Identification and characterization of three chemosensory receptor families in the cotton bollworm Helicoverpa armigera
- Nai-Yong Liu†1, 2,
- Wei Xu†1,
- Alexie Papanicolaou†1,
- Shuang-Lin Dong2Email author and
- Alisha Anderson1Email author
© Liu et al.; licensee BioMed Central Ltd. 2014
Received: 31 October 2013
Accepted: 9 July 2014
Published: 15 July 2014
Chemosensory receptors including olfactory receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) play a central role in sensing chemical signals and guiding insect behaviours, and are potential target genes in insect pest control. The cotton bollworm Helicoverpa armigera is one of the most destructive pest species that can feed on over 200 different plant species. This diversity of host plants is likely linked to a complex chemosensory system. Here we built on previous work to characterize crucial chemosensory tissues linked to environmental interactions including larval antennae, larval mouthparts and larval fat bodies, as well as male and female adult heads, male and female adult tarsi, and female abdomens.
Using transcriptome sequencing, Trinity RNA-seq assemblies and extensive manual curation, we identified a total of 91 candidate chemosensory receptors (60 candidate ORs, 10 GRs and 21 IRs). Thirty-five of these candidates present full-length transcripts. First, we performed in silico differential expression analysis on different sequenced tissues. Further, we created extensive expression profiles using reverse transcription (RT)-PCR on a variety of adult and larval stages. We found that the expression profile of HarmOR51 was limited to adult male antenna suggesting a role in mating that was further supported by a phylogenetic analysis clustering it into the pheromone receptor clade. HarmOR51 in calcium imaging analysis did not show responses to either of the two H. armigera sex pheromone components (Z9-16:Ald or Z11-16:Ald) inviting a future detailed study. In addition, we found four novel HarmORs (OR1, 53, 54 and 58) that appeared to be larvae-antennal specific. Finally, our expression profiling showed that four “divergent” HarmIRs (IR2, 7d.1, 7d.2 and 7d.3) were expressed in both adult and larval antennae, suggesting a functional divergence from their Drosophila homologues.
This study explored three chemoreceptor superfamily genes using a curated transcriptomic approach coupled with extensive expression profiling and a more limited functional characterization. Our results have now provided an extensive resource for investigating the chemoreceptor complement of this insect pest, and meanwhile allow for targeted experiments to identify potential molecular targets for pest control and to investigate insect-plant interactions.
The chemosensory system is critical in guiding insect feeding, mating and oviposition behaviours . Hair-like chemosensory sensilla distributed over the surface of chemosensory tissues including antennae, palps, mouthparts and tarsi, are used to detect chemical signals from the external environment [2, 3]. Chemosensory receptors of olfactory receptors (ORs) , gustatory receptors (GRs)  and ionotropic receptors (IRs)  are localized on the dendrite of chemosensory neurons housed in these sensilla. These receptors play a central role in helping insects detect chemical signals and regulate their behaviours , and are also important molecular targets for designing and developing new pest control strategies.
Even though Drosophila melanogaster is the main model insect for these genes [4–6], the availability of new genomic and transcriptomic sequences from other species is consistently extending both the phylogenetic coverage and the number of orthologs identified. The D. melanogaster genome hosts 60 OR genes that encode 62 ORs by alternative splicing . Interestingly, insect ORs are highly diverse, share no sequence similarity and have an inverted membrane topology compared with mammalian OR genes, suggesting that insect ORs are not G-Protein Coupled Receptors (GPCRs) . Drosophila GR genes have been classified into “CO2” , “sugar” , “GR43a-like”  and “bitter” clades . GRs promote insect survival by detecting nutritious compounds and help avoid toxic ones . Like ORs, these receptors share no sequence similarity with vertebrate GRs and their topology is inverted compared to the classic GPCRs [14, 15]. More recently, a variant sub-family of ionotropic glutamate receptors (iGluRs), the ionotropic receptor family, was identified as a new class of chemosensory receptors in Drosophila. On the basis of their expression and sequence characteristics, Drosophila IRs were further distinguished into two sub-families: conserved “antennal IRs” involved in olfaction and species-specific “divergent IRs” that are expressed in other tissues including gustatory organs and may possibly be associated with gustation .
