Sexual-biased gene expression of olfactory-related genes in the antennae of Conogethes pinicolalis (Lepidoptera: Crambidae)

doi:10.21203/rs.2.9614/v2

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Sexual-biased gene expression of olfactory-related genes in the antennae of Conogethes pinicolalis (Lepidoptera: Crambidae)

https://doi.org/10.21203/rs.2.9614/v2

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published 19 Mar, 2020

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Background Conogethes pinicolalis (Lepidoptera: Crambidae), is similar to Conogethes punctiferalis (yellow peach moth) and its host plant is gymnosperms, especially for masson pine. So far, less literature was reported on this pest. In present study, we sequenced and characterized the antennal transcriptomes of male and female C. pinicolalis for the first time. Results Totally, 26 odorant-binding proteins (OBP) genes, 19 chemosensory proteins (CSP) genes, 55 odorant receptors (OR) genes and 20 ionotropic receptors (IR) genes were identified from the C. pinicolalis antennae transcriptome and most of them were olfactory genes, amino sequences were annotated against homologs of C. punctiferalis. The neighbor-joining tree indicated that the amino acid sequence of olfactory related genes is highly homologous with C. punctiferalis. Furthermore, the reference genes were selected, and we strongly recommended the GAPDH or RP49 to verify gene expression for larvae development and RP49 or RPL13 for adult tissues. Conclusions Our study provides a starting point on the molecular level characterization between C. pinicolalis and C. punctiferalis, which might be a supportive for pest management studies in future.

Epigenetics & Genomics

Conogethes pinicolalis

Conogethes punctiferalis

yellow peach moth

transcriptomics

OBP

GOBP

PBP

RNA-Seq

Transcriptome

Olfaction system plays a key role in insects, which includes kin recognition, mediating foraging, aggregation, toxic compound avoidance and oviposition behaviors. However, the olfaction is a complex network that contains odorant-binding proteins (OBP), odorant receptors (OR), chemosensory proteins (CSP), sensory neuron membrane proteins (SNMPs), ionotropic receptors (IR) and odorant degrading enzymes (ODEs). They form a functional network with each other in detecting different odorants types, thus complete the odorants recognition process [1, 2]. In Lepidoptera, OBPs are composed of pheromone-binding proteins (PBPs), general odorant-binding proteins (GOBPs) and antennal binding proteins (ABPs), and they combined to detect a wide range of odors and transport hydrophobic odorants to the ORs or IRs [3]. The functions of CSPs are also similar to OBPs, localized in the lymph of trochoid sensilla [4]. IRs or ORs are localized on the dendrite of the chemosensory neuron, which can transform the chemical signals from OBPs or CSPs into an electric signal and transmit to the brain [5, 6]. The SNMPs and ODEs are regarded to trigger ligand delivery to the receptor and terminate the signal stimulation, respectively [6].

Conogethes pinicolalis (Lepidoptera: Crambidae), is a sibling species of Conogethes punctiferalis (Lepidoptera: Crambidae). Morphological features of C. pinicolalis egg, larva, pupa and adult resemble those of C. punctiferalis and it is considered as same species. Koizumi firstly identified and classified the C. pinicolalis as another type of yellow peach moth and commonly known as pinaceae-feeding type (PFT) in 1963 [7]. Later, Honda and Mitsuhashi identified and distinguished the difference between these pests in the adults, larvae and pupal stages [8]; Konno et al. reported that they were different species from their response to different spectra of host-plant constituents [9]; In the end, the pinaceae-feeding type was named as C. pinicolalis in 2006 [10]. Though these studies have provided important information regarding the identification of species, it is not entirely reliable because these insect groups were undergoing speciation, genomic changes, or evolving into new taxon [11]. Therefore, for its high reliability, molecular characterization technique can serve as a complementary method for further analysis. Especially, DNA sequencing and mitochondrial DNA (mtDNA) have been successfully used to deal with the species uncertainty in morphological taxonomy [12-14]. For example, Shashank integration of conventional taxonomy, DNA bar code and others methods successfully confirmed the difference in populations of Conogethes which reared on castor and cardamom in India [11]. Furthermore, Wang et al. used mitochondrial DNA sequencing technique to verify C. pinicolalis and C. punctiferalis were significantly different species [15].

