319.92

531.37

Unigene_35755

OR24

1206

GEDO010000047.1

gb|BAR43452.1|olfactory receptor 10 [Ostrinia furnacalis]

424

1e142

56%

1.37

2.80

Continued

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Gene name

ORF

(bp) Accession number BLASTx annotation Score E-value Identify

RPKM Value

Male Female

Unigene

Unigene_22804

OR25*

1245

GEDO010000048.1

gb|ADB89180.1| olfactory receptor 3 [Ostrinia furnacalis]

308

1e96

37%

0.57

1.98

Unigene_29130

OR26*

1278

GEDO010000049.1

gb|AIT71991.1| olfactory receptor 22 [Ctenopseustis obliquana]

303

1e94

43%

233.32

4.77

Unigene_33708

OR27

1005

GEDO010000050.1

gb|BAR43475.1| putative olfactory receptor 33 [Ostrinia furnacalis]

533

0.0

82%

3.95

5.46

Unigene_36273 OR28 1170 GEDO010000051.1 gb|AII01045.1| odorant

receptor [Dendrolimus houi] 354 2e115 44% 2.08 5.45

Unigene_9909 OR29 306 GEDO010000052.1 gb|BAJ61939.1| odorant

receptor [Ostrinia nubilalis] 75.1 1e13 41% 0 1.84

Unigene_34694

OR30

1203

GEDO010000053.1

gb|BAR43467.1| putative olfactory receptor 25 [Ostrinia furnacalis]

410

3e137

51%

7.64

12.86

Unigene_29284

OR31

1224

GEDO010000054.1

gb|BAR43494.1| putative olfactory receptor 52 [Ostrinia furnacalis]

491

1e168

56%

1.99

6.58

Unigene_35553

OR32

1224

GEDO010000055.1

gb|BAR43494.1| putative olfactory receptor 52 [Ostrinia furnacalis]

508

4e175

61%

3.33

5.20

Unigene_31835

OR33

984

GEDO010000056.1

gb|BAR43484.1| putative olfactory receptor 42 [Ostrinia furnacalis]

414

2e140

63%

1.30

4.47

Unigene_37901

OR34

1260

GEDO010000057.1

gb|BAR43458.1| putative olfactory receptor 16 [Ostrinia furnacalis]

502

2e172

60%

5.95

16.00

Unigene_30980

OR35

1230

GEDO010000058.1

gb|KOB74670.1|Odorant receptor 50 [Operophtera brumata]

479

6e164

53%

1.46

3.59

Unigene_32663

OR36

1194

GEDO010000059.1

gb|BAR43480.1| putative olfactory receptor 38 [Ostrinia furnacalis]

411

1e137

49%

11.15

14.14

Unigene_32039 OR37 1188 GEDO010000060.1 gb|NP001166611.1| olfactory

receptor 59 [Bombyx mori] 363 1e118 47% 20.16 42.48

Unigene_30358

OR38

1167

GEDO010000061.1

gb|BAR43453.1| putative olfactory receptor 11 [Ostrinia furnacalis]

494

1e170

68%

6.25

6.33

Unigene_35167

OR39

1248

GEDO010000062.1

gb|BAR43456.1| putative olfactory receptor 14 [Ostrinia furnacalis]

548

0.0

64%

8.24

22.00

Unigene_29815

OR40

1203

GEDO010000063.1

gb|BAR43469.1| putative olfactory receptor 27 [Ostrinia furnacalis]

647

0.0

86%

7.23

8.41

Unigene_34345

OR41

1215

GEDO010000064.1

gb|BAR43481.1| putative olfactory receptor 39 [Ostrinia furnacalis]

248

4e74

34%

5.11

10.70

Unigene_34297

OR42*

969

GEDO010000065.1

gb|AII01110.1|odorant receptor [Dendrolimus kikuchii]