For this work we focused on the cotton bollworm (Helicoverpa armigera Hübner 1809; Lepidoptera: Noctuidae), an economically important species feeding on a wide range of host plants. H. armigera is one of the most polyphagous and cosmopolitan pest species with larvae that feed on numerous important cultivated crops such as cotton, peanuts, soybeans and maize. In a previous study, forty-seven OR genes were identified from H. armigera adult antennal transcriptomes but only one GR and 12 IR genes . Only 22 full-length open reading frames were identified in this study, limiting any functional characterizations of these receptors. This previous transcriptome sequencing was restricted to adult antennae and did not consider other chemosensory tissues. Here, we conducted additional sequencing on other potential chemosensory tissues, including taste organs, not only in adults but also in larvae. Such transcriptomic data greatly improved the description of chemosensory receptors in H. armigera and will also assist in defining gene models for future genome studies.
Indeed, some chemosensory gene families have a low amino acid identity with their homologues and in our experience de-novo gene predictors have a limited capacity in annotating them. Even though genome sequencing will eventually provide a framework for full ascertainment, an extensive manual effort is required to curate complex genes such as GRs and IRs. Moreover, in this work we first used the expression profile and phylogenetic analysis to associate each gene with putative functions, and then characterized the functions of candidate pheromone receptors by the Sf9 calcium imaging technique. In addition, we outlined a path forward for an integrated study of the insect chemosensory system that can proceed solely based on transcriptomic information.
Analysis of H. armigeratranscriptome
We prepared 10 RNA-Seq libraries which after sequencing and quality control provided us with 239,276,681 read pairs of up to 100 bp in length (Additional file 1: Table S1). Our Trinity RNA-Seq assembly produced 68,100 contigs which included redundancy due to alternative transcription or high polymorphism and non-coding regions. After an initial protein prediction using a combination of a 5th order Markov chain and the Pfam database, we created a subset of 30,111 contigs with coding potential and annotated them with controlled vocabulary terms using the Uniprot database. These open reading frames are available via the NCBI BioProject PRJNA244590 (http://www.ncbi.nlm.nih.gov/bioproject/PRJNA244590). The full dataset, annotations such as Gene Ontology, Enzyme Classification, and visualizations can be explored at http://annotation.insectacentral.org/Liu2013. After extensive manual curation, we verified or reconstructed 91 chemosensory receptors.
In silico differential expression profile of candidate H. armigerachemosensory receptors
To provide global gene expression profiles in different sequenced libraries, we surveyed the differential expression of all these open reading frames identified in this study. The complete result dataset is available at the CSIRO Data Portal (http://dx.doi.org/10.4225/08/535DB2C141C8A). As expected, all these genes (94 genes including iGluRs) were detected in at least one library. Most of these genes were expressed in male and/or female heads, where the antenna, proboscis and labial palp of crucial chemosensory organs, are located suggesting a functional role of these genes in olfaction or gustation. Specially, some genes appeared to be expressed only in male and/or female heads. For example, in the GR family HarmGR1; in the IR family HarmIR1, 41a, 75p.2 and 87a; and in the OR family five of seven pheromone receptors including the newly identified pheromone receptor HarmOR51 were detected only in male heads. In summary, from 10 sequenced libraries we found the transcriptomic support of nearly all predicted chemosensory receptor genes.
The H. armigeracandidate olfactory receptors
The H. armigeracandidate gustatory receptors
Expression profiles were investigated with RT-PCR on larval antenna, larval mouthpart, male and female adult head, as well as male and female adult tarsi tissues. All of the sugar, CO2 and GR43a-like receptor genes were detected in the male and female adult heads, HarmGR5 and 7 were expressed in male and female adult tarsi, and HarmGR2, 4 and 8 were expressed only in female adult tarsi. Only HarmGR3 was detected in larval antennae while none of GRs were detected in larval mouthparts (Figure 2).
The H. armigeracandidate ionotropic receptors
Functional studies of candidate H. armigerapheromone receptors in Sf9 cells
In this work we built upon previous studies to investigate an array of tissues that play an important role in insect-plant interactions [28, 29]: larval antennae, larval mouthparts, larval fat bodies, as well as female abdomens, adult tarsi and adult heads with antenna, proboscis and labial palp. Our research interest primarily focused on three crucial repertoires of chemosensory receptor superfamilies (ORs, GRs and IRs) due to their significance as potential target genes for designing and developing new pest control strategies and addressing the insect host-plant interactions. After the extensive sequencing and assembly using Trinity RNA-Seq, we manually curated every transcriptome so that the community now has accessed to a repertoire of 60 ORs, 10 GRs and 21 IRs. We found that H. armigera has more IRs than in any other lepidopteran species reported to date . Further, nine H. armigera GRs were found in the transcriptomic data, although gustatory receptors have been reported to exhibit extremely low expression in gustatory organs [5, 31]. We were also able to fully reconstruct the transcripts of the newly predicted H. armigera chemosensory receptors from sequenced libraries.