C. pinicolalis is a typical oligophagous pest that can only feed on Pinus massoniana (masson pine) and few pine trees. However, as a sibling species, C. punctiferalis, is a polyphagous pest that can infest hundreds of plants [9, 16]. High-throughput sequencing technology can provide us with a lot of data and it has greatly promoted the research on entomology [17, 18]. In this study, we analyzed the difference of male and female antennae transcriptome and identified the olfactory genes from gene ontology (GO) annotation as well as sets of putative OBPs, CSPs, ORs and IRs in C. pinicolalis. Furthermore, we compared the difference of the gene with C. punctiferalis. These results may help better to understand about these pests and its response to different odorants.

Overall sequence analysis

A total of 78,199,136 and 75,969,652 raw readings were obtained from male and female antennas, respectively. We obtained 77,254,390 and 74,994,240 clean-reads from male and female antennae after trimming adapter sequences, eliminating low-quality reads, and N represented sequences. A total of 98,214 unigenes were obtained with an average length of 815 bp and with a N50 of 2,968 (Table 1).The raw reads of the C. pinicolalis are available from the SRA database (accession number: SRX5250688, SRX5250689, SRX5250690, SRX5250691, SRX5250692 and SRX5250693).

Functional annotation of the C. pinicolalis antennal unigenes

In total. 98,214 unigenes were successfully annotated in all databases (Table 2), including 47,089 (47.94%) unigenes matched to known proteins and 33,852 unigenes (34.46%) in the Swiss-Prot database. GO analysis was used to classify the biological process, molecular function and cellular components (Additional file 1: Figure. S1A). Under the molecular function category, the genes expressed in the antennae were mostly related to binding, catalytic activity and transporter activity (Additional file 1: Figure. S1B). From the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, 10,298 unigenes were classified into five groups, cellular processes, environmental information processing, genetic information processing, metabolism and organismal systems (Additional file 1: Figure. S1C).

Olfactory-related genes in the C. pinicolalis antennae

Totally, 26 OBP genes, 19 CSP genes, 55 OR genes and 20 IR genes were identified from the C. pinicolalis antennae (Additional file 3: Table S1). Among the identified OBP genes, we found 4 PBP, 2 GOBP and 20 other kinds of OBP genes. Furthermore, OBP and CSP genes are highly detected in male and female antennae and showed the significant differences in genes abundance (P < 0.05) (Fig. 1). Interestingly, PBP2, OBP13 and OBP15 are male biased expression, whereas the other PBPs (PBP1, PBP3 and PBP4), as well as GOBPs (GOBP1 and GOBP2) are female bias expression. Furthermore, two of the other OBPs (OBP7 and OBP9) remained female biased expression (Fig. 1A). CSP genes (CSP4, CSP5, CSP14, CSP11 and CSP17) showed female biased expression and significantly different from the male (Fig. 1B), Other insignificantly expressed genes were shown in Additional file 3: Table S1.

In OR gene sets, 7 pheromones receptors (PRs) and 47 other ORs were identified in male and female antennae. Three PR genes (OR1, OR3 and OR6), as well as OR34, showed significantly higher expression in male antennae. However, a large number of ORs (about 18 genes) hadsignificantly higher expression in female antennae. Especially the OR48 and OR53, are highly expressed in female antennae with differential fold change (FC) >5. Six ORs with 2.0 < FC < 5.0 (P < 0.05) and eight ORs with 1.5 < FC < 2.0 (P < 0.05) (Fig. 2A). Three IR genes (IR75p2, IR75d and IR4) showed female biased expression (p<0.05) and other four genes (IR2, IR75p2, IR75p, and IR64a) had a male biased expression (p<0.05) (Fig. 2B).

Significantly expressed genes were confirmed by quantitative real-time PCR (RT-qPCR) (Additional file 2: Figure. S2). Expressions of female biased genes from class OBP (PBP1, PBP3, PBP4, GOBP1, GOBP2, OBP6, OBP7 and OBP9) were extremely consistent with the transcripts per kilobase million (TMP) values.. The same results were obtained in the expression of CSPs, ORs and IRs (Additional file 2: Figure. S2).