386

4e129

53%

0.05

9.88

Unigene_37409

OR43

1275

GEDO010000066.1

gb|NP001292415.1|odorant receptor 13a-like [Plutella xylostella]

307

3e96

40%

6.69

17.05

Unigene_33544

OR44*

1200

GEDO010000067.1

gb|BAR43461.1| putative olfactory receptor 19 [Ostrinia furnacalis]

280

4e86

43%

46.14

4.43

Unigene_36203 OR45 1275 GEDO010000068.1 gb|ADB89183.1| odorant

receptor 6 [Ostrinia nubilalis] 342 1e109 42% 9.31 12.66

Unigene_35759

OR46*

1191

GEDO010000069.1

gb|BAR43470.1| putative olfactory receptor 28 [Ostrinia furnacalis]

446

1e150

50%

86.74

4.77

Table 4. Candidate OR genes in Conogethes punctiferalis antennae.

have a male antennae-specic expression, whereas other 4 ORs (OR5, OR16, OR25 and OR42) have a female antennae-enriched expression (Fig.6). This male-biased transcription also appears to be retained among the B. mori orthologs OR3, 4, 5 and 650. Comparative genomic analyses suggested that male-biased expression and female pheromone receptor function is retained in OR subfamily in B. mori, and female-biased transcription of OR gene family members is predicted among transcripts in both B. mori50,51 and O. furnacalis18.

A neighbor-joining tree of 130 OR sequences was built from three dierent Lepidoptera species, including C. punctiferlis, B. mori and O. furnacalis (Fig.7). The ORco (OR23) was clustered with other Lepidoptera ORco

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Figure 6. Conogethes punctiferalis OR transcript levels in dierent tissues as measured by RT-qPCR.

MA: male antennae; FA: female antennae; MB: male body with antennae cut o; FB: female body with antennae cut o. The internal controls -actin was used to normalize transcript levels in each sample. The standard error is represented by the error bar, and the dierent letters (ac) above each bar denote signicant dierences (p<0.05).

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Figure 7. Neighbor-joining dendrogram based on protein sequences of candidate odorant receptor proteins (ORs). The protein names and sequences of ORs used in this analysis are listed in Supplementary Table 4.

sequences (OfurOR2). Most ORs from C. punctiferlis and O. furnacalis appear in pairs on the dendrogram, according with the fact that they belong to the same family of Crambidae. Especially to be mentioned, four male-biased ORs (OR22, OR26, OR44 and OR46) were clustered together with OfurOR4 and OfurOR6, which were suggestive of a functional role in male pheromone response18. However, the female-biased ORs (OR5, OR16, OR25 and OR42) were stretched in dierent branches. Given that several B. mori female-biased ORs are capable to respond to host plant volatiles51,52, it is conceivable that C. punctiferalis orthologs may have retained similar functions, but further studies are required to investigate any potential evolutionary conservation of function. However, based on the dierent expression proles of these ORs in male and female antennae, we suggest that these male antennae-enriched expressed ORs are involved in sex pheromone detection, whereas female antennae-enriched expressed ORs play important roles in locating suitable host plants and oviposition sites.

Candidate ionotropic receptors in the C. punctiferalis IRs were recently discovered as another class of receptors involved in chemoreception53. Since IRs have been identied throughout protostome lineages, they belong to an ancient chemosensory receptor family54. To date, 15 IRs in Cy. Pomonella28, 24 IRs in Ag. Ipsilon42, and 12 IRs in H. armigera44 have been identied. In the present study, 7 IR genes were rst identied from the C. punctiferalis antennal transcriptomes. Among these, two IRs (IR2 and IR6) had intact ORF, whereas the other 5 candidate IRs were represented as partial ORFs. The BLASTx results indicated that all of these 7 identied CpunIRs shared relatively high amino acid identities (6781%) with Lepidoptera IRs at NCBI (Table5). Compared with the number of IRs in above mentioned three species, the scarcity of divergent IRs in C. punctiferalis antennal transcriptomes may due to some IRs only expressed in other tissues. For example, the expression of divergent IRs was detected only in gustatory organs in Drosophila melanogaster53,54. It is generally reported that in insects, the antennal IR subfamily constitutes only a portion of the total number of IRs49. In particular 15 D. melanogaster IRs53, 10 H. armigera IRs44 and 7 S. littoralis IRs55 were expressed exclusively in the antennae.

