A mutation in the second transmembrane domain of the GABA receptor subunit, Rdl, is associated with resistance to insecticides such as dieldrin and fipronil. Molecular cloning of Rdl cDNA from a strain of the malaria mosquito, Anopheles gambiae, which is highly resistant to dieldrin revealed this mutation (A296G) as well as another mutation in the third transmembrane domain (T345M). Wild-type, A296G, T345M and A296G + T345M homomultimeric Rdl were expressed in Xenopus laevis oocytes and their sensitivities to fipronil, deltamethrin, 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT), imidacloprid and spinosad were measured using twoelectrode voltage-clamp electrophysiology. Spinosad and DDT had no agonist or antagonist actions on Rdl. However, fipronil, deltamethrin and imidacloprid decreased GABAevoked currents. These antagonistic actions were either reduced or abolished with the A296G and the A296G + T345M mutations while T345M alone appeared to have no significant effect. In conclusion, this study identifies another mutation in the mosquito Rdl that is associated with insecticide resistance. While T345M itself does not affect insecticide sensitivity, it may serve to offset the structural impact of A296G. The present study also highlights Rdl as a potential secondary target for neonicotinoids and pyrethroids.

The insect GABA receptor, RDL (resistance to dieldrin), plays central roles in neuronal signalling and is the target of several classes of insecticides. To study the GABA receptor from an important pollinator species, we cloned Rdl cDNA from the honey bee, Apis mellifera. Three Rdl variants were identified, arising from differential use of splice acceptor sites in the large intracellular loop between transmembrane regions 3 and 4. These variants were renamed from previously, as Amel_RDLvar1, Amel_RDLvar2 and Amel_RDLvar3. When expressed in Xenopus laevis oocytes, the three variants showed no difference in sensitivity to the agonist, GABA, with ECs of 29μM, 20μM and 29μM respectively. Also, the potencies of the antagonists, fipronil and imidacloprid, were similar on all three variants. Fipronil IC values were 0.18μM, 0.31μM and 0.20μM whereas 100μM imidacloprid reduced the GABA response by 17%, 24% and 31%. The possibility that differential splicing of the RDL intracellular loop may represent a species-specific mechanism leading to insensitivity to insecticides is discussed.

The present study focused on the toxicity of the aphid anti-feedant flonicamid and its main metabolite, 4-trifluoromethylnicotinamide (TFNA-AM) to Aedes aegypti and Anopheles gambiae mosquitoes.The compounds were toxic to both species via topical application, resulting in un-coordinated locomotion and leg splaying, with a favorable An. gambiae LD 50value of 35 ng/mg for TFNA-AM, but no significant lethality to Ae. aegypti at 10 μg/female.There was mild cross resistance in the Akron-kdr (Akdr) strain of An. gambiae. Both compounds were non-toxic to intact larvae (LC 50 >300 ppm); however, headless Ae. aegypti larvae displayed spastic paralysis, with PC 50 values of 2-4 ppm, indicating that the cuticle is a significant barrier to penetration. TFNA-AM showed low mammalian toxicity, with an LD 50 of >2000 mg/kg in mice. Electrophysiological experiments showed larval Aedes muscle depolarization and Kv2 channel blocking activity that required near mM concentrations, suggesting that this potassium channel is not the main target for flonicamid nor its metabolite.However, TFNA-AM was a potent blocker of evoked body wall sensory discharge in dipteran larvae, suggesting that some component of the chordotonal organ system may be involved in its toxicity. Finally, flonicamid and TFNA-AM showed about 2-fold synergism of permethrin toxicity against An. gambiae adult females whose mechanism should become more clear once the mode of action of these compounds is better defined.

The insect GABA receptor, RDL, is the target of several classes of pesticides. The peptide sequences of RDL are generally highly conserved between diverse insects. However, RNA A-to-I editing can effectively alter amino acid residues of RDL in a species specific manner, which can affect the potency of GABA and possibly insecticides. We report here that RNA A-to-I editing alters the gene products of Rdl in three mosquito disease vectors, recoding five amino acid residues in RDL of Aedes aegypti and six residues in RDLs of Anopheles gambiae and Culex pipiens, which is the highest extent of editing in RDL observed to date. Analysis of An. gambiae Rdl cDNA sequences identified 24 editing isoforms demonstrating a considerable increase in gene product diversity. RNA editing influenced the potency of the neurotransmitter, GABA, on An. gambiae RDL editing isoforms expressed in Xenopus laevis oocytes, as demonstrated by ECs ranging from 5 ± 1 to 246 ± 41 μM. Fipronil showed similar potency on different editing isoforms, with ICs ranging from 0.18 ± 0.08 to 0.43 ± 0.09 μM. In contrast, editing of An. gambiae RDL affected the activating, potentiating and inhibiting actions of ivermectin. For example, ivermectin potentiated currents induced by GABA at the EC concentration in the unedited isoform but not in the fully edited variant. Editing of a residue in the first transmembrane domain or the cys-loop influenced this potentiation, highlighting residues involved in the allosteric mechanisms of cys-loop ligand-gated ion channels. Understanding the interactions of ivermectin with molecular targets may have relevance to mosquito control in areas where people are administered with ivermectin to treat parasitic diseases.

The resistance to dieldrin (RDL) receptor is an insect γ-aminobutyric acid (GABA) receptor, characterized by the dieldrin resistance mutation that was pivotal to understanding target based insecticide resistance. RDL is the target for various non-competitive antagonists, including dieldrin and fipronil, as well as novel acting compounds such as the meta-diamides and isoxazolines. Therefore the RDL receptor has returned to center stage as a relevant and effective insecticide target. Our understanding of the function of RDL in vivo is still unfolding, with the discovery of species specific post-transcriptional modifications such as alternative splicing and RNA editing, modifications shown to influence the pharmacology of the receptor. Exposing these receptors to insecticides also evokes ever evolving mechanisms of mutagenesis, and a number of contributory mutations have been identified both in field and laboratory resistant insects, occurring in parallel to the dieldrin resistance mutation. We present an overview of these variations and discuss the impact on the pharmacology of GABA and various insecticides.