Phylogenetic analysis

Phylogenetic trees were constructed using 95 OBPs, 157 ORs, 89 CSPs and 59 IRs sequences from diﬀerent species of Lepidoptera (Fig. 3; Additional file 2: Figure. S3).. The GOBP/PBP genes sequences include six subgroups (GOBP1 and 2, PBP1-4) formed a conserved order (Fig 3). Furthermore, OBPs, CSPs, ORs and IRs showed a very close relationship with C. punctiferlis, only a few CSPs and IRs clustered with other insects (Fig. 3; Additional file 2: Figure. S3). Most of the olfactory related genes showed more than 90% identity. Moreover, 4 OBP, 5 OR, 2 IR and 2 CSP genes had 99% sequence similarity with the C. punctiferlis (Table. 3). ORs and IRs genes indicated the Ostrinia furnacalis is the next close neighbor in the same clade. On the other hand, OBPs and CSPs genes showed Cnaphalocrocis medinalisin in the same clade as a close neighbor after C. punctiferlis. Olfactory-related genes in Bombyx mori showed gene divergence when compared with these two sibling species.

Reference genes selection

The gene stability results obtained from both the software seems to be similar (Fig. 4). In the adult tissues (antanna, head, throax, abdomen, leg and wings) RP49 and RPL13 gene showed more stability than GADPH gene, and Actin gene was unstable (Fig. 4B and D). However, RPL13 performed unstable in different development stages of the C. pinicolalis. The results of GeNorm software showed that Actin and GAPDH are the most stable gene (Fig. 2A); while NormFinder software considered RP49 to be the most stable gene (Fig. 4B).

The application of next-generation sequencing technology in the field of entomology has greatly promoted the efficiency and quantity of gene annotation [19]. Meantime, a lot of antennal transcriptomes olfactory-related genes were identified [20-22]. In this research, we identified 26 OBP genes, 19 CSP genes, 55 OR genes and 20 IR genes from the C. pinicolalis antennal transcriptome, and all the genes are reported for the first time. C. pinicolalis is a sibling species of C. punctiferlis, and has ever been recognized as the same species [10]. In C. punctiferlis, totally 25 OBPs, 15 CSPs, 62 ORs and 10 IRs were identified from antennae transcriptome [23], and the numbers of OBPs, CSPs and ORs are similar with C. pinicolalis, whereas more IRs were identified from the C. pinicolalis antennal transcriptome dataset. The sequence similarity of olfactory-related genes was analyzed and shown in the evolution tree (Fig. 3, Table 3), OBP, CSP, OR and IR genes sequences showed high similarity with C. punctiferlis. Most of the identities are more than 90%, 4 OBP, 5 OR, 2 IR and 2 CSP genes had 99% sequence similarity with the C. punctiferlis (Table 3). These two pests were first identified by Koizumi et al. [7] and classified into pinaceae-feeding type (PFT) and fruit-feeding type (FFT) based on their feeding habits and morphological characters. They were later named as C. pinicolalis and C. punctiferalis [10]. Further investigation revealed their behaviors, morphologies, and feeding patterns, and indicated reproductive isolation between these two types [9,16,18]. Recently, using mitochondrial cytochrome c oxidase subunits I, II and cytochrome b gene sequencing, Wang et al. proved that the C. pinicolalis was different from C. punctiferalis [15]. The phylogenetic tree also revealed an evolutionary relationship with other Lepidopteran species. The GOBP/PBP genes sequences include six subgroups (GOBP1 and 2, PBP1-4) formed a conserved order (Fig. 3). ORs and IRs genes indicated the Ostrinia furnacalis is the next close neighbor in the same clade (Additional file 2: Figure. S3). On the other hand, OBPs and CSPs genes showed Cnaphalocrocis medinalisin in the same clade as a close neighbor after C. punctiferlis. Olfactory-related genes in Bombyx mori showed gene divergence when compared with these two sibling species.

Menken et al [25] suggested the two major transitions in the evolution of larval (Lepidoptera) feeding, switching from litter-feeding to herbivory. Larvae feeding on leaf-litter from a single dominant tree species would have been the main precursor for evolving from litter-feeding to leaf-mining type. In the course of evolution, leaf-mining type gained the new type of enzymatic system to digest the nutritious freshly fallen leaves. Once this evolved niche had been acquired the ability of leaf-mining and with the special digestive system could apparently exploit the diversity more and larval feeding mode had evolved in searching of new host-plants [26]. Insects olfaction system allows them to recognize and track the volatile cues from host-plant, mating and evade from their predators. The polyphagous insects significantly adapted to recognize, digest and detoxify a large variety of host-plants. Polyphagous insects must handle the defensive toxic molecules (secondary metabolites) produced by the host-plant. Genes from the moth pheromone glands could have evolved and altered the normal fatty acid metabolism [27]. In a previous study, experiments proved the major change in the pheromone blend in various moth species, the existence of different desaturase from mRNA in the moth pheromone gland [28]. In Spodoptera frugiperda, due to tandem duplications within a single region of the genome 10 OBP genes expansion was observed when compared with B. mori. In the same study, the author showed a difference in IRs gene count between the strains, S. frugiperda corn strain had 42 IRs and rice strain had 43 IRs [29]. Similarly, in our study C. pinicolalis had 10 more IRs when compared with C. punctiferlis. Evidently, the selection of host plant is also a reason that leads to gene duplications, insertions or deletions when there is a need to adapt to an environment.

