Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

The developmental transcriptome of the bamboo snout beetle Cyrtotrachelus buqueti and insights into candidate pheromone-binding proteins

  • Hua Yang ,

    Contributed equally to this work with: Hua Yang, Ting Su

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Key Laboratory of Ecological Forestry Engineering of Sichuan Province, College of Forestry, Sichuan Agricultural University, Chengdu, China

    ORCID http://orcid.org/0000-0002-7523-5862

  • Ting Su ,

    Contributed equally to this work with: Hua Yang, Ting Su

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization

    Affiliation Key Laboratory of Ecological Forestry Engineering of Sichuan Province, College of Forestry, Sichuan Agricultural University, Chengdu, China

  • Wei Yang ,

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Supervision

    ywei0218@aliyun.com

    Affiliation Key Laboratory of Ecological Forestry Engineering of Sichuan Province, College of Forestry, Sichuan Agricultural University, Chengdu, China

  • Chunping Yang,

    Roles Conceptualization, Investigation, Software

    Affiliation Key Laboratory of Ecological Forestry Engineering of Sichuan Province, College of Forestry, Sichuan Agricultural University, Chengdu, China

  • Lin Lu,

    Roles Conceptualization, Investigation, Software

    Affiliation Key Laboratory of Ecological Forestry Engineering of Sichuan Province, College of Forestry, Sichuan Agricultural University, Chengdu, China

  • Zhangming Chen

    Roles Software

    Affiliation Key Laboratory of Ecological Forestry Engineering of Sichuan Province, College of Forestry, Sichuan Agricultural University, Chengdu, China

The developmental transcriptome of the bamboo snout beetle Cyrtotrachelus buqueti and insights into candidate pheromone-binding proteins

  • Hua Yang, 
  • Ting Su, 
  • Wei Yang, 
  • Chunping Yang, 
  • Lin Lu, 
  • Zhangming Chen
PLOS
x

Abstract

Cyrtotrachelus buqueti is an extremely harmful bamboo borer, and the larvae of this pest attack clumping bamboo shoots. Pheromone-binding proteins (PBPs) play an important role in identifying insect sex pheromones, but the C. buqueti genome is not readily available for PBP analysis. Developmental transcriptomes of eggs, larvae from the first instar to the prepupal stage, pupae, and adults (females and males) from emergence to mating were built by RNA sequencing (RNA-Seq) in the present study to establish a sequence background of C. buqueti to help understand PBPs. Approximately 164.8 million clean reads were obtained and annotated into 108,854 transcripts. These were assembled into 24,338, 21,597, 24,798, 21,886, 24,642, and 83,115 unigenes for eggs, larvae, pupae, females, males, and the combined datasets, respectively. Unigenes were annotated against NCBI non-redundant protein sequences, NCBI non-redundant nucleotide sequences, Gene Ontology (GO), Protein family, Clusters of Orthologous Groups of Proteins/ Clusters of Eukaryotic Orthologous Groups (KOG), Swiss-Prot, and KEGG Orthology databases. A total of 17,213 unigenes were annotated into 55 sub-categories belonging to three main GO categories; 10,672 unigenes were classified into 26 functional categories by KOG classification, and 8,063 unigenes were classified into five functional KEGG categories. RSEM software for RNA sequencing showed that 4,816, 3,176, 3,661, 2,898, 4,316, 8,019, 7,273, 5,922, 5,844, and 4,570 genes were differentially expressed between larvae and males, larvae and eggs, larvae and pupae, larvae and females, males and females, males and eggs, males and pupae, females and eggs, females and pupae, and eggs and pupae, respectively. Of these, three were confirmed to be significantly differentially expressed between larvae, females, and males. Furthermore, PBP Cbuq7577_g1 was highly expressed in the antenna of males. A comprehensive sequence resource of a desirable quality was constructed from developmental transcriptomes of C. buqueti eggs, larvae, pupae, and adults. This work enriches the genomic data of C. buqueti, and facilitates our understanding of its metamorphosis, development, and response to environmental change. The identified candidate PBP Cbuq7577_g1 might play a crucial role in identifying sex pheromones, and could be used as a targeted gene to control C. buqueti numbers by disrupting sex pheromone communication.

Introduction

The bamboo snout beetle Cyrtotrachelus buqueti Guerin-Meneville (Coleoptera: Curculionidae) is widely distributed in China, Vietnam, Burma, Thailand, and other Southeast Asian countries [1, 2]. C. buqueti mainly attacks the shoots of 28 bamboo species, including Bambusa, Dendrocalamopsis, and Dendrocalamus, while the larvae bore into the shoots of clumping bamboo species such as Phyllostachys pubescens, Neosinocalamus affinis, Bambusa. textiles, Bambusa. pervariabilis, and Bambusa. oldhamii [3].

In Sichuan Province, China, nearly 67,000 hm2 of forests are affected by C. buqueti every year. The damage rate is typically 50%–80%, although in severe cases this may reach 100%. C. buqueti is therefore a major forest pest, and the severity of the damage caused has become an important factor restricting the development of bamboo for paper making [4]. In April 2003, the State Forestry Administration of the People’s Republic of China released a list of 156 harmful forest organisms, which included C. buqueti among other pests and harmful mites.

