Functional Evolution of a Multigene Family: Orthologous and Paralogous Pheromone Receptor Genes in the Turnip Moth, Agrotis segetum

Abstract

Lepidopteran pheromone receptors (PRs), for which orthologies are evident among closely related species, provide an intriguing example of gene family evolution in terms of how new functions may arise. However, only a limited number of PRs have been functionally characterized so far and thus evolutionary scenarios suffer from elements of speculation. In this study we investigated the turnip moth Agrotis segetum, in which female moths produce a mixture of chemically related pheromone components that elicit specific responses from receptor cells on male antennae. We cloned nine A. segetum PR genes and the Orco gene by degenerate primer based RT-PCR. The nine PR genes, named as AsegOR1 and AsegOR3-10, fall into four distinct orthologous clusters of known lepidopteran PRs, of which one contains six paralogues. The paralogues are under relaxed selective pressure, contrasting with the purifying selection on other clusters. We identified the receptors AsegOR9, AsegOR4 and AsegOR5, specific for the respective homologous pheromone components (Z)-5-decenyl, (Z)-7-dodecenyl and (Z)-9-tetradecenyl acetates, by two-electrode voltage clamp recording from Xenopus laevis oocytes co-expressing Orco and each PR candidate. These receptors occur in three different orthologous clusters. We also found that the six paralogues with high sequence similarity vary dramatically in ligand selectivity and sensitivity. Different from AsegOR9, AsegOR6 showed a relatively large response to the behavioural antagonist (Z)-5-decenol, and a small response to (Z)-5-decenyl acetate. AsegOR1 was broadly tuned, but most responsive to (Z)-5-decenyl acetate, (Z)-7-dodecenyl acetate and the behavioural antagonist (Z)-8-dodecenyl acetate. AsegOR8 and AsegOR7, which differ from AsegOR6 and AsegOR1 by 7 and 10 aa respectively, showed much lower sensitivities. AsegOR10 showed only small responses to all the tested compounds. These results suggest that new receptors arise through gene duplication, and relaxed evolutionary constraints or positive selection among paralogues allow functional divergence to occur in spite of purifying selection being the norm.

Sensory genes identification with head transcriptome of the migratory armyworm, Mythimna separata

“…The female M. separata moths emit the main component of the sex pheromone, Z11–16:OAc78. Lepidoptera sex pheromones produced by females may attract males for mating opportunities798081. Based on phylogenetic tree analyzes, six M. separata ORs (MS|comp813906_c0, MS|comp533077_c0, MS|comp3974_c0, MS|comp90072_c0, MS|comp152487_c0 and MS|comp157094_c0) clustered in a conserved clade of PRs found in Lepidopteran insects (Fig.…”

Putative ligand binding sites of two functionally characterized bark beetle odorant receptors

“…Stage V-VII oocytes were co-injected with cRNAs from the ItypOrco and ORs (50 ng of each), and then incubated in Ringer's buffer (96 mM NaCl, 2 mM KCl, 5 mM MgCl2, 0.8 mM CaCl2, 5 mM HEPES, pH 7.6) containing 550 mg/L sodium pyruvate and 100 mg/L gentamicin at 18 °C for at least 3 days. The previously described two-electrode voltage clamp electrophysiological set-up was used to record whole-cell inward currents from oocytes in good condition (3-6 days post-injection) at a holding potential of -80 mV 66 . Test compounds were applied to the oocyte chamber by means of a computer-controlled perfusion system at a rate of 2 ml/min for 20 s with extensive washing with Ringer's buffer at 4 ml/min between stimulations.…”

Mate discrimination among subspecies through a conserved olfactory pathway

“…Functional analysis of receptor genes in Xenopus oocytes. Oocyte injections and two-electrode voltage clamp recordings were described previously (Zhang and Lofstedt, 2013). Briefly, the open reading frames of D. moj.…”