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Unigene

Gene name

ORF

(bp) Accession number BLASTx annotation Score E-value Identify

RPKM Value

Male Female

Unigene_32271

IR1

1278

GEDO01000001.1

gb|ADR64682.1| putative chemosensory ionotropic receptor IR68a [Spodoptera littoralis]

499

2e167

77%

1.25

1.91

Unigene_39471

IR2

2727

GEDO01000002.1

gb|BAR64796.1| ionotropic receptor [Ostrinia furnacalis]

1493

0.0

81%

14.74

54.32

Unigene_33510

IR3

1899

GEDO01000003.1

gb|BAR64800.1| ionotropic receptor [Ostrinia furnacalis]

874

0.0

71%

1.25

6.29

Unigene_36510

IR4

1923

GEDO01000004.1

gb|BAR64803.1| ionotropic receptor [Ostrinia furnacalis]

821

0.0

68%

7.07

20.83

Unigene_37845

IR5

1926

GEDO01000005.1

gb|BAR64808.1| ionotropic receptor [Ostrinia furnacalis]

994

0.0

78%

2.35

6.38

Unigene_35392

IR6

2556

GEDO01000006.1

gb|BAR64797.1| ionotropic receptor [Ostrinia furnacalis]

1352

0.0

81%

15.61

17.66

Unigene_30586

IR7

1644

GEDO01000007.1

gb|BAR64809.1| ionotropic receptor [Ostrinia furnacalis]

875

0.0

77%

13.63

45.88

Table 5. Candidate IR genes in Conogethes punctiferalis antennae.

The RPKM value analysis revealed almost no dierences between male and female IRs, which was validated by RT-qPCR results (Table5, Fig.8). Therefore we speculated that the IRs were relatively highly conserved. Similarly to the ORs, the RPKM value analysis revealed that all of the 7 IRs showed a relative low expression level (RPKM value ranged from 1 to 54) compared with the OBPs. Our RT-qPCR results also indicated that all of the 7 C. punctiferlis IRs were highly expressed in the antennae. The antennae-enriched IRs may play important roles in odorant detection. The IR tree from four lepidopteran insects was similar to that from ORs, with most of IRs from C. punctiferlis and O. furnacalis appearing in pairs on the dendrogram, concordant with the fact that they belong to the same family of Crambidae (Fig.9).

Conclusion

Olfaction is a primary sensory modality in insects. In the present study we performed a comprehensive analysis of the antennal transcriptome of C. punctiferalis. As a result, three major gene families (OBPs, ORs and IRs) that encode olfactory-related proteins were annotated for the rst time, and their expression levels were measured based on the transcriptomic data, and validated by RT-qPCR. The expression prole analysis revealed that 15 OBPs, 46 ORs and 7 IRs are uniquely or primarily expressed in the male and female antennae. The results from the present study will be fundamental for future functional studies of olfactory-related genes in C. punctiferalis. Connection of the molecular information presented here and the available chemical and ecological knowledge will clarify the olfactory mechanisms of C. punctiferalis, and provide new targets for pest management in the future.

Materials and Methods

The mature larvae of C. punctiferalis were collected from cornelds of the Agricultural Experiment Station of Beijing University of Agriculture on October 9th, 2009, and the insects had been maintained for about 25 generations on maize in climate incubators (RTOP-B, Zhejiang Top Instrument Co., Ltd.) at 231C, RH 75 2%, 16L/8D photoperiod, and 3500 lux light intensity. Adult moths were provided with 58% honey solution aer emergence2. Antennae were excised from 3-days-old male and female moths, frozen immediately and stored in liquid nitrogen until use.