Current studies of C. buqueti are mainly focused on its biological characteristics and chemical control methods. Wang et al. [4] conducted a preliminary study on its reproductive behavior, while Chen et al. [5] analyzed the harmful activity and pest control of C. buqueti. Yang et al. [3] studied the relationship between C. buqueti larval density and wormhole number and bamboo shoot damage. A later study [6] investigated the behavioral and electroantennogram (EAG) responses of C. buqueti adults to host volatiles and their body extracts, revealing that pheromones released by both male and female C. buqueti strongly attract members of the opposite sex, and that the addition of host plants can strengthen the attraction between sexes. On this basis, the main components of bamboo shoot volatiles and EAG responses of C. buqueti to bamboo shoot volatiles were examined [7], and Mang et al. [1] extracted and identified the cuticular semiochemical components of C. buquti adults.

Insect odorant binding proteins (OBPs) are mainly divided into PBPs, general OBPs (GOBPs), and antennae-binding proteins. The main function of PBPs is to bind and transport sex pheromones, while GOBPs may be associated with the general binding of biogenic volatile organic compounds [8]. Because PBPs selectively recognize sex pheromone components of very similar structures [9], they play an important role in the exchange of information between male and female insects and in reproductive isolation.

Researchers have identified several PBPs in a single species that are encoded by different genes [10, 11]. PBP genes have been cloned from Sesamia nonagrioides [12], Bombyx mori [13], Spodoptera litura [14], Spodoptera exigua [15], Antherea polyphemus [16], Leucophea maderea [17], Drosophila melanogaster [18], and Apis mellifera [19], including PBP1, PBP2, and PBP3. The homology of the encoded amino acid sequences is between 32% and 92% [20]. The identification of functional olfactory molecules will also facilitate the development of attractants for baits in management systems.

In the present study, we used RNA sequencing (RNA-seq) to identify developmental stage-specific genes by building transcriptomes of eggs, larvae from the first instar to the prepupal stage, pupae, and adults (females and males) from emergence to mating (3 days old). We identified differentially expressed genes among eggs, larvae, pupae, females, and males by comparative transcriptome analysis. We also screened C. buqueti candidate PBPs because the olfactory system is crucial to sexual communication and reproductive isolation in insects. Finally, differentially expressed candidate PBPs underwent transcriptome data validation.

Material and methods

Insect rearing and collection

Eggs, larvae, and pupae of C. buqueti were collected in July 2015 from in the bamboo planting base of Lushan City, Sichuan Province, China (102°91′N,30°13′E). The field studies did not involve endangered or protected species, and no specific permission was required for the research activity at this location. Pupae were reared in our laboratory at 25°C ± 1°C and 70 ± 10% relative humidity, with a 12L: 12D photoperiod. Adults were used in the experiment 3 days after emergence [21]. Female and male adults were placed on ice and were quickly dissected into the head (without antenna), thorax (without thoracic legs), abdomen, antenna, and thoracic legs for qRT-PCR analysis. All samples were immediately frozen in liquid nitrogen and stored at −80°C until use. Each sample contained eggs, larvae, pupae, males, females and adult tissues from at least five insects, respectively. After mixed sample, three biological replicates were conducted for each treatment.

RNA extraction and sequencing

Eggs, mixed larvae from the first instar to the prepupal stage, pupae, and adults (females and males) from emergence to mating (3 days old) were prepared for RNA extraction. Total RNA was extracted using TRIzol reagent (Qiagen, China Shanghai). The concentration of total RNA was quantified using a spectrophotometer (Implen, Westlake Village, CA), and the RNA integrity was tested using 1.5% agarose gel electrophoresis. After RNA extraction, mRNAs were purified using a Poly A T tract mRNA isolation system and collected using an RNeasy RNA reagent. Mixed mRNAs were fragmented into 300–800 bp pieces using RNA Fragment reagent (Illumina), and the pieces were collected using an RNeasy RNA cleaning kit (Qiagen). Subsequently, RNA fragments were copied into first strand cDNA using MMLV reverse transcriptase (TaKaRa, Dalian Liaoning, Chinese) and random primers. Second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. The Illumina HiSeq2000 system and 100 paired-end reads were used for sequencing. Clean reads were obtained by removing adaptors, low-quality reads, and contaminated reads from raw sequence reads. Statistical analysis of the sequence length was performed to ensure sequence purity.

Assembly and functional annotation

Raw sequence data reads in fasta format were firstly processed through in-house Perl scripts [22]. In this step, clean data (clean reads) were obtained by removing reads containing adapter, ploy-N and low-quality reads form raw read data [23, 24]. At the same time, Q20, Q30, GC-content, and sequence duplication level of the clean data were calculated [24]. All downstream analyses were based on clean high-quality data.

A flow chart of transcriptome assembly as described by Grabherret et al. [25] was used in the present analyses. A Perl pipeline as described by Haas et al. [22] was used for analyzing sequence data. As suggested by Haas et al.[22], if multiple sequencing runs are conducted for a single experiment, these reads may be concatenated into two files in the case of paired-end sequencing. The left files (read 1 files) from all samples were pooled into a single large left.fq file, and right files (read 2 files) into a single large right.fq file. Transcriptome assembly was accomplished based on the left.fq and right.fq using Trinity (http://trinityrnaseq.github.io) with min_kmer_cov set to two by default and all other parameters set default. The assembled unigenes were annotated by BLASTX and ESTScan against Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO, and GO databases (E < 10−5), and the best annotations were selected [23, 24, 26] (S1 Table). Differentially expressed genes were selected by log2 fold change > 1 and q value < 0.005 according the method of DESeq[27]. The nucleotide sequences of each identified PBP gene are listed in S2 Table.