In addition to verifying our transcript reconstructions, an extensive sex-, tissue- and stage-specific expression profile using RT-PCR for the newly identified ORs and GRs, as well as all the IRs allowed us to further characterize these genes. We found four larvae-specific HarmORs which may be involved in larvae-specific behaviours. It has been reported that BmorOR56, expressed in silkworm larvae, mediated larval responses to cis-jasmone that may be emitted from mulberry leaves as an attractant . These larvae-specific HarmORs may be candidate targets for future larval control. Phylogenetic analysis showed that our newly identified HarmOR51 was clustered into the pheromone receptor sub-family, and shared a high identity to other noctuid PRs. Thus we believe that it is possibly a new candidate of H. armigera pheromone receptors, leading to a total of seven pheromone receptors reported in a lepidopteran species for the first time.
Further, we used the Sf9 calcium technique to measure the binding of HarmOR13 and 51 to the H. armigera sex pheromone components (Z11-16:Ald or Z9-16:Ald). This system is likely to more closely resemble in vivo H. armigera olfactory receptor functions, since insect Sf9 cells are derived from a lepidopteran species S. frugiperda, and express a native co-receptor (Orco, a highly-conserved OR across different insect taxa) to assist HarmORs in ligand detection. We identified a strong response of HarmOR13 to Z9-16:Ald, as seen in another functional study . The HarmOR51 receptor, however, did not elicit a response to either component tested, and needs to be investigated in a comprehensive future analysis. Although our calcium imaging did not help identify the ligands for HarmOR51, it did work very well in the analysis of HarmOR13 to Z11-16:Ald, suggesting that the assay could be applied in future analyses to seek potential ligands for other ORs.
Four novel divergent HarmIRs were detected in both larval and adult antennae, suggesting a different function to the Drosophila divergent IRs that are not expressed in the antennae of any stages . Our extensive expression profile showed that most HarmIRs were expressed in antennae (at least 19 in adult and 18 in larva), providing a more extensive repertoire of antennal IRs compared to 16 of D. melanogaster [6, 30]. Some HarmIRs were detected in adult proboscis and larval mouthpart, indicating that these IRs may also have a gustatory function as previously indicated in D. melanogaster  and S. littoralis .
In this study, we sequenced crucial chemosensory tissues, and utilized an integrated approach to probe three chemosensory receptor repertoires in our transcriptome. We identified a total of 91 chemosensory receptor genes comprised of 60 ORs, 10 GRs and 21 IRs (an increase of 13 ORs, nine GRs and nine IRs compared to the previous work). In particular, the novel HarmOR51, the orthologous genes of which have been never found in any other lepidopteran species, was identified as a new potential pheromone receptor. We also carried out, for the first time, an extensive sex-, tissue- and stage- expression profile for all H. armigera IRs that showed a widespread expression of HarmIRs in non-antennal tissues, implying that in this species IRs have a more complex function. Together, this study greatly complements the information of chemosensory receptors in H. armigera, and shows the utility of combining transcriptomic, phylogenetic and functional assays to elucidating the function and evolution of the insect chemosensory system.
Insect rearing and tissue collection
H. armigera were fed an artificial diet in the laboratory of CSIRO under conditions described previously . Approximately 200 larval antennae, 200 larval mouthparts and two larval fat bodies were dissected from fifth instar larvae. Five heads with antenna, proboscis and labial palp, 50 tarsi and two female abdomens were collected from 1 to 5-day-old male and female moths. In expression profile studies, all adult tissues were collected from 3-day-old male and female moths. All collected tissues were immediately stored in RNAlater (Invitrogen, USA).
