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Int J Mol Sci
2018 Jul 31;198:. doi: 10.3390/ijms19082235.
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Cloning and Functional Characterisation of the Duplicated RDL Subunits from the Pea Aphid, Acyrthosiphon pisum.
Del Villar SG
,
Jones AK
.
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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.
Figure 1. Protein sequence alignment of A. pisum cysLGIC subunits. D. melanogaster RDLbd (RDL) is included for comparison. N-terminal signal leader peptides are shown in gray shading and white text. Loops implicated in ligand binding (LpA–F) are indicated, as well as the four transmembrane (TM) domains. The two cysteines forming the cys-loop are highlighted in black shading, and putative N-glycosylation sites are boxed. Potential cAMP, PKC, CK2 and tyrosine kinase phosphorylation sites are shown in gray shading. The sequences presented in this alignment can be found in the Supplementary Material.
Figure 2. Tree showing relationships of A. pisum, A. mellifera, D. melanogaster and T. castaneum cysLGIC subunits. Numbers at each branch signify bootstrap values with 1000 replicates, and the scale bar represents substitutions per site. A. pisum cysLGICs are shown in boldface type.
Figure 3. Splice variants of A. pisum and D. melanogaster RDL. Alternative splicing of exons 3 and 6. Acyrthosiphon residues that differ from those of the orthologous Drosophila exon are highlighted in bold. N-glycosylation sites are boxed and Loops C and F, which contribute to ligand binding, are indicated.
Figure 4. Tree showing relationships of RDL protein sequences from insects of various species. ELIC, which is an ancestral cysLGIC from the bacterium Erwinia chrysanthemi [29], was used as an outgroup. Numbers at each node signify bootstrap values with 1000 replicates, and the scale bar represents substitutions per site. A. pisum RDLs are shown in boldface type.
Figure 5. Responses to GABA in X. laevis oocytes expressing Apisum RDL. (a) Representative current trace of a GABA concentration response curve showing responses to GABA from 0.1–250 μM for Apisum RDL1bd; (b) GABA concentration response curves obtained for Apisum RDL1ad, Apisum RDL1bd, and Apisum RDL2b variant 1. Data were normalised to the maximal response (250 μM). Data is the mean ± SEM from n = 4–5 oocytes from ≥ 3 different frogs.
Figure 6. Effects of fipronil on currents activated by GABA at EC50 in X. laevis oocytes expressing either Apisum RDL1ad, Apisum RDL1bd or Apisum RDL2b variant 1. (A) Representative current traces showing the effect of 0.001–10 μM fipronil on the GABA response for Apisum RDL1bd. (B) Fipronil inhibition curves for Apisum RDL1ad, Apisum RDL1bd or Apisum RDL2b variant 1. Each data point was normalised to the maximum GABA response. Data are the mean ± SEM from n = 5 oocytes from ≥3 different frogs.
Figure 7. Representative current traces showing effects of 100 μM (a) imidacloprid, (b) clothianidin and (c) thiacloprid on GABA-induced currents (at EC50 concentration) in X. laevis expressing Apisum RDL1bd.
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