Homology analysis

A neighbor-joining (NJ) tree was constructed with MEGA version 5.0 [28] and the Jones-Taylor-Thornton model. The olfactory gene sequences of other coleopteran insects were first transcribed into their amino acid sequences using the ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Olfactory genes of other coleopteran species were obtained from the NCBI databases. Bootstrap support values were based on 1000 replicates. All of the candidate olfactory genes were named according to the nomenclature system described previously [29, 30].

RT-PCR and qRT-PCR validation

Specific primer pairs were derived from the transcriptome data, and primer pairs for each gene were designed to amplify 100–200 bp products, which were verified by sequencing. A conventional RT-PCR (Bio-Rad S1000, US) analysis was performed for each primer pair using rTaq DNA polymerase (TaKaRa, Dalian, Liaoning, China) before the qRT-PCR (Bio-Rad CFX96, US) analysis to ensure that the correct products were amplified and no primer dimers were present. The qRT-PCR analysis was carried out using an Mx 3000P detection system (Stratagene, La Jolla, CA) as described previously, with thermal cycler parameters of 1 min at 95°C, then 40 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. GAPDH of C. buqueti (GenBank accession number: SAMN06176790) was used as the housekeeping gene. A standard curve was derived from 10-fold serial dilutions of plasmid containing the target DNA segment to determine the PCR efficiency and for quantifying the amount of target mRNA. All primers tested gave amplification efficiencies of 90%–100%. For each treatment, three biological replicates were conducted. qRT-PCR data were analyzed by the 2-ΔΔCT method[31]. The primers used in this experiment were designed with Primer premier 5.0 and Oligo 6.0 and are listed in S3 Table. The qRT-PCR data were analyzed and output as PDF files using Graphpad 5.0.

Results

Illumina sequencing and assembly

Clean reads were obtained from raw reads after the removal of those reads with low quality, adapters, duplicated, and ambiguous. This resulted in a total of 31,469,916, 36,773,825, 32,128,345, 33,070,448, and 31,434,121 clean reads in eggs, larvae, pupae, females, and males of C. buqueti, respectively. All clean reads were assembled into transcripts by Trinity software; the longest copy of redundant transcripts was regarded as a unigene [22, 24, 25]. A total of 108,854 transcripts were obtained and assembled into 83,115 unigenes. Many unigenes were 200–1000 bp in length (Table 1), while approximately 14.7% unigenes exceeded 1000 bp, and 7.2% exceeded 2000 bp (Table 1).

Annotation of unigenes

The assembled unigenes were annotated against NCBI non-redundant protein sequences (Nr), NCBI non-redundant nucleotide sequences (Nt), KEGG Orthology (KO), Swiss-Prot, Protein family (Pfam), Gene Ontology (GO), and Clusters of Eukaryotic Orthologous Groups/Clusters of Orthologous Groups of Proteins (KOG/COG) databases. A total of 24,798 unigenes were annotated in C. buqueti pupae (CP), 24,338 in eggs (CE), 21,597 in larval (CP), 24.642 in male (CM), 21,886 in female (CF), 1,387 in CP-specific, 1,296 in CE-specific, 801 in CL-specific, 1,051 in CM-specific, 735 in CF-specific, 8,989 in Common, 83,115 in CP-CE-CL-CF-CM Combined datasets. (Table 2). The number and percentage of unigenes annotated in these databases were counted. The Nr database had the best match against the CP-CE-CL-CF-CM Combined dataset (21,058, 25.33%) (Table 2), while Swiss-Prot (14,748, 17.74%), Pfam (17,105, 20.57%), and GO (17,213, 20.70%) shared similar quantities (Table 2) (S1S15 Texts).

After functional annotation, the numbers of sequences from different species that matched the bamboo snout beetle unigenes were calculated from the annotation characteristics. As displayed in Fig 1, the five species were Dendroctonus ponderosae (28.8%), Tribolium castaneum (16.4%), Harpegnathos saltator (4.7%), Acromyrmex echinatior (3.7%), and Lasius niger (2.8%), representing around 56% of all the species that were annotated.

Functional annotation results

A total of 17,213 unigenes were annotated into 55 sub-categories belonging to three main GO categories: biological process (BP), cellular component (CC), and molecular function (MF) (Fig 2). There were 25 sub-categories in BP, 20 in CC, and 10 in MF. The top 10 sub-categories were cellular process (10,055 unigenes), metabolic process (9,255 unigenes), single organism process (7,651 unigenes), biological regulation (3,728 unigenes), cell (5,827 unigenes), cell part (5,827 unigenes), organelle (3,914 unigenes), macromolecular complex (3,682 unigenes), binding (9,624 unigenes), and catalytic activity (7,481 unigenes) (S16 Text).

KOG classification placed 10,672 unigenes into 26 functional categories (Fig 3). The cluster of ‘general function prediction only’ was the largest group (1,940 unigenes), followed by ‘signal transduction mechanisms’ (1,362 unigenes), and ‘posttranslational modification, protein turnover, chaperons’ (1,144 unigenes). The top three categories had 41.7% of unigenes annotated to the KOG database (S17 Text).

