Analysis of the Antennal Transcriptome and Insights into Olfactory Genes in Hyphantria cunea (Drury)

Abstract

Hyphantria cunea (Drury) (Lepidoptera: Arctiidae) is an invasive insect pest which, in China, causes unprecedented damage and economic losses due to its extreme fecundity and wide host range, including forest and shade trees, and even crops. Compared to the better known lepidopteran species which use Type-I pheromones, little is known at the molecular level about the olfactory mechanisms of host location and mate choice in H. cunea, a species using Type-II lepidopteran pheromones. In the present study, the H. cunea antennal transcriptome was constructed by Illumina Hiseq 2500TM sequencing, with the aim of discovering olfaction-related genes. We obtained 64,020,776 clean reads, and 59,243 unigenes from the analysis of the transcriptome, and the putative gene functions were annotated using gene ontology (GO) annotation. We further identified 124 putative chemosensory unigenes based on homology searches and phylogenetic analysis, including 30 odorant binding proteins (OBPs), 17 chemosensory proteins (CSPs), 52 odorant receptors (ORs), 14 ionotropic receptors (IRs), nine gustatory receptors (GRs) and two sensory neuron membrane proteins (SNMPs). We also found many conserved motif patterns of OBPs and CSPs using a MEME system. Moreover, we systematically analyzed expression patterns of OBPs and CSPs based on reverse transcription PCR and quantitative real time PCR (RT-qPCR) with RNA extracted from different tissues and life stages of both sexes in H. cunea. The antennae-biased expression may provide a deeper further understanding of olfactory processing in H. cunea. The first ever identification of olfactory genes in H. cunea may provide new leads for control of this major pest.

Fig 1. Phylogenetic tree of putative odorant binding protein (OBP) genes from H. cunea and other lepidopteran insects.

The tree was constructed with MEGA5.0, which was based on amino acid sequence alignments by using the ClustalX2.0. Aips: A. ipsilon; Bmor: B. mori; Dple: D. plexippus; Harm: H. armigera; Hass: H. assulta; Msex: M. sexta; Sexi: S. exigua; Sinf: S. inferens; Slit: S. litura.

Fig 2. Phylogenetic tree of putative chemosensory protein (CSP) genes from H. cunea and other lepidopteran insects.

Aips: A. ipsilon; Bmor: B. mori; Harm: H. armigera; Sexi: S. exigua; Sinf: S. inferens.

Fig 3. Phylogenetic tree of putative odorant receptor (OR) genes from H. cunea and other lepidopteran insects.

Bmor: B. mori; Harm: H. armigera; Sinf: S. inferens; Obru: O. brumata; Aseg: A. segetum.

Fig 4. Phylogenetic tree of putative gustatory receptor (GR) genes from H. cunea and other lepidopteran insects.

Aips: A. ipsilon; Bmor: B. mori; Dple: D. plexippus; Harm: H. armigera; Hass: H. assulta; Msex: M. sexta; Sexi: S. exigua.

Fig 5. Phylogenetic tree of putative ionotropic receptor (IR) genes from H.cunea and other lepidopteran insects.

Bmor: B. mori; Cpom: C. pomonella; Dhou: D. houi; Dkik: D. kikuchii; Dmel: D. melanogaster; Sinf: S. inferens.

Fig 6. Phylogenetic tree of putative sensory neuron membrane protein (SNMP) genes from H. cunea and other lepidopteran insects.

Aips: A. ipsilon; Bmor: B. mori; Harm: H. armigera; Hass: H. assulta; Hvir: H. virescens; Msex: M. sexta; Sexi: S. exigua; Sinf: S. inferens; Slit: S. litura.

Fig 7. Motif analysis of OBPs in H. cunea.

The upper parts listed the eight motifs discovered in 76 OBPs. The lower parts indicate approximate locations of each motif on the protein sequence. The numbers in the colored boxes correspond to the numbered motifs in the upper part of the figure. The small number represents high conservation. The numbers on the bottom show the approximate locations of each motif on the protein sequence, starting from the N-terminal. This figure just listed 11 relatively common motif patterns including 54 OBPs.

Fig 8. Motif analysis of CSPs in H. cunea.

The upper parts listed the eight motifs discovered in 43 CSPs. The lower parts indicate approximate locations of each motif on the protein sequence. The numbers in the colored boxes correspond to the numbered motifs in the upper part of the figure. The small number represents high conservation. The numbers on the bottom show the approximate locations of each motif on the protein sequence, starting from the N-terminal. This figure just listed 11 relatively common motif patterns including 30 CSPs.

Fig 9. H. cunea OBPs transcript levels in different tissues and life stages as measured by RT-PCR.

A: antennae; Th: thoraces; Ab: abdomens; L: legs; W: wings; P: pupae; La: larvae; NC: no template control; ♀: female; ♂: male. EF1-a was used as a reference gene for each cDNA template.

Fig 10. H. cunea CSPs transcript levels in different tissues and life stages as measured by RT-PCR.

A: antennae; Th: thoraces; Ab: abdomens; L: legs; W: wings; P: pupae; La: larvae; NC: no template control; ♀: female; ♂: male. EF1-a was used as a reference gene for each cDNA template.

Fig 11. H. cunea OBPs transcript levels in different tissues and life stages as measured by RT-qPCR.

Relative mRNA level in different tissues and life stages were analyzed with Duncan's new multiple range method. The standard errors are represented by error bars, different letters (a, b, c, d) above bars denote significant difference between different tissues and life stages, at the 0.05 level; FA: female antennae; MA: male antennae; L: legs; W: wings; FP: female pupae; MP: male pupae; La: larvae.

Fig 12. H. cunea CSPs transcript levels in different tissues and life stages as measured by RT-qPCR.

Relative mRNA level in different tissues and life stages were analyzed with Duncan's new multiple range method. The standard errors are represented by error bars, different letters (a, b, c, d) above bars denote significant difference between different tissues and life stages, at the 0.05 level; FA: female antennae; MA: male antennae; L: legs; W: wings; FP: female pupae; MP: male pupae; La: larvae.