As in other insects [30-32] OBPs and CSPs were highly detected in the antennae of both male and female (Additional file 3: Table S1). Among these genes, many of them were sexual biased genes (Fig. 1). PBPs were widely thought to be sex pheromone binding function, normally insects have 3-5 PBP genes. Many research reported at least one of the PBPs could bind well with the sex pheromones [33-35]. In our analysis, PBP2 showed significantly male biased expression, and PBP1, PBP3 and PBP4 showed significantly female biased expression. In male moth, the main assignment is to trail the sex pheromones to find a female moth for mating. We speculated the PBP2 might play a critical role in pheromone binding. Females are often selective when deciding to mate they find a healthy counterpart. GOBP1 and GOBP2 genes, as well as OBP6, OBP7 and OBP9, were also highly expressed in female, this may play some important roles and need for further study. GOBPs are proposed to detect host plants volatiles, food and oviposition sites and PBPs play a key role in detecting sex pheromones [36-38]. However, some studies have demonstrated that GOBPs were also strongly bound with sex pheromones and possibly responsible for conducting the function [39]. Sex pheromones interaction with GOBPs provides us with an important vision, adaptive evolution (gene expansion) might occur in C. pinicolalis. Although the transcriptome of C. pinicolalis and C. punctiferlis possess higher similarity, the C. pinicolalis adult rely on fresh masson pine branches for laying eggs, which the case is very different in C. punctiferlis adult, they have a wide variety of host plants selection. Therefore, both GOBPs and PBPs from C. pinicolalis and C. punctiferlis might have a greater interest in future research.

CSPs were found in insect contact lymph and olfactory sensilla, but members showed peculiar functions. Binding activity of volatile compounds has been described in a similar way as OBPs. In C. pinicolalis antennae transcriptome, we totally identified 19 putative CSPs, and found the transcript per kilobase million (TPM) values of five CSPs (CSP4, CSP5, CSP11, CSP14, and CSP17) were significantly higher in female antennae (Fig. 1B). MsepCSP8 of Mythimna separate was specially expressed in female antennae and showed less sensitive to plant volatiles after RNAi [40]. Also in Locusta migratoria, nearly 17 CSPs abundantly expressed in the female reproductive organs [41]. The above results provide a valuable understanding that CSPs may have an important role in female, especially when it comes to tracking the volatile cues from host-plants and oviposit.

OR or IR genes are responsible for receiving and detecting odor molecules sent by OBP during the recognition process. Totally there were 55 OR genes identified from male and female antennal transcriptome dataset, among them 22 ORs showed a significant difference in TPM ratio (Table S1). In Lepidoptera, OR1 and OR3-8 were identified as pheromone receptors. Our result obviously showed OR1, OR3 and OR6 were specially expressed in male antennae, this may suggest OR1, OR3 and OR6 genes focus on sex pheromones recognition. PRs in other Lepidoptera were reported to bind with sex pheromones [42]. OR34 also performed biased expression in male antennae, but till now, the function is unknown. More numbers of ORs were highly expressed in female antennae (Fig. 2), this is also discovered in mosquitos [43], female need more receptors for host seeking, especially during oviposition. In Bombyx mori, more female biased ORs suggested having function of oviposition cues or male-produced courtship pheromones [44]. This indicated more OR bias in female C. Pinicolalis might provide more receptors for the detection of correct host plants and sex pheromones as well.