In total, 8,063 unigenes were classified into five KEGG functional categories (Fig 4): cellular process (1,110 unigenes, 11.44% of which were annotated to the KEGG database), environmental information processing (1,083, 11.16%), genetic information processing (1,762, 18.15%), metabolism (3,739, 38.52%), and organismal system (2,012, 20.73%) (S18 Text). The top three subcategories out of a total of 32 were ‘signal transduction’, ‘translation’, and ‘carbohydrate metabolism’.

thumbnail
Fig 4. Histogram of KEGG classification of unigenes.

a Cellular Processes. b Environmental Information Processing. c Genetic Information Processing. d Metabolism. e Organismal Systems.

https://doi.org/10.1371/journal.pone.0179807.g004

CDS prediction

A total of 21,102 (25.39%) unigenes were predicted via BLASTX with an E-value threshold of 10−5 in the Nr, and the Swiss-Prot database (Figs 5 and 6). Among these, 14,998 unigenes were in the length of more than 300 bp (Fig 5). Furthermore, 17,703 (21.30%) unigenes were then predicted using ESTScan, which identified 3,415 unigenes of more than 300 bp in length (Fig 6).

thumbnail
Fig 5. Length distribution of unigenes predicted protein coding sequence (CDS) using BLAST.

https://doi.org/10.1371/journal.pone.0179807.g005

thumbnail
Fig 6. Length distribution of assembled unigenes predicted protein coding sequence (CDS) using ESTScan.

https://doi.org/10.1371/journal.pone.0179807.g006

Differentially expressed genes

A total of 7,273, 3,661, 4,570, and 5,844 genes were differentially expressed between pupae and males, pupae and larvae, pupae and eggs, and pupae and females, respectively, with 1,484 common to pupae, males, larvae, eggs, and females (Fig 7A). A total of 8,019, 3,176, 4,570, and 5,922 genes were differentially expressed between eggs and males, eggs and larvae, eggs and pupae, and eggs and females, respectively, with 1,110 common to eggs, males, females, pupae, and larvae (Fig 7B). A total of 4,316, 5,922, 5,844, and 2,898 genes were differentially expressed between females and males, females and eggs, females and pupae, and females and larvae, respectively, with 580 common to females, males, eggs, pupae, and larvae (Fig 7C). A total of 4,316, 8,019, 7,273, and 4,816 genes were differentially expressed between males and females, males and eggs, males and pupae, and males and larvae, respectively, with 2,043 common to males, females, eggs, pupae, and larvae (Fig 7D). A total of 4,816, 3,176, 3,661, and 2,898 genes were differentially expressed between larvae and males, larvae and eggs, larvae and pupae, and larvae and females, respectively, with 539 common to larvae, males, eggs, pupae, and females (Fig 7E) (S19S28 Texts).

thumbnail
Fig 7. Venn diagram of the number of differentially expressed genes in CE, CL, CP, CM, and CF.

CE: C. buqueti eggs, CL: C. buqueti larvae, CP: C. buqueti pupae, CM: C. buqueti males, CF: C. buqueti females.

https://doi.org/10.1371/journal.pone.0179807.g007

More genes were shown to be expressed in eggs than in pupae, in females than in pupae, in females than in eggs, in females than in larvae, in larvae than in pupae, in larvae than in eggs, in males than in pupae, in males than in eggs, in males than in females, and in males than in larvae (2,576, 3,223, 3,080, 1,554, 2,129, 1,737, 4,563, 4,705, 2,706, and 3,266, respectively; Fig 8). Conversely, fewer genes were shown to be expressed in eggs than in pupae, in females than in pupae, in females than in eggs, in females than in larvae, in larvae than in pupae, in larvae than in eggs, in males than in pupae, in males than in eggs, in males than in females, and in males than in larvae (1,994, 2,621, 2,842, 1,344, 1,532, 1,439, 2,710, 3,314, 1,610, and 1,550, respectively; Fig 8).

thumbnail
Fig 8. Volcano plot of differentially expressed genes in eggs, larvae, pupae, males, and females.

a Volcano plot of differentially expressed genes between CE and CP. b Volcano plot of differentially expressed genes between CF and CP. c Volcano plot of differentially expressed genes between CF and CE. d Volcano plot of differentially expressed genes between CF and CL. e Volcano plot of differentially expressed genes between CL and CP. f Volcano plot of differentially expressed genes between CL and CE. g Volcano plot of differentially expressed genes between CM and CP. h Volcano plot of differentially expressed genes between CM and CE. i Volcano plot of differentially expressed genes between CM and CF. j Volcano plot of differentially expressed genes between CM and CL. Splashes represent different genes. Blue splashes means genes without significant different expression. Red splashes mean significantly upregulated genes. Green splashes mean significantly downregulated genes. CE, CL, CP, CM, and CF represent eggs, larvae, pupae, males, and females of C. buqueti, respectively.

https://doi.org/10.1371/journal.pone.0179807.g008

Phylogenetic analysis of candidate PBP

We constructed a phylogenetic tree comparing Cbuq7577_g1 (Gene name: CbuqPBP1) and the olfactory genes from 28 coleopteran insects (Fig 9) (S29 Text). In this tree, the olfactory genes are well separated with strong bootstrap support. Cryptolaemus montrouzieri CmonOBP2 and Colaphellus bowringi CbowOBP26, along with CbuqPBP1, were located on the same clade.

thumbnail
Fig 9. Neighbor-joining tree of CbuqPBP1.

