Identification and characterization of an antennae-specific aldehyde oxidase from the navel orangeworm.

Antennae-specific odorant-degrading enzymes (ODEs) are postulated to inactivate odorant molecules after they convey their signal. Different classes of insect ODEs are specific to esters, alcohols, and aldehydes--the major functional groups of female-produced, hydrophobic sex pheromones from moth species. Esterases that rapidly inactive acetate and other esters have been well-studied, but less is known about aldehyde oxidases (AOXs). Here we report cloning of an aldehyde oxidase, AtraAOX2, from the antennae of the navel orangeworm (NOW), Amyelois transitella, and the first activity characterization of a recombinant insect AOX. AtraAOX2 gene spans 3,813 bp and encodes a protein with 1,270 amino acid residues. AtraAOX2 cDNA was expressed in baculovirus-infected insect Sf21 cells as a ≈280 kDa homodimer with 140 kDa subunits. Recombinant AtraAOX2 degraded Z11Z13-16Ald and plant volatile aldehydes as substrates. However, as expected for aldehyde oxidases, recombinant AtraAOX2 did not show specificity for Z11Z13-16Ald, the main constituent of the sex pheromone, but showed high activity for plant volatile aldehydes. Our data suggest AtraAOX2 might be involved in degradation of a diversity of aldehydes including sex pheromones, plant-derived semiochemicals, and chemical cues for oviposition sites. Additionally, AtraAOX2 could protect the insect's olfactory system from xenobiotics, including pesticides that might reach the sensillar lymph surrounding the olfactory receptor neurons.

Silent, generic and plant kairomone sensitive odorant receptors from the Southern house mosquito.

The Southern house mosquito Culex quinquefasciatus has the largest repertoire of odorant receptors (ORs) of all mosquitoes and dipteran species whose genomes have been sequenced to date. Previously, we have identified and de-orphanized two ORs expressed in female antennae, CquiOR2 and CquiOR10, which are sensitive to oviposition attractants. In view of a new nomenclature for the Culex genome (VectorBase) we renamed these ORs as CquiOR21 (formerly CquiOR10) and CquiOR121 (CquiOR2). In addition, we selected ORs from six different phylogenetic groups for deorphanization. We cloned four of them by using cDNA from female antennae as a template. Attempts to clone CquiOR87 and CquiOR110 were unsuccessful either because they are pseudogenes or are not expressed in adult female antennae, the main olfactory tissue. By contrast, CquiOR1, CquiOR44, CquiOR73, and CquiOR161 were highly expressed in female antennae. To de-orphanize these ORs, we employed the Xenopus oocyte recording system. CquiORx-CquiOrco-expressed oocytes were challenged with a panel of 90 compounds, including known oviposition attractants, human and vertebrate host odorants, plant kairomones, and naturally occurring repellents. While CquiOR161 did not respond to any test compound in two different laboratories, CquiOR1 showed the features of a generic OR, with strong responses to 1-octen-3-ol and other ligands. CquiOR44 and CquiOR73 showed preference to plant-derived terpenoids and phenolic compounds, respectively. While fenchone was the best ligand for the former, 3,5-dimethylphenol elicited the strongest responses in the latter. The newly de-orphanized ORs may be involved in reception of plant kairomones and/or natural repellents.

Moth sex pheromone receptors and deceitful parapheromones.

The insect's olfactory system is so selective that male moths, for example, can discriminate female-produced sex pheromones from compounds with minimal structural modifications. Yet, there is an exception for this "lock-and-key" tight selectivity. Formate analogs can be used as replacement for less chemically stable, long-chain aldehyde pheromones, because male moths respond physiologically and behaviorally to these parapheromones. However, it remained hitherto unknown how formate analogs interact with aldehyde-sensitive odorant receptors (ORs). Neuronal responses to semiochemicals were investigated with single sensillum recordings. Odorant receptors (ORs) were cloned using degenerate primers, and tested with the Xenopus oocyte expression system. Quality, relative quantity, and purity of samples were evaluated by gas chromatography and gas chromatography-mass spectrometry. We identified olfactory receptor neurons (ORNs) housed in trichoid sensilla on the antennae of male navel orangeworm that responded equally to the main constituent of the sex pheromone, (11Z,13Z)-hexadecadienal (Z11Z13-16Ald), and its formate analog, (9Z,11Z)-tetradecen-1-yl formate (Z9Z11-14OFor). We cloned an odorant receptor co-receptor (Orco) and aldehyde-sensitive ORs from the navel orangeworm, one of which (AtraOR1) was expressed specifically in male antennae. AtraOR1•AtraOrco-expressing oocytes responded mainly to Z11Z13-16Ald, with moderate sensitivity to another component of the sex pheromone, (11Z,13Z)-hexadecadien-1-ol. Surprisingly, this receptor was more sensitive to the related formate than to the natural sex pheromone. A pheromone receptor from Heliothis virescens, HR13 ( = HvirOR13) showed a similar profile, with stronger responses elicited by a formate analog than to the natural sex pheromone, (11Z)-hexadecenal thus suggesting this might be a common feature of moth pheromone receptors.