A Candidate Pheromone Receptor and Two Odorant Receptors of the Hawkmoth Manduca sexta

Abstract

In this study, we cloned and characterized three Manduca sexta odorant receptors (ORs). One receptor is a putative pheromone receptor expressed exclusively in a cell associated with male-specific type-I trichoid sensilla. We describe the results of real-time PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) experiments that show MsextaOR1 is expressed only in male antennae. In situ hybridization labels a single cell associated with type-1 trichoid sensilla, which houses two neurons that have been previously determined to respond to the major components of the pheromone blend. The second receptor, MsextaOR2, was discovered using degenerate primers designed to conserved motifs of a unique group ORs that share as much as 88% identity. Comparison of RT-PCR, qRT-PCR, and in situ hybridization results with those of ORs in the Drosophila melanogaster Or83b subfamily shows a strong sequence and expression pattern similarity. The third receptor, MsextaOR3, was found by 5'-end sequencing of a normalized and subtracted cDNA library from male M. sexta antennae. RT-PCR and qRT-PCR show that this receptor is expressed only in male and female antennae. These are the first ORs, including a putative pheromone receptor, to be described from M. sexta.

Antennal transcriptome of Manduca sexta

“…Because of their high sequence variability, the most common method to identify ORcoding genes is to analyze genomic sequence databases. For Manduca, the coreceptor (MsexOR-2) and five OR-coding genes have been identified (46,47). Transcripts for all except MsexOR-3 were present in our data; MsexOR-2 and MsexOR-6 could also be extended to full-length.…”

Alternative splicing produces two transcripts encoding female-biased pheromone subfamily receptors in the navel orangeworm, Amyelois transitella

“…Amino acid sequences for ORs identified from B. mori (Wanner et al, 2007) and H. virescens (Krieger et al, 2002(Krieger et al, , 2004 along with odorant receptor members of the pheromone receptor subfamily from Ostrinia nubialis (Wanner et al, 2010), Manduca sexta (Patch et al, 2009), Cydia pomonella (Bengtsson et al, 2012;Garczynski, unpublished), Plutella xylostella (Mitsuno et al, 2008), Mythimna separata (Mitsuno et al, 2008), Diaphania indica (Mitsuno et al, 2008), and A. transitella (this study) were aligned using the Clustal W program (Thompson et al, 1994) built into the MEGA5 software. The bootstrap consensus tree was inferred from 500 replicates (Felsenstein, 1985) and branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed.…”

“…Subsequently, members of this subfamily have been referred to as male-biased sex pheromone receptor clade (Jordan et al, 2009), sex pheromone receptor subfamily (Miura et al, 2009(Miura et al, , 2010Wanner et al, 2010), pheromone receptors (Patch et al, 2009;Große-Wilde et al, 2010), and male-specific OR subfamily (Mitsuno et al, 2008). To gain a better understanding of how lepidopteran sex pheromones work at the molecular level, research to identify and characterize pheromone receptors in males has been the topic of much research (Krieger et al, 2004(Krieger et al, , 2005Sakurai et al, 2004;Nakagawa et al, 2005;Große-Wilde et al, 2007Wanner et al, 2007Wanner et al, , 2010Mitsuno et al, 2008;Jordan et al, 2009;Miura et al, 2009Miura et al, , 2010Patch et al, 2009;Xu et al, 2012). However, some members of the pheromone receptor subfamily are also activated by plant kairomones (Jordan et al, 2009;Bengtsson et al, 2014), indicating that not all of these ORs respond to pheromones.…”

Perfusion with cAMP analogue affects pheromone-sensitive trichoid sensilla of the hawkmothManduca sextain a time-dependent manner

“…Another potential target for cAMP, which might increase SP amplitude, could be the ubiquitous OR83b analogue MsextaOR2 (Patch et al, 2009;Wicher et al, 2008). It remains to be investigated whether MsextaOR2 is cAMP sensitive and involved in pheromone transduction of M. sexta.…”

“…Same lower case letters denote no significant differences between tested groups of mean values. cAMP affects ORNs time dependently Furthermore, 8bcAMP might affect the spontaneous activity via direct or indirect interaction with the putative cAMP-sensitive OR83b analogue MsextaOR2 (Patch et al, 2009;Wicher et al, 2008), because olfactory sensilla lacking OR83b receptors showed very little or no spontaneous AP activity (Larsson et al, 2004). However, the physiological relevance of the observed changes in the spontaneous AP frequency remains to be determined.…”