Values indicated at the nodes are bootstrap values based on 1000 replicates; scale bar = 0.1. Cbuq: Cyrtotrachelus buqueti; Rdom: Rhyzopertha dominica; Dhel: Dastarcus helophoroides; Dpon: Dendroctonus ponderosae; Hele: Hylamorpha elegans; Rfer: Rhynchophorus ferrugineus; Acor: Anomala corpulenta; Malt: Monochamus alternatus; Tmol: Tenebrio molitor; Rpal: Rhynchophorus palmarum; Aosa: Anomala osakana; Pjap: Popillia japonica; Hpar: Holotrichia parallela; Hobl: Holotrichia oblita; Tcas: Tribolium castaneum; Darm: Dendroctonus armandi; Cmon: Cryptolaemus montrouzieri; Cbow: Colaphellus bowringi; Lory: Lissorhoptrus oryzophilus; Bhor: Batocera horsfieldi; Bpra: Brachysternus prasinus; Haxy: Harmonia axyridis; Aruf: Anomala rufocuprea; Asch: Anomala schonfeldti; Hpic: Heptophylla picea; Pdiv: Phyllopertha diversa; Aoct: Anomala octiescostata; Acup: Anomala cuprea; Eori: Exomala orientalis. The olfactory genes from different species were marked with different colors (S32 Text).

https://doi.org/10.1371/journal.pone.0179807.g009

Expression profiles of pheromone-binding proteins

We identified 19 candidate PBPs through the Nr database (nucleotide sequences are listed in S30 Text). Of these, significant differences in expression profiles were identified in 13 candidate PBPs in male adults and larvae (Table 3), 10 candidate PBPs in female adults and larvae (Table 4), and three candidate PBPs in female and male adults (Table 5).

Validation of transcriptome data

To validate the transcriptome result, we selected 12 significant differentially expressed genes from Tables 35 for quantitative reverse transcriptase-PCR (qRT-PCR) confirmation. Six PBPs transcripts which have demonstrated by RNA-seq to be enriched in larvae were confirmed by qRT-PCR (Fig 10) (S31 Text). Additionally, Cbuq7577_g1 had significantly higher transcriptional level in male than in female and larvae with 5.36 and 85.19 fold exchanges. Moreover, to further explore tissue- and sex-specific expression, we selected Cbuq7577_g1 for qRT-PCR confirmation. We observed the highest expression of PBP Cbuq7577_g1 in male antennae, followed by male heads, compared with low levels of expression in female antennae and heads (Fig 11).

thumbnail
Fig 10. qPCR results of differentially expressed genes in larvae, male and female adults.

The expression levels of the mix-aged larvae, male and female adults were showed by black, red and green bar, respectively by the results of 2-ΔΔCT method with three biological repeats. Sub-caption A to L indicate the identified different expressed genes between the larvae, male and female adults (A: Cbuq12614_g1 B: Cbuq16395_g1 C: Cbuq25979_g1 D: Cbuq29237_g1 E: Cbuq37516_g1 F: Cbuq74007_g1 G: Cbuq67219_g1 H: Cbuq7577_g1 I: Cbuq85742_g1 J: Cbuq97376_g1 K: Cbuq97535_g1 L: Cbuq74056_g1)

https://doi.org/10.1371/journal.pone.0179807.g010

thumbnail
Fig 11. Tissue- and sex-dependent expression patterns of Cbuq7577_g1.

The expression levels of Cbuq7577_g1 in various tissues are shown for males (green) and females (red) based on the 2-ΔΔCT method for three biological repeats.

https://doi.org/10.1371/journal.pone.0179807.g011

Discussion

Overview of transcriptome data

The transcriptome is the complete set of expressed RNA transcripts in one or more cells [32], and its analysis enables the study of gene transcription and the characteristics of transcriptional regulation. High-throughput sequencing technology has been applied to the transcriptome study of many species in class Insecta, such as Phyllotreta striolata [33], D. melanogaster [34], Biston betularia [35], Aedes aegypti [36], Brugia malayi [37], and Bemisia tabaci [38].

In the present study, developmental transcriptomes were established of C. buqueti eggs, mixed-age larvae, pupae, and male and female adults, providing a relatively comprehensive gene pool. The numbers of clean reads from egg, larval, pupal, female and male transcriptomes were 31,469,916, 36,773,825, 32,128,345, 33,070,448, and 31,434,121, respectively. All these clean reads were assembled into 108,854 transcripts by Trinity software. A total of 83,115 unigenes were annotated by Nr, Nt, KO, Swiss-Prot, PFAM, and KOG/COG. Thousands of differentially expressed genes were identified, which facilitates developmental and evolutionary studies of C. buqueti, and contributes to future work in bamboo snout beetle comparative genomics.

Pheromone-binding proteins

Previously reported physiological functions of PBP include: binding specificity, transporting specific pheromone molecules, and filtering odorant molecules entering the sensor chamber [39]; acting as a carrier to transport pheromones through the hemolymph to the receptor [40]; forming the PBP–pheromone complex for receptor recognition [41], cascade initiation, and deactivation to restore receptor sensitivity [42]; and protecting pheromones from enzymatic degradation [41]. Identifying the developmental transcriptome of C. buqueti provides an opportunity to understand the physiological function of PBPs. A total of 19 candidate PBPs were identified in the present study.

The alignment of CbuqPBP1 and 27 known coleopteran insect olfactory gene sequences, and a phylogenetic tree indicated that CbuqPBP1, CmonOBP2 and CbowOBP26 are on the same clade. Therefore, it was speculated that such genes may have the same ancestral gene, but were differentiated by adaptation to different types of environmental chemical factors during evolution, and perform the same or similar functions among different species.