We have identified 20 IRs in pinicolalis that is one-fold higher than the IRs reported in punctiferlis. Indeed, the expression of IR may have a certain specificity. For example, there are some IRs were expressed exclusively in Spodoptera littoralis and Helicoverpa armigera [45, 46]. Also, different IR genes were detected in gustatory organs in Drosophila melanogaster [47]. However, in this study the IR gene family from transcriptome data analyzed only from the C. pinicolalis antennae and compared with C. punctiferlis antennal dataset. Based on the transcriptome data analysis, we cannot conclude that there are only 20 (C. pinicolalis) and 11 (C. punctiferlis) [23] IR isoforms in C. pinicolalis and C. punctiferlis antenna. The identified IR isoforms in C. pinicolalis could help to study gene expansion/deletion and existence of other possible IR isoforms in the C. punctiferlis antenna and evolutionary relationship between these two species. NormFinder and geNorm programs are commonly used to screen and optimize the number of internal reference genes for qRT-PCR analysis [48, 49]. At the same time, the difference between reference genes can be compared, but only one optimal gene can be screened when using the NormFinder [50]. In this research, we used both methods to screen the reference gene. The GeNorm result showed Actin and GAPDH were more stable during different development stages of the C. pinicolalis, and NormFinder showed the RP49 as a stable reference gene. This variation may be due to different algorithms coded in this software. Different software were used for calculating the reference gene stability at different developmental stages in the yellow peach moth, RP49 and GAPDH was found to be more stable [51]. Since the expression of the reference gene differs for different developmental stage and tissue, therefore the selection of two or more reference genes is useful to calibrate the expression level of the gene of interest. Cardoso et al. [52] reported three different reference genes (Actin, 60S ribosomal protein L3, RPL13; and peptidylprolyl isomerase, PPI) for different developmental stages in Aphidius gifuensis [53]. Also, Actin, GAPDH and RP49 reported being the most stable reference gene in the Calliphoridae family [54]. According to our results, it is recommended to use GAPDH or RP49 at different developmental stages of the C. pinicolalis. On another hand, ribosomal proteins are involved in translation and protein synthesis, this recommended us to use RP49 and RPL13 for different tissues in yellow peach moth [51]. Similarly, our findings indicate that both RP49 and RPL13 are the best reference genes for the different body part of the adult.

Furthermore, the female bias genes expression level of OBPs (PBP1, PBP3, PBP4, GOBP1, GOBP2, OBP6, OBP7 and OBP9) were verified by RT-qPCR and extremely consistent with the TMP values obtained from the transcriptome dataset. In addition, the fold change expression results of CSPs, ORs and IRs are consistent with the TMP values (Additional file 2: Figure. S2). Therefore, we compared these olfactory-related gene expression levels of C. pinicolalis with C. punctiferalis, reported by Ge xing et al., 2016 [23]. Astonishingly, gene expression pattern reported from C. punctiferalis mostly differs from our study. Noteworthy, most of the ORs (OR2, OR3, OR5, OR6, OR13 and OR15) were significantly expressed in male antenna, whereas in C. punctiferalis the ORs were highly expressed in female antenna. At this point, we suggest these ORs might be functionally active in male moths when comparing with C. punctiferalis males. On the other hand, OBPs (OBP2, 5 and 6) and GOBPs (GOBP1 and 2) expression patterns were similar to that of C. punctiferalis. Exclusively, PBP (PBP1, 2, 3 and 4) genes expression was highly recorded in the C. punctiferalis male antenna [23]. In contrast, PBP1, 3 and 4 genes were significantly expressed in C. pinicolalis female antenna, only PBP3 had a similar expression pattern. However, most of the gene expression patterns of these olfactory-related proteins were different when compared with C. punctiferalis dataset [23], since C. pinicolalis is a monophagous pest that mainly feeds on Masson pines.

We mainly performed a comprehensive analysis of the antennal transcriptome of C. pinicolalis and mined many sexual bias expression olfactory related genes. Meanwhile, transcriptome data analysis revealed that most of the olfactory related genes had more than 90% identity with the C. punctiferlis. Noteworthy, 4 OBP, 5 OR, 2 IR and 2 CSP genes had 99% sequence similarity with its sibling species C. punctiferalis. This study provides a starting point to understand the genetic difference at the molecular level and further intensive studies are required to understand the genetic drift between these two species.

Insects rearing and antennae collection

C. pinicolalis larvae were collected from the masson pine in Quanjiao County (32.07 N 117.54 E), Anhui Province, China. Fresh masson pine branch was used to feed the larvae under ambient conditions 27 ± 0.5 °C, with 70–75% relative humidity (RH) and a photo period of 16:8 h light: dark (L:D). After emergence, the moths were feed on 10% honey solution [55]. Three days old moths were selected from both sex (20 moths/sex) and the antennae were excised for RNA extraction.

