Identification of Putative Olfactory Genes from the Oriental Fruit Moth <i>Grapholita molesta</i> via an Antennal Transcriptome Analysis

<div><p>Background</p><p>The oriental fruit moth, <i>Grapholita molesta</i>, is an extremely important oligophagous pest species of stone and pome fruits throughout the world. As a host-switching species, adult moths, especially females, depend on olfactory cues to a large extent in locating host plants, finding mates, and selecting oviposition sites. The identification of olfactory genes can facilitate investigation on mechanisms for chemical communications.</p><p>Methodology/Principal Finding</p><p>We generated transcriptome of female antennae of <i>G</i>.<i>molesta</i> using the next-generation sequencing technique, and assembled transcripts from RNA-seq reads using Trinity, SOAPdenovo-trans and Abyss-trans assemblers. We identified 124 putative olfactory genes. Among the identified olfactory genes, 118 were novel to this species, including 28 transcripts encoding for odorant binding proteins, 17 chemosensory proteins, 48 odorant receptors, four gustatory receptors, 24 ionotropic receptors, two sensory neuron membrane proteins, and one odor degrading enzyme. The identified genes were further confirmed through semi-quantitative reverse transcription PCR for transcripts coding for 26 OBPs and 17 CSPs. OBP transcripts showed an obvious antenna bias, whereas CSP transcripts were detected in different tissues.</p><p>Conclusion</p><p>Antennal transcriptome data derived from the oriental fruit moth constituted an abundant molecular resource for the identification of genes potentially involved in the olfaction process of the species. This study provides a foundation for future research on the molecules involved in olfactory recognition of this insect pest, and in particular, the feasibility of using semiochemicals to control this pest.</p></div

Sequences alignment of candidate GmolOBPs.

<p>The six conserved cysteine residues were marked with “☆”. As GmolOBP17, 18, 19, 20, 21, 22 and 23 are not intact sequences, those sequences are not included in the multisequence alignment.</p

Tissue and sex specific expressions of <i>G</i>. <i>molesta</i> OBPs and CSPs.

<p>Fa: female antennae, Ma: male antennae, Fb: female boby (without antennae), Mb: male boby (without antennae), NTC: No template control.</p

Unigenes of candidate odorant binding proteins.

<p>Unigenes of candidate odorant binding proteins.</p

Gene Ontology (GO) analysis of <i>G</i>. <i>molesta</i> antennal transcripts.

<p>GO terms assigned to biological process, cellular component and molecular functions.</p

RSEM-EVAL evaluating <i>de novo</i> assemblies from Trinity, SOAPdenovo-Trans and Abyss-trans.

<p>RSEM-EVAL evaluating <i>de novo</i> assemblies from Trinity, SOAPdenovo-Trans and Abyss-trans.</p

Unigenes of candidate ordoart receptors and gustatory receptors.

<p>Unigenes of candidate ordoart receptors and gustatory receptors.</p

Neighbor-joining tree of candidate ionotropic receptor (IR) genes from <i>G</i>. <i>molesta</i> and other insects.

<p>The tree was drawn using Adobe Photoshop CS5, based on an unrooted tree constructed using the BioNJ algorithm in Seaview v.4. The unrooted tree was constructed based on a sequence alignment produced using MAFFT version 6. Gmol, <i>Grapholita molesta</i>, Msex, <i>Manduca sexta</i>, Cpom, <i>Cydia pomonella</i>, Slit, <i>Spodoptera Iittoralis</i>, Bmor, <i>Bombyx mori</i>, Dmel, <i>Drosophila melanogaster</i>, Amel, <i>Apis mellifera</i>, Tcas, <i>Tribolium castaneum</i>.</p

Neighbor-joining tree of candidate OBP genes from <i>G</i>. <i>molesta</i> and other Lepidoptera.

<p>The tree was drawn using Adobe Photoshop CS5, based on the unrooted tree constructed using BioNJ algorithm in Seaview v.4. The unrooted tree was constructed based on the sequence alignment obtained using MAFFT version 6. Gmol, <i>Grapholita molesta</i>, Cpom, <i>Cydia pomonella</i>, Dple, <i>Danaus plexippus</i>, Hvir, <i>Heliothis virescens</i>, Msex, <i>Manduca sexta</i>, Harm, <i>Helicoverpa armigera</i>, Aips, <i>Agrotis ipsilon</i>. The clade in blue indicates the GOBP1 gene clade; the clade in red indicates the GOBP2 clade, the clade in fuchsia indicates the PBPs clade, the yellow indicates other OBPs clade.</p

TransRate assembly scores.

<p>(A) Number of contigs for three representative assemblies from RNA-seq data of <i>G</i>.<i>molesta</i>. (B) Proportion of reads that map to each assembly. (C) Percentage of transcripts best represented in the assembly. (D) Final TransRate assembly scores for the three different assemblies.</p