Thirteen candidate PBPs in male adults and larvae (Table 3), 10 candidate PBPs in female adults and larvae (Table 4), and three candidate PBPs in female and male adults (Table 5) showed significant differences in expression, with Cbuq7577_g1 demonstrating significant differences in expression among larvae, male adults, and female adults. Cbuq7577_g1 showed 49% identity with the OBP of Dendroctonus ponderosae (AGI05175), and 47% identity with the OBP of Lissorhoptrus oryzophilus (AHE13794). AGI05175 and AHE13794 were previously functionally annotated as insect pheromone/OBP domains. Cbuq7577_g1 in C. buqueti showed very low similarity to genes in the NCBI database, which likely reflects the limited research that has been carried out into Curculionidae and Lepidoptera PBPs. To research gene function, a PBP gene of C. buqueti (CbuqPBP1) was cloned in this study for prokaryotic expression. Using N-phenyl-1-naphthylamine as the fluorescent probe in a competitive binding assay, the ability of CbuqPBP1 to bind 12 sex pheromone analogs and three volatiles of Neosinocalamus affinis shoots was examined. These results will be published later.

qRT-PCR results of the present study showed that candidate PBP Cbuq7577_g1 in C. buqueti is expressed in male and female adult antennae, which is consistent with the expression pattern of PBPs in most insects, such as Heliothis virescens [43], Manduca sexta [44], Spodoptera exigua [45], and B. mori [46]. PBP expression in adult females may enable the identification of hydrophobic pheromones in the male or the monitoring of pheromones released by the female, as well as transporting pheromones and general odorant molecules. Although it is generally believed that insect PBPs are only expressed in the antennae, researchers have also documented their expression in the head, feet, wings, and other body parts [46, 47]. Zhang et al. [48]found HarmPBP2 of Helicoverpa armigera was expressed in female’s maxillary palp, but the highest expression in the antennae. The candidate PBP Cbuq7577_g1 was mainly expressed in antennae (97.07%). Its expression level in male antennae was 14.43 times that in female antennae. Cbuq7577_g1 may play an important role in the identification of odorant molecules, specifically those involved in identifying females from the external environment through C. buqueti antennae.

Conclusions

We constructed a comprehensive, good-quality sequence resource from the developmental transcriptomes of C. buqueti eggs, larvae, pupae, and female and male adults. This resource enriches what is known about C. buqueti genomics, thus facilitating our understanding of metamorphosis, development, and fitness to environmental change. The identified candidate PBP Cbuq7577_g1 might play a crucial role in identifying sex pheromones, and could be used as a target to control C. buqueti as a pest by disrupting sex pheromone communication.

Supporting information

S29 Text. Deduced amino acid sequences of Cbuq7577_g1.

https://doi.org/10.1371/journal.pone.0179807.s029

(DOCX)

S30 Text. Deduced amino acid sequences of PBP genes.

https://doi.org/10.1371/journal.pone.0179807.s030

(DOCX)

S31 Text. The Genbank number and open reading frame of all identified candidate PBP genes.

https://doi.org/10.1371/journal.pone.0179807.s031

(DOCX)

S32 Text. The dataset and accession number.

https://doi.org/10.1371/journal.pone.0179807.s032

(DOCX)

S1 Table. Transcriptome software and parameters list.

https://doi.org/10.1371/journal.pone.0179807.s033

(DOCX)

S2 Table. Nucleotide sequences of all identified candidate PBP genes.

https://doi.org/10.1371/journal.pone.0179807.s034

(DOCX)

S3 Table. Primer used in RT-PCRs and qRT-PCRs.

https://doi.org/10.1371/journal.pone.0179807.s035

(DOCX)

Acknowledgments

We acknowledge Novogene (Beijing, China) for its assistance in original data processing and related bioinformatics analysis. We are extremely grateful to the members of our laboratory for the collection of materials. The materials used in this work were supported by the Sichuan Provincial Key Laboratory of Ecological Forestry Engineering, Sichuan Agricultural University, Chengdu, China.

