Cloning and Functional Characterisation of the Duplicated RDL Subunits from the Pea Aphid, Acyrthosiphon pisum.

Silvia del Villar,Andrew Jones

doi:10.3390/ijms19082235

Silvia del Villar, Andrew Jones

Open Access

https://doi.org/10.3390/ijms19082235

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Abstract

The insect GABA receptor, RDL (resistance to dieldrin), is a cys-loop ligand-gated ion channel (cysLGIC) that plays a central role in neuronal signaling, and is the target of several classes of insecticides. Many insects studied to date possess one Rdl gene; however, there is evidence of two Rdls in aphids. To characterise further this insecticide target from pests that cause millions of dollars’ worth of crop damage each year, we identified the complete cysLGIC gene superfamily of the pea aphid, Acyrthosiphon pisum, using BLAST analysis. This confirmed the presence of two Rdl-like genes (RDL1 and RDL2) that likely arose from a recent gene duplication. When expressed individually in Xenopus laevis oocytes, both subunits formed functional ion channels gated by GABA. Alternative splicing of RDL1 influenced the potency of GABA, and the potency of fipronil was different on the RDL1bd splice variant and RDL2. Imidacloprid and clothianidin showed no antagonistic activity on RDL1, whilst 100 μM thiacloprid reduced the GABA responses of RDL1 and RDL2 to 55% and 62%, respectively. It was concluded that gene duplication of Rdl may have conferred increased tolerance to natural insecticides, and played a role in the evolution of insect cysLGICs.

Highlights

The insect γ-aminobutyric acid (GABA) receptor, known as RDL, plays a central role in neuronal signaling, and is involved in various processes, including regulation of sleep [1], aggression [2], and olfactory or visual learning [3,4]
The GABA receptor is a member of the cys-loop ligand-gated ion channel superfamily, which, in insects, includes nicotinic acetylcholine receptors, histamine-gated chloride channels (HisCls), and glutamate-gated chloride channels (GluCls) [5]
We report here that the two Rdl genes in A. pisum encode for GABA-gated ion channels, upon which the insecticides fipronil and thiacloprid act as antagonists

Summary

Introduction

The insect γ-aminobutyric acid (GABA) receptor, known as RDL (resistant to dieldrin), plays a central role in neuronal signaling, and is involved in various processes, including regulation of sleep [1], aggression [2], and olfactory or visual learning [3,4]. In the genomic DNA of the model organism, Drosophila melanogaster, a mutation resulting in an alanine to serine substitution located in TM2 of Rdl was identified, which underlies resistance to several insecticides, including dieldrin, picrotoxin and fipronil [8,9]. This alanine to serine mutation, found as alanine to glycine or to asparagine [10], has since been associated with insecticide resistance in various species, ranging from disease vectors (the malaria mosquito Anopheles gambiae [11,12]), to pests afflicting livestock (the horn fly Haematobia irritans [13]) or domesticated animals (the cat flea Ctenocephalides felis [14]) and crop pests (e.g., the planthopper Laodelphax striatellus [15]). It was concluded that the duplicated Rdl in A. pisum may represent diversification, leading to the evolution of novel cysLGIC subunits in higher insects

Results

Materials and Methods

Sequence Analysis

Data Analysis