Extraction of total RNA and first-strand cDNA synthesis
Total RNA was purified using RNeasy (Qiagen, USA) or RNAqueous (Ambion, USA) kits according to the manufacturer's protocol. The purified RNA was then treated with DNaseI (Ambion, USA) at 37°C for 30 min along with the manufacturer’s protocol, quantified and qualified by NanoDrop ND-2000 (Thermo Scientific, USA) as well as 2100 Bioanalyzer (Agilent, USA).
cDNA templates were prepared from purified RNA samples of larval and adult tissues using SuperScript™ III Reverse Transcriptase (Invitrogen, USA), according to the manufacturer's manual. Briefly, the first-strand cDNA was synthesized with Oligo(dT)20 primer at 65°C for 5 min, and then at 50°C for 60 min. The reaction was stopped at 70°C for 15 min. The templates were stored at -20°C until use.
Sequencing and transcriptome analysis
The prepared RNA samples from larval antennae, larval mouthparts, larval fat bodies, male adult tarsi, female adult tarsi and female abdomens were sent to BGI-Tech for generating Paired-End Illumina TruSeq libraries and sequencing with an Illumina HiSeq 2000 platform. To help identify the chemosensory genes which are lowly expressed, we further sequenced the sample libraries of male and female adult heads that were treated by using the Duplex‒Specific thermostable nuclease (DSN) enzyme (Evrogen) according to the DSN normalization protocol (Illumina). The RNA samples from male and female adult heads as well as male and female adult heads with DSN treatment were used to prepare Paired-End Illumina TruSeq libraries according to the manufacturer’s protocol and sent to ACRF Biomolecular Resource Facility for Illumina sequencing service (http://brf.anu.edu.au/).
All RNAseq data were then pre-processed using the default settings of Just_Preprocess_My_Reads (http://justpreprocessmyreads.sourceforge.net) which conducted a mild quality control and trimming, pooled and assembled using Trinity-RNASeq using the default settings as per . Trinity RNA-Seq is highly capable of overcoming quality and polymorphism issues due to bubble popping algorithms in each of the three modules, Inchworm, Chrysalis and Butterfly. Open reading frames (ORFs) were predicted using the TranscriptDecoder software in Trinity-RNASeq (http://sourceforge.net/projects/transdecoder) with the Pfam option. The raw sequence data and coding sequences of the transcriptome assembly have been deposited in the National Center for Biotechnology Information (NCBI) (accession number: PRJNA244590; http://www.ncbi.nlm.nih.gov/bioproject/PRJNA244590).
In silico expression profiles were generated using DEW (http://dew.sourceforge.net/) which is an automated pipeline that: 1) used Bowtie2  to align the reads of each library against a sequence file comprised of our assembly; 2) post-processed the alignments with eXpress  to account for isoforms and paralogues; 3) performed Trimmed Mean Normalization using edger and estimated Fragments Per Kilobase Per Million reads (FPKM) ; and 4) visualized the results using ggplot2  and canvasXpress (http://canvasxpress.org/). From these data, certain cutoffs were used to make a true/false decision on whether a gene was expressed: a gene had to have at least four RNA-Seq reads aligned against it and covering at least 0.30 of its length. Functional annotations were performed and visualised using Just_Annotate_My_Proteins (JAMp; http://sourceforge.net/projects/jamps) which is an automated pipelines that 1) used HHblits  to search against Hidden Markov Models derived from the curated Uniprot archive; 2) assigned controlled vocabulary terms (e.g. GO, KEGG etc.) linked to a Uniprot accession only if an actual experiment provides evidence (i.e. we did not use any ‘inference via electronic similarity’ evidence); 3) visualized them using ExtJS4 and canvasXpress. Up to 10 HHblits alignments that passed the following JAMp cutoffs were used: homology probability: >= 80%; homology e-value: <= 1e-10; homology p-value: <= 1e-12; HHblits score: >= 70. These assemblies were then subsequently mined and curated using Geneious 5 to create a non-redundant set of the Helicoverpa chemosensory genes.
Identification of candidate chemosensory receptors
TBLASTN searches were carried out with previously identified H. armigera, B. mori and Drosophila melanogaster ORs [4, 28], GRs [5, 39] and IRs [6, 17]. In order to verify these receptors, amino acid sequences of all identified candidate receptors were searched against NCBI non-redundant protein database using BLASTX based on the identity and similarity to orthologous genes from other insect species. These gene sequences were finally compared to previously described chemosensory receptors  to identify novel genes. All newly identified H. armigera OR, GR, as well as iGluR and IR amino acid sequences are available in GenBank as ACCESSION (Additional file 2: Table S2).