References

  1. 1. Mang DZ, Luo QH, Shu M, Wei W. Extraction and identification of cuticular semiochemical components of Cyrtotrachelus buqueti Guerin-Meneville (Coleoptera: Curculionidae). Acta Entomol Sin. 2012;55(3):291–302.
  2. 2. Ju RT, Xiao CH, Xu JH, Xu Y, Chi XZ, Li YZ. Cyrthotrachelus buqueti in Shanghai. Forest Pest and Disease. 2005;24(2):7–9.
  3. 3. Yang YJ, Wang SF, Gong JW, Liu C, Mu C, Qin H. Relationships among Cyrtotrachelus buqueti larval density and wormhole number and bamboo shoot damage degree. Chinese J Appl Ecol. 2009;20(8):1980–85.
  4. 4. Wang WD, Chen FZ, Wang XQ. Reproductive behavior of Cyrtotrachelus buqueti. Sichuan J Zool. 2005;24(4):540–41.
  5. 5. Chen FZ, Wang WD, Wang XQ, Li SH, Gong JW. Occurrence and control of Cyrtotrachelus buqueti Guer. Plant Prot. 2005;31(2):89–90.
  6. 6. Yang H, Yang MF, Yang W, Yang CP, Zhu TH, Huang Q, et al. Behavioral and EAG responses of Cyrtotrachelus buqueti Guerin-Meneville (Coleoptera: Curculionidae) adults to host volatiles and their body extracts. Acta Entomol Sin. 2010;53(3):286–92.
  7. 7. Yang YJ, Qin H, Wang SF, Wang YP, Liao H, Li SG. Antennal ultrastructure and electroantennogram responses of Cyrtotrachelus buqueti Guerin-Meneville (Coleoptera: Curculionidae) to volatiles of bamboo shoot. Acta Entomol Sin. 2010;53(10):1087–96.
  8. 8. Steinbrecht RA L M, Ziegelberger G. Immunolocalization of pheromone-binding protein and general odorant-binding protein in olfactory sensilla of the silk moths Antheraea and Bombyx. Cell Tissue Res. 1995;282(2):203–17.
  9. 9. Hansson B. Olfaction in Lepidoptera. Experientia. 1995;51(11):1003–27.
  10. 10. Picimbon JF, Gadenne C. Evolution of noctuid pheromone binding proteins: identification of PBP in the black cutworm moth, Agrotis ipsilon. Insect Biochem Mol Biol. 2002;32(8):839–46. Epub 2002/07/12. pmid:12110291.
  11. 11. Bette S, Breer H, Krieger J. Probing a pheromone binding protein of the silkmoth Antheraea polyphemus by endogenous tryptophan fluorescence. Insect Biochem Mol Biol. 2002;32(3):241–6. Epub 2002/01/24. pmid:11804795.
  12. 12. de Santis F, Francois MC, Merlin C, Pelletier J, Maibeche-Coisne M, Conti E, et al. Molecular cloning and in Situ expression patterns of two new pheromone-binding proteins from the corn stemborer Sesamia nonagrioides. J Chem Ecol. 2006;32(8):1703–17. Epub 2006/08/11. pmid:16900426.
  13. 13. Forstner M, Gohl T, Breer H, Krieger J. Candidate pheromone binding proteins of the silkmoth Bombyx mori. Invert Neurosci. 2006;6(4):177–87. Epub 2006/11/04. pmid:17082917.
  14. 14. Xiu WM, Zhou YZ, Dong SL. Molecular characterization and expression pattern of two pheromone-binding proteins from Spodoptera litura (Fabricius). J Chem Ecol. 2008;34(4):487–98. Epub 2008/03/19. pmid:18347871.
  15. 15. Xiu WM, Dong SL. Molecular characterization of two pheromone binding proteins and quantitative analysis of their expression in the beet armyworm, Spodoptera exigua Hubner. J Chem Ecol. 2007;33(5):947–61. Epub 2007/03/30. pmid:17393279.
  16. 16. Mohanty S, Zubkov S, Gronenborn AM. The solution NMR structure of Antheraea polyphemus PBP provides new insight into pheromone recognition by pheromone-binding proteins. J Mol Biol. 2004;337(2):443–51. Epub 2004/03/09. pmid:15003458.
  17. 17. Lartigue A, Gruez A, Spinelli S, Riviere S, Brossut R, Tegoni M, et al. The crystal structure of a cockroach pheromone-binding protein suggests a new ligand binding and release mechanism. J Biol Chem. 2003;278(32):30213–8. Epub 2003/05/27. pmid:12766173.
  18. 18. Kruse SW, Zhao R, Smith DP, Jones DN. Structure of a specific alcohol-binding site defined by the odorant binding protein LUSH from Drosophila melanogaster. Nat Struct Biol. 2003;10(9):694–700. Epub 2003/07/26. pmid:12881720;
  19. 19. Lartigue A, Gruez A, Briand L, Blon F, Bezirard V, Walsh M, et al. Sulfur single-wavelength anomalous diffraction crystal structure of a pheromone-binding protein from the honeybee Apis mellifera L. J Biol Chem. 2004;279(6):4459–64. Epub 2003/11/05. pmid:14594955.
  20. 20. Abraham D, Lofstedt C, Picimbon JF. Molecular characterization and evolution of pheromone binding protein genes in Agrotis moths. Insect Biochem Mol Biol. 2005;35(10):1100–11. Epub 2005/08/17. pmid:16102416.
  21. 21. Yang H, Yang W, Yang CP, Cai Y, Pu YF, Fu YW, et al. Mating behavior of Cyrtotrachelus buqueti (Coleoptera: Curculionidae). Acta Entomol Sin. 2015;58(1):60–7.
  22. 22. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc. 2013;8(8):1494–512. Epub 2013/07/13. pmid:23845962;
  23. 23. Pei MS, Niu JX, Li CJ, Cao FJ, Quan SW. Identification and expression analysis of genes related to calyx persistence in Korla fragrant pear. BMC Genomics. 2016;17(1):132–50.
  24. 24. Wang X, Xiong M, Lei C, Zhu F. The developmental transcriptome of the synanthropic fly Chrysomya megacephala and insights into olfactory proteins. BMC Genomics. 2015;16(1):1–12. Epub 2015/01/24. pmid:25612629;