200 antennae from each sex were pooled for total RNA extraction using RNeasy Plus Mini Kit (Qiagen GmbH, Hiden, Germany) following the manufacturers instructions. During which, the DNA could be eliminated automatically. The quantity and concentration of RNA samples were determined using 1.2% aga-rose electrophoresis and a Qubit RNA Assay Kit in a Qubit 2.0 Fluorometer (Life Technologies, CA, USA),

respectively. The integrity of RNA samples was assessed using a RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

Firstly, mRNA was puried from total RNA using Oligo (dT) magnetic beads. mRNA was fragmented in fragmentation buer into 200700 nucleotides sections. The rst cDNA was synthesized using random hexamer primer with the fragmented mRNA as templates. Secondstrand cDNA were synthesized using DNA Polymerase I, dNTPs and RNaseH (Invitrogen, Carlsbad, CA, USA). Short fragments were puried using QiaQuik PCR Extraction Kit (Qiagen, Hilden, Germany) and eluted with ethidium bromide (EB) buer for end-repair, poly (A) addition, then linked to sequencing adapters. The suitable fragments, as judged by agarose gel electrophoresis, were selected as templates for PCR amplication. The cDNA library of

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Figure 8. Conogethes punctiferalis IR transcript levels in dierent tissues as measured by qRT-PCR.

MA: male antennae; FA: female antennae; MB: male body with antennae cut o; FB: female body with antennae cut o. The internal controls -actin was used to normalize transcript levels in each sample. The standard error is represented by the error bar, and the dierent letters (ac) above each bar denote signicant dierences (p<0.05).

Figure 9. Neighbor-joining dendrogram based on protein sequences of candidate ionotropic receptors (IRs). The protein names and sequences of IRs used in this analysis are listed in Supplementary Table 5.

C. punctiferalis was sequenced on Illumina HiSeq 2500 using PE125 technology in a single run by Beijing

Biomake Company.

The raw reads were cleaned by removing adapter sequences, low-quality sequences (reads with ambiguous bases N), and reads with >10% Q < 20 bases. Cleaned reads

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shorter than 60 bases were removed because short reads might represent sequencing artifacts56. The quality reads were assembled into unigenes using short reads assembling program Trinity (Trinityrnaseq_r2013-11-10)57.

The assembled sequences were annotated using BLASTn (version 2.2.14) with an E-value<105 and BLASTx (E-value<105) programs against the NCBI nr database58,59. To annotate the assembled sequences with GO terms, the Swiss-Prot BLAST results were imported into BLAST2GO, a soware package that retrieves GO terms, allowing gene functions to be determined and compared60. The COG database was also used to predict and classify functions of the unigene sequences61. Kyoto Encyclopedia of Genes and Genome (KEGG) pathways were assigned to the assembled sequences using the online KEGG Automatic Annotation Server (KAAS) used to determine pathway annotations for unigenes62. Finally, the best matches were used to identify coding regions and to determine the sequence direction.

All candidate OBPs, ORs and IRs were manually checked by the BLASTx program at the National Center for Biotechnology Information (NCBI). For contigs with hits against genes of interest, open reading frames (ORFs) were identied and the annotation veried OBPs, ORs and IRs protein sequences and orthologs in other species of Lepidoptera and model insects to analyze the characteristics of olfactory genes in C. punctiferalis. The nucleotide sequences of all olfactory genes that were identied from C. punctiferalis antennal transcriptomes were named according to sequence homology analysis and numbered arbitrarily. Of which, the genes of OBP1, OBP2, PBP1, PBP4, GOBP1, GOBP2, and ABP were numbered according to blast results, whereas other OBPs, and all ORs and IRs were numbered arbitrarily. In addition, we use the prex CpunOBP, CpunOR or CpunIR to reect that the gene is a putative member belonging to yellow peach moth OBP, OR or IR-like family (Tables3, 4 and 5).

