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. 2012 Jul;42(7):499-505.
doi: 10.1016/j.ibmb.2012.03.007. Epub 2012 Apr 5.

Sequencing and Characterizing Odorant Receptors of the Cerambycid Beetle Megacyllene Caryae

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Sequencing and Characterizing Odorant Receptors of the Cerambycid Beetle Megacyllene Caryae

Robert F Mitchell et al. Insect Biochem Mol Biol. .
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Abstract

Odorant receptors (Ors) are a unique family of ligand-gated ion channels and the primary mechanism by which insects detect volatile chemicals. Here, we describe 57 putative Ors sequenced from an antennal transcriptome of the cerambycid beetle Megacyllene caryae (Gahan). The male beetles produce a pheromone blend of nine compovnents, and we functionally characterized Ors tuned to three of these chemicals: receptor McOr3 is sensitive to (S)-2-methyl-1-butanol; McOr20 is sensitive to (2S,3R)-2,3-hexanediol; and McOr5 is sensitive to 2-phenylethanol. McOr3 and McOr20 are also sensitive to structurally-related chemicals that are pheromones of other cerambycid beetles, suggesting that orthologous receptors may be present across many cerambycid species. These Ors are the first to be functionally characterized from any species of beetle and lay the groundwork for understanding the evolution of pheromones within the Cerambycidae.

Figures

Fig. 1
Fig. 1
Phylogram showing peptide sequences of odorant receptors from Tribolium castaneum (TcOrs) and Megacyllene caryae (McOrs). Partial sequences of at least 200 amino acids are included in the tree and denoted by the suffix “PAR”. Receptors are organized into groups 1–6 defined by Engsontia et al. (2008), but including a group described only from M. caryae (7). Receptors from Groups 4–6, not present in the antennal transcriptome of M. caryae, are represented as black triangles to indicate the number of omitted receptors. Receptors of M. caryae that are sensitive to pheromone components (and the Orco) are indicated by “+”, those insensitive to components are indicated by “−”, and those that were not tested (ligands unknown) are indicated by “•”. The tree is rooted with Orco proteins from both species (McOr1, TcOr1).
Fig. 2
Fig. 2
Change in electric current induced by candidate odorant receptors, expressed in Xenopus oocytes, in response to pheromone components of Megacyllene caryae. Frog oocytes that expressed receptors A) McOr3, B) McOr5, and C) McOr20 were sequentially challenged with 30 µM of each of the following pheromone components (applied for 20 s): 1. (2R,3R)-2,3-hexanediol, 2. (2S,3S)-2,3-hexanediol, 3. (2R,3S)-2,3-hexanediol, 4. (2S,3R)-2,3-hexanediol, 5. (R)-3-hydroxyhexan-2-one, 6. (S)-3-hydroxyhexan-2-one, 7. (S)-(−)-limonene, 8. (R)-(+)-limonene, 9. (−)-α-terpineol, 10. 2-phenylethanol, 11. citral, 12. (R)-2-methylbutan-1-ol, and 13. (S)-2-methylbutan-1-ol. Diagonal hatching indicates short breaks in recording.
Fig. 3
Fig. 3
Relationships between concentrations of pheromone components and the responses of Xenopus oocytes that are expressing pheromone receptors of Megacyllene caryae. A) An example of a trace used to generate concentration-response curves (described in section 2.6): an oocyte expressing McOr3 is challenged with 20-s applications of (S)-2-methylbutan-1-ol at a range of doses. Concentration-response curves of B) McOr3 challenged with (R)- and (S)-2-methyl-1-butanol (normalized to the response of each ooc yte to 1 µM [S]-2-methylbutan-1-ol; n = 5–15); C) McOr5 challenged with 2-phenylethanol (normalized to 100 µM 2-phenylethanol; n = 6); D) McOr20 challenged with three isomers of 2,3-hexanediol and (R)-hydroxyhexan-2-one (normalized to 30 µM [2S,3R]-2,3-hexanediol; n = 5–7).

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