Sequence and phylogenetic analysis
Alignments of amino acid sequences were performed by ClustalW2  and were visualized by Jalview 2.7 . Phylogenetic trees were constructed by PhyML  based on Jones-Taylor-Thornton (JTT) model with Nearest Neighbour Interchange (NNI). Branch support was estimated by approximate likelihood ratio test (Chi2). In the OR data set, we selected the ORs with available genomic databases from B. mori , D. plexippus  and H. melpomene . Due to the small number of HarmGRs, we only selected GRs from two species, a moth B. mori  and a butterfly D. plexippus . In the IR data set, we selected IRs from lepidopteran species including B. mori , C. pomonella , D. plexippus , M. sexta , S. littoralis  and S. nonagrioides  but also IRs and iGluRs from a model insect D. melanogaster . Trees were viewed and edited using iTol [48, 49]. Networks using the protein identity were generated using custom scripts and analyzed with CytoScape  and clusterMarker .
To verify expression of candidate HarmOR, GR and IR genes identified from our transcriptome and to investigate sex- and tissue- expression profiles, RT-PCR was performed using gene-specific primers (Additional file 3: Table S3) as follows: 95°C for 3 min; 35 cycles at 95°C for 30 s, 60°C for 30 s and 72°C for 40 s; and final extension at 72°C for 7 min. H. armigera actin gene (GenBank accession number: X97614) was used as control to check the quality of cDNA templates. For each RT-PCR amplification, negative controls using sterile water as the template were performed. Each gene PCR reaction was repeated at least twice. PCR products were analysed using 1.5% agarose gels. The expected products of the genes randomly selected were sequenced to confirm the identity with their original sequence identified.
The sex pheromone Z11-16:Ald was purchased from Sigma-Aldrich (95% purify), and another component Z9-16:Ald was synthesized from Yick-Vic Chemicals & Pharmaceulicals (Hong Kong, China) (>95% purify). In the calcium imaging experiment, sex pheromones were dissolved in dimethyl sulfoxide (DMSO) as 1 M stock solution and stored at -20°C according to previously reported protocols . For dose-dependent assays, pheromone solutions were diluted from stock solution to the desired concentration with HBSS buffer .
HarmOR13 and HarmOR51 ORFs were cloned with 35 cycles and an annealing temperature of 60°C using gene-specific primers (Additional file 3: Table S3). The PCR products were ligated into pBluescript vector, previously digested by EcoRV. Positive clones were sequenced. The verified plasmids were digested with KpnI and SacII for HarmOR13, and HindIII and XbaI for HarmOR51. And the digested target genes were ligated into PIB/V5-His vector, previously digested by the same enzymes. The constructed plasmids were further sequenced to confirm the orientation and sequence. Next, Sf9 cells were plated into 12-well plates and left to settle for about 20 min. Finally, the cells were transfected with 500 ng of plasmid construct PIB/HarmORs or PIB/V5-His vector (negative control) and 3 μL of Fugene HD transfection reagent (Promega, USA) in 100 μL of medium per well. After 48 h post-transfection, the methods of cell treatment and functional assay were the same as previously described [14, 53]. All data were analyzed by GraphPad 5.0.
Availability of supporting data
All supporting data are included as additional files. The data of transcriptome assembly were deposited in the National Center for Biotechnology Information (NCBI) (accession number: PRJNA244590; http://www.ncbi.nlm.nih.gov/bioproject/PRJNA244590). The transcriptome dataset and annotations can be found at http://annotation.insectacentral.org/Liu2013. The dataset of gene expression profiles is available at the CSIRO Data Portal (http://dx.doi.org/10.4225/08/535DB2C141C8A). Phylogenetic data were deposited in the CSIRO Data Portal (http://dx.doi.org/10.4225/08/538FD6C5C9897).
We are grateful to Joel Armstrong for providing H. armigera and Peter Campbell for help with tissue dissection. We also thank all our colleagues who provided critical feedback. Nai-Yong Liu was supported by the China Scholarship Council (CSC) under a joint PhD program with CSIRO Ecosystem Sciences, Australia. This work was supported by Commonwealth Scientific and Industrial Research Organisation (CSIRO) Julius Award (R-00094-01-005) and the CSIRO Office of the Chief Executive (OCE) Postdoctoral Fellowship (R-01479-1).
During the revision of our manuscript, a related work titled “Sequence similarity and functional comparisons of pheromone receptor orthologs in two closely related Helicoverpa species” identified HarmOR51 gene that was described as HarmOR14b in this paper .
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