RNA extraction and first-strand cDNA synthesis

Total RNA from male and female antennae was isolated using the Quick-RNA^TMMicroPrep Kit (ZYMO Research, USA) according to the manufacturer’s protocol. Ten pairs of antenna was excised from both the sexes. Three biological replicate was maintained (10 pairs/replication). The integrity of the total RNA was analyzed using 1.5% agarose gel electrophoresis [56]. The quality and concentration were analyzed on NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The cDNA was synthesized by following the instructions from RT^TM All-in-One Master Mix Kit (Herogen Biotech, USA). The first strand cDNA synthesis reaction was carried out from 1 μg of total RNA. Anchored oligo (dT) from the kit is used and cDNA was synthesized by following the manufacturer’s protocol. The final cDNA samples were stored at −20 °C until further analysis.

Illumina sequencing

Transcriptome sequencing was performed at Novogen Co., Ltd. Beijing, China, and the RNA samples (including 3 biological replicate) were sequenced on the Illumina Hiseq 4000 platform. The raw reads were curated by removing adaptor sequences and reads of low quality, then assembled into unigenes using Trinity v2.4.0 [57, 58]. 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 reads. Sequences redundancy were minimized using CD-HIT program to obtain longest transcript contigs. Annotation-based metrics was adapted for the study. DESeq2 v1.6.3 was used to calculate the identified candidate genes differential expression levels (log2 fold change, P <0.05).

Unigenes annotation and classification.

The unigenes were searched using BLASTX against the non-redundant (nr) NCBI protein database [59]. Using Blast2Go [60], we predicted and classified functions of unigenes by EuKaryotic of orthologous groups (KOG) database [61]. In addition, the online KEGG Automatic Annotation Server (KAAS) was employed for KEGG pathway enrichment analysis following the procedure pathway annotations for unigenes [62, 63].

Identification of Olfactory genes and phylogenetic analyses

The candidate OBPs, ORs and IRs olfactory genes were analyzed using BLASTX, open reading frames (ORFs) were also identified. Phylogenetic tree based on amino acids of these genes was performed with MEGA7.0 software with the neighbour-joining (NJ) method by 1000 replication.

Analysis of differential gene expression

In order to investigate the expression bias in the antennae of both male and female of C. pinicolalis adults, we compared and reported the transcript abundance in units of TPM in both sexes. In the whole dataset of the transcriptome, we identified the interested candidate genes according to their FC, as assessed using corrected p-value (P) of <0.05 (n = 3). Genes were considered as interesting bias expressed at a FC ≥ 2 and of potential interest if the genes exhibited 1.5 ≤ FC< 2, both with P< 0.05.

Reference genes selection in C. pinicolalis

To obtain the stably expressed gene as a reference gene for quantitative real-time PCR (RT-qPCR) and provide a useful message in C. pinicolalis study, we selected β-actin gene (Actin), glyceraldehyde 3- GAPDH, RP49 and ribosomal protein L13 gene (RPL13) as candidate reference genes based on reference genes in other insect species. The candidate reference gene expression pattern in different development stages (egg, larva, pupa and adult) and the different body part of the adult (antanna, head, throax, abdomen, leg and wings) of the C. pinicolalis were assessed by RT-qPCR. Ct values were evaluated by using the GeNorm and NormFinder method to identify the stable reference gene for specific tissues (Additional file 3: Table S2).

RT-qPCR analysis

The RT-qPCR analysis was performed on select genes to verify the fold changes expression explained in transcriptome data. The primers for RT-qPCR were designed using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) (Additional file 3: Table S3). The primers efficiency was tested by using 10-fold diluted cDNA samples and the standard curve was generated. The Ct values are plotted against the Log of the cDNA dilutions, efficiency percentage and R² values are within the acceptable range [64]. Quantitative PCR was performed using SYBR green (Bestar SybrGreen qPCR Mastermix, DBI Bioscience, Germany), according to manufacturers’ protocol on ABI 7500 Fast (Applied Biosystems, USA) by using the following two-step program: denatured for 2 min at 95 °C followed by 40 cycles: 10 s at 95 °C; 30 s at 60 °C; melting curve analysis was performed from 60 °C to 95 °C to determine the specificity of PCR products. All experiments were repeated independently 3 times in both biological and technical replicates. Three independent biological replicates were maintained for all the sample and four technical replicates were performed form each biological sample. The 2^−ΔΔCT method was used to calculate relative fold change expression [65]. Fold change expression was analysed using t-test, software package SPSS v20.0.