Phylogenetic reconstruction for analysis of OBPs, ORs and IRs was performed with MEGA5.0 soware63,

with construct consensus phylogenetic trees using neighbour-joining (NJ) method. Bootstrap analysis of 1000 replications was performed to evaluate the branch strength of each tree.

To compare the dierential expression of chemosensory genes in the C. punctiferalis male and female antennal transcriptomes, the read number for each chemosensory gene between male and female antennae was converted to RPKM (Reads Per Kilobase of exon model per Million mapped reads)64. The RPKM method eliminates the inuence of gene length and sequencing depth on the calculation of gene expression, and is currently the most commonly used method for estimating gene expression levels. Thus, the calculated gene expression can be directly used to compare gene expression between samples.

To verify the quantification of gene expression levels in transcriptome sequencing, the RT-qPCR for dierent tissue and sex samples was performed. Two biological samples each with 80 male antennae or 80 female antennae, and another two samples each with one male or one female moth body with antennae cut o, were used for RNA extraction using RNeasy Plus Mini Kit (Qiagen GmbH, Hiden, Germany) following the manufacturers instructions. cDNAs from antennae and other body part of both sexes were synthesized using the SMARTTMPCR cDNA synthesis kit(Clontech, Mountain View, CA, USA).

An equal amount of cDNA (100 ng) was used as RT-qPCR templates. For each sample, the -actin gene (GenBank JX119014) of C. punctiferalis was used as an internal control gene. The primers were designed using the Primer Premier 5.0 program (Primer Bioso International, Palo Alto, CA, USA) (Supplementary Table 2). The RT-qPCR was performed in an iCycler iQ2 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with SYBR green dye bound to double strand DNA at the end of each elongation cycle. Each RT-qPCR reaction was conducted in a 20.0l reaction mixture containing 10.0l of 2 SYBR Green PCR Master Mix, 0.4l of each primer, 2.0l of cDNA sample (100ng/l), and 7.2l sterilized ultrapure H2O. The cycling parameters were: 95C for 3min, 40 cycles at 95C for 10sec, and 60C for 30 sec to measure the dissociation curves. Blank controls with sterilized ultrapure H2O instead of template were included in each experiment. To check reproducibility, each

RT-qPCR reaction for each sample was carried out in three technical replicates and three biological replicates.

The Relative quantication analyses among four samples were performed using comparative 2Ct method65. The comparative analyses of each target gene among dierent tissues were determined with one-way nested analysis of variance (ANOVA), followed by Least-signicant dierence (LSD) test using SPSS Statistics 19.0 (SPSS Inc., Chicago, IL, USA).

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We thank Dr. S. H. Gu for discussion and comments on the manuscript and, J. Wang and D. Y. Li for their help in raising insects. We appreciate S. D. Hao for his help in directing RNA extraction. This work was supported by the Science and Technology Fund of Beijing Municipal Commission of Education (KZ201210020019), and the National Natural Science Foundation of China (41271492).

Author Contributions

Y.L.D. conceived the idea, designed the experiments and directed implementation. X.J.J., H.X.W., M.Z.Z., C.H.W., X.C.Q. and W.R.J. performed the experiments. X.J.J., Z.G.Y. and Y.L.D. analyzed the data. X.J.J., Y.L.D. and P.F. wrote the paper.

Supplementary information accompanies this paper at http://www.nature.com/srep

Competing nancial interests: The authors declare no competing nancial interests.

How to cite this article: Jia, X.-J. et al. Antennal transcriptome and dierential expression of olfactory genes in the yellow peach moth, Conogethes punctiferalis (Lepidoptera: Crambidae). Sci. Rep. 6, 29067; doi: 10.1038/ srep29067 (2016).

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