  25. 25. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29(7):644–52. Epub 2011/05/17. pmid:21572440;
  26. 26. Zhang LW, Ming R, Zhang JS, Tao AF, Fang PP, Qi JM. De novo transcriptome sequence and identification of major bast-related genes involved in cellulose biosynthesis in jute (Corchorus capsularis L.). BMC Genomics. 2015;1(16):1062–5.
  27. 27. Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol. 2010;11(10):R106. Epub 2010/10/29. pmid:20979621;
  28. 28. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28(10):2731–9. Epub 2011/05/07. pmid:21546353;
  29. 29. Glusman G, Bahar A, Sharon D, Pilpel Y, White J, Lancet D. The olfactory receptor gene superfamily: data mining, classification, and nomenclature. Mamm Genome. 2000;11(11):1016–23. Epub 2000/11/04. pmid:11063259.
  30. 30. Wang YL, Chen Q, Zhao HB, Ren BZ. Identification and comparison of candidate olfactory genes in the olfactory and non-olfactory organs of elm pest Ambrostoma quadriimpressum (Coleoptera: Chrysomelidae) based on transcriptome analysis. PLoS One. 2016;11(1–28).
  31. 31. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8. Epub 2002/02/16. pmid:11846609.
  32. 32. Etebari K, Palfreyman RW, Schlipalius D, Nielsen LK, Glatz RV, Asgari S. Deep sequencing-based transcriptome analysis of Plutella xylostella larvae parasitized by Diadegma semiclausum. BMC Genomics. 2011;12(2):446. Epub 2011/09/13. pmid:21906285;
  33. 33. He HL, Bin SY, Wu ZZ, Lin JT. Transcriptome characteristics of Phyllotreta striolata (Fabricius) (Coleoptera: Chrysomelidae) analyzed by using Illumina's Solexa sequencing technology. Acta Entomol Sin. 2012;55(1):1–11.
  34. 34. Graveley BR, Brooks AN, Carlson JW, Duff MO, Landolin JM, Yang L, et al. The developmental transcriptome of Drosophila melanogaster. Nature. 2011;471(7339):473–9. Epub 2010/12/24. pmid:21179090;
  35. 35. van't Hof AE, Saccheri IJ. Industrial melanism in the peppered moth is not associated with genetic variation in canonical melanisation gene candidates. PLoS One. 2010;5(5):e10889. Epub 2010/06/09. pmid:20526362
  36. 36. Biedler JK, Tu Z. Evolutionary analysis of the kinesin light chain genes in the yellow fever mosquito Aedes aegypti: gene duplication as a source for novel early zygotic genes. BMC Evol Biol. 2010;10(3):206. Epub 2010/07/10. pmid:20615250
  37. 37. Xiao S, Jia J, Mo D, Wang Q, Qin L, He Z, et al. Understanding PRRSV infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing. PLoS One. 2010;5(6):e11377. Epub 2010/07/09. pmid:20614006;
  38. 38. Wang XW, Luan JB, Li JM, Bao YY, Zhang CX, Liu SS. De novo characterization of a whitefly transcriptome and analysis of its gene expression during development. BMC Genomics. 2010;11(7):400. Epub 2010/06/25. pmid:20573269;
  39. 39. Du G, Prestwich GD. Protein structure encodes the ligand binding specificity in pheromone binding proteins. Biochemistry. 1995;34(27):8726–32. Epub 1995/07/11. pmid:7612612.
  40. 40. Prestwich GD, Du G, LaForest S. How is pheromone specificity encoded in proteins? Chem Senses. 1995;20(4):461–9. Epub 1995/08/01. pmid:8590031.
  41. 41. Klusak V, Havlas Z, Rulisek L, Vondrasek J, Svatos A. Sexual attraction in the silkworm moth. Nature of binding of bombykol in pheromone binding protein—an ab initio study. Chem Biol. 2003;10(4):331–40. Epub 2003/05/03. pmid:12725861.
  42. 42. Kaissling KE. Chemo-electrical transduction in insect olfactory receptors. Annu Rev Neurosci. 1986;9(1):121–45. Epub 1986/01/01. pmid:3518584.
  43. 43. Krieger J, Ganssle H, Raming K, Breer H. Odorant binding proteins of Heliothis virescens. Insect Biochem Mol Biol. 1993;23(4):449–56. Epub 1993/06/01. pmid:8508187.
  44. 44. Robertson HM, Martos R, Sears CR, Todres EZ, Walden KK, Nardi JB. Diversity of odourant binding proteins revealed by an expressed sequence tag project on male Manduca sexta moth antennae. Insect Mol Biol. 1999;8(4):501–18. Epub 2000/01/05. pmid:10620045.
  45. 45. Callahan FE, Vogt RG, Tucker ML, Dickens JC, Mattoo AK. High level expression of "male specific" pheromone binding proteins (PBPs) in the antennae of female noctuiid moths. Insect Biochem Mol Biol. 2000;30(6):507–14. Epub 2000/05/10. pmid:10802242.
  46. 46. Krieger J, von Nickisch-Rosenegk E, Mameli M, Pelosi P, Breer H. Binding proteins from the antennae of Bombyx mori. Insect Biochem Mol Biol. 1996;26(3):297–307. Epub 1996/03/01. pmid:8900598.
  47. 47. Calvello M, Brandazza A, Navarrini A, Dani FR, Turillazzi S, Felicioli A, et al. Expression of odorant-binding proteins and chemosensory proteins in some Hymenoptera. Insect Biochem Mol Biol. 2005;35(4):297–307. Epub 2005/03/15. pmid:15763466.
  48. 48. Zhang S, Zhang YJ, Su HH, Gao XW, Guo YY. Cloning, expression and tissue-specific expression of cDNA encoding pheromone binding protein PBP2 in Helicoverpa armigera (Hübner). Scientia Agricultura Sinica. 2009;42(7):2359–65.