OBPs: odorant-binding proteins; ORs: odorant receptors; CSPs: chemosensory proteins; SNMPs: sensory neuron membrane proteins; IRs: ionotropic receptors; ODEs: odorant degrading enzymes; PBPs: pheromone-binding proteins; PRs: pheromones receptors; GOBPs: general odorant-binding proteins; GO: gene ontology; KOG: EuKaryotic of orthologous groups; KEGG: Kyoto Encyclopedia of Genes and Genomes; Actin:β-actin gene; GAPDH: glyceraldehyde 3-phosphate dehydrogenase gene; RP49: ribosomal protein 49 gene; RPL13: ribosomal protein L13 gene; FC: differential fold change; TPM: transcript per kilobase million; RT-qPCR: Quantitative real-time PCR;

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors approved for publication.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

DJ and TZ conceived of the study and led data management; DJ wrote the paper; DJ and SB analyzed the data and produced the figures; DJ, Kh and JL discussed the results; ZW, TZ and SP revised reviewed the article.

Funding

This research was funded by the National Key Research and Development Program of China (2018YFD0200602) and the China Agriculture Research System (CARS-02). The funding is designed to support the research on odorant binding proteins which plays a key role in tracking the host volatiles and mating. The techniques employed in this study will accelerate the understanding of insect odorant binding proteins as the targets for bioactive semio-chemical molecules in the field of pest control management.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary materials. All Illumina data have been deposited in NCBI’s Sequence Read Archive (SRA) under accession number SRX5250688, SRX5250689, SRX5250690, SRX5250691, SRX5250692 and SRX5250693.

Acknowledgements

Not applicable.

Author details

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
College of Plant Protection, Shenyang Agricultural University, Shenyang 110161, China.

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Table 1 Summary of assembled contigs and unigenes

Type (bp)	Contigs	Unigenes
Total number	121,650	98,214
Total length	160,640,609	154,441,888
Min length	201	201
Mean length	568	815
Maximum length	25,856	25,856
N50	2,825	2,968
N90	467	612

Table 2 Summary of annotations of unigenes

Type (bp)	Number of Unigenes	Percentage (%)
Annotated in NR	47,089	47.94
Annotated in NT	31,124	31.68
Annotated in KO	18,774	19.11
Annotated in SwissProt	33,852	34.46
Annotated in PFAM	37,710	38.39
Annotated in GO	37,882	38.57
Annotated in KOG	19,474	19.82
Annotated in all Databases	8,967	9.13
Annotated in at least one Database	59,764	60.85
Total Unigenes	98,214	100

Table 3 Percentage identity of OBP, OR, IR and CSP gene family in C. pinicolalis with the sibling C. punctiferalis

Gene family

Gene names

C. pinicolalis access No.

C. punctiferalis access No.

Score

E-value

% Identity

Gene family

Gene names

C. pinicolalis access No.

C. punctiferalis access No.

Score

E-value

% Identity

Odorant-binding proteins

OBP2

MK458342

KF026055

306

3e-102

Odorant receptors

OR27

MK458386

KX084477

740

OBP3

MK458343

KF026056

210

5e -67

OR28

MK458387

KX084478

586

OBP4

MK458344

KP985222

278

2e-91

OR29

MK458388

KX084479

734

OBP5

MK458345

KP985223

180

6e-94

OR31

MK458390

KX084481

564

OBP6

MK458346

KP985224

249

2e-79

OR32

MK458391

KX084482

712

OBP7

MK458347

ALC76547

288

2e-95

OR33

MK458392

KX084483

774

OBP8

MK458348

KP985226

193

1e-94

OR34

MK458393

KX084484

444

1e-153

OBP9

MK458349

KY130463

330

2e-112

OR35

MK458394

KX084485

882

OBP10

MK458350

KY130464

251

1e-82

OR36

MK458395

KX084486

409

3e-98

OBP11

MK458351

KY130465

280

4e-89

OR37

MK458396

KX084487

735

OBP12

MK458352

KY130466

221

2e-50

OR38

MK458397

KX084488

657

OBP13

MK458353

KY130467

124

1e-34

OR41

MK458400

KX084491

644

OBP14

MK458354

KY130469

271

2e-34

OR42

MK458401

KX084492

686

OBP15

MK458355

KY130470

297

1e-97

OR43

MK458402

KX084493

581

OBP16

MK458356

KY130472

226

3e-72

OR44

MK458403

KX084494

684

OBP17

MK458357

KY130473

307

3e-104

OR45

MK458404

KX084495

508

3e-175

OBP18

MK458358

KY130474

353

5e-115

OR47

MK458406

KX084497

299

4e-100

OBP19

MK458359

KY130475

252

1e-82

OR48

MK458407

KX084498

437

1e-148

GOBP1

MK458335

KY130468

297

3e-100

OR49

MK458408

KX084499

114

3e-23

GOBP2

MK458336

KT983812

191

4e-57

OR50

MK458409

KX084500

800

PBP1

MK458337

MH006604

192

2e-59

OR51

MK458410

KX084501

647

PBP2

MK458338

KP985228

190

5e-33

OR52

MK458411

KX084502

728

PBP3

MK458339

KP985229

338

1e-100

OR53

MK458412

KX084503

691

PBP4

MK458340

KP985227

329

3e-106

OR54

MK458413

KX084504

853

Odorant receptors

OR1

MK458361

KX084452

890

OR55

MK458414

KX084505

839

OR2

MK458362

KX084453

952

OR56

MK458415

KX084506

690

OR3

MK458363

KX084454

641

ionotropic receptors

IR3

MK458418

KX084511

1299

OR4

MK458364

KX084455

868

IR4

MK458419

KX084512

1057

OR5

MK458365

KX084456

758

IR5

MK458420

KX084513

1484

OR6

MK458366

KX084457

805

IR6

MK458421

KX084514

1348

OR7

MK458367

KX084458

555

IR7

MK458422

KX084515

1089

OR8

MK458368

KX084459

339

3e-110

IR25a

MK458424

KX094508

1797

OR10

MK458369

KX084461

656

5e-165

Chemosenso-ry proteins

CSP1

MK574125

KF026049

154

1e-41

OR11

MK458370

KX084462

683

CSP2

MK574126

KF026050

259

8e-78

OR12

MK458371

KX084463

664

CSP3

MK574127

KY130477

191

1e-60

OR13

MK458372

KX084464

752

CSP4

MK574128

KF026057

226

5e-69

OR14

MK458373

KX084465

798

CSP5

MK574129

KF026058

246

1e-78

OR15

MK458374

KX084466

673

CSP6

MK574130

KF026051

228

1e-67

OR16

MK458375

KX084467

794

CSP7

MK574131

KF026052

201

1e-59

OR17

MK458376

KX084468

790

CSP8

MK574132

KF026053

172

3e-53

OR18

MK458377

KX084469

786

CSP9

MK574133

KY130480

241

5e-78

OR19

MK458378

KX084470

726

CSP10

MK574134

KY130479

197

8e-71

OR20

MK458379

KX084471

729

CSP11

MK574135

KY130480

219

2e-59

OR21

MK458380

KX084472

536

3e-178

CSP13

MK574137

KY130482

206

2e-64

OR23

MK458382

KX084473

730

CSP14

MK574138

KY130483

228

3e-71

OR24

MK458383

KX084474

897

CSP15

MK574139

KY130484

237

4e-76

OR25

MK458384

KX084475

833

Additional file 1: Table S1. Candidate OBPs, CSPs, ORs and IRs genes in Conogethes pinicolalis antennae. Table S2. Candidate reference genes in Conogethes pinicolalis antennae. Table S3. Primers for candidate genes by qRT-PCR.

Additional file 2: Figure S1. A) functional annotation of assembled sequences based on gene ontology (GO) categorization; b) EuKaryotic of orthologous groups (KOG) classification; c) is KEGG pathway annotation of the transcriptome. Figure S2. Quantitative RT-qPCR expression levels of olfactory genes from female and male moth of C. pinicolalis. The expression levels were statistically significant (t-test, *P< 0.05, **P< 0.01, ***P< 0.001, NS: not significant). Figure S3. Phylogenetic relationship of olfactory-related gene from C. pinicolalis and other insects. A: ORs, B: IRs, C: CSPs. Red font represents the genes from C. pinicolalis; Cpun, Ofur, Bmor, Cmed, Ehip, Harm and Mcin are the abbreviation of C. punctiferalis, O. furnacalis, B. mori, Cnaphalocrocis medinalis, Eogystia hippophaecolus, Helicoverpa armigera, Macrocentrus cingulum, respectively.

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Sexual-biased gene expression of olfactory-related genes in the antennae of Conogethes pinicolalis (Lepidoptera: Crambidae)

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