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Voltage-gated sodium channels (VGSCs) are a major target site for the action of pyrethroid insecticides and resistance to pyrethroids has been ascribed to mutations in the VGSC gene. VGSCs in insects are encoded by only one gene and their structural and functional diversity results from posttranscriptional modification, particularly, alternative splicing. Using whole cell patch clamping of neurons from pyrethroid susceptible (wild-type) and resistant strains (s-kdr) of housefly, Musca domestica, we have shown that the V50 for activation and steady state inactivation of sodium currents (INa+) is significantly depolarised in s-kdr neurons compared with wild-type and that 10 nM deltamethrin significantly hyperpolarised both of these parameters in the neurons from susceptible but not s-kdr houseflies. Similarly, tail currents were more sensitive to deltamethrin in wild-type neurons (EC15 14.5 nM) than s-kdr (EC15 133 nM). We also found that in both strains, INa+ are of two types: a strongly inactivating (to 6.8% of peak) current, and a more persistent (to 17.1% of peak) current. Analysis of tail currents showed that the persistent current in both strains (wild-type EC15 5.84 nM) was more sensitive to deltamethrin than was the inactivating type (wild-type EC15 35.1 nM). It has been shown previously, that the presence of exon l in the Drosophila melanogaster VGSC gives rise to a more persistent INa+ than does the alternative splice variant containing exon k and we used PCR with housefly head cDNA to confirm the presence of the housefly orthologues of splice variants k and l. Their effect on deltamethrin sensitivity was determined by examining INa+ in Xenopus oocytes expressing either the k or l variants of the Drosophila para VGSC. Analysis of tail currents, in the presence of various concentrations of deltamethrin, showed that the l splice variant was significantly more sensitive (EC50 42 nM) than the k splice variant (EC50 866 nM). We conclude that in addition to the presence of point mutations, target site resistance to pyrethroids may involve the differential expression of splice variants.

Recent work has shown that two bumblebee (Bombus terrestris) cytochrome P450s of the CYP9Q subfamily, CYP9Q4 and CYP9Q5, are important biochemical determinants of sensitivity to neonicotinoid insecticides. Here, we report the characterisation of a third P450 gene CYP9Q6, previously mis-annotated in the genome of B. terrestris, encoding an enzyme that metabolises the N-cyanoamidine neonicotinoids thiacloprid and acetamiprid with high efficiency. The genomic location and complete ORF of CYP9Q6 was corroborated by PCR and its metabolic activity characterised in vitro by expression in an insect cell line. CYP9Q6 metabolises both thiacloprid and acetamiprid more rapidly than the previously reported CYP9Q4 and CYP9Q5. We further demonstrate a direct, in vivo correlation between the expression of the CYP9Q6 enzyme in transgenic Drosophila melanogaster and an increased tolerance to thiacloprid and acetamiprid. We conclude that CYP9Q6 is an efficient metaboliser of N-cyanoamidine neonicotinoids and likely plays a key role in the high tolerance of B. terrestris to these insecticides., Graphical abstract Image 1, Highlights • A cytochrome P450 gene CYP9Q6 is identified as a potential N-cyanoamidine neonicotinoid metaboliser in Bombus terrestris. • CYP9Q6 efficiently metabolized N-cyanoamidine neonicotinoid insecticides when expressed in vitro in insect cell lines. • Transgenic D. melanogaster expressing CYP9Q6 in vivo had increased tolerance to N-cyanoamidine neonicotinoids. • Amino acid substitutions in the P450 active site may be important determinants of binding and metabolism of these compounds.

Knockdown resistance (kdr) in insects, caused by inherited nucleotide polymorphisms in the voltage-gated sodium channel (VGSC) gene, is a major threat to the efficacy of pyrethroid insecticides. Classic kdr, resulting from an L1014F substitution in the VGSC is now present in numerous pest species. Two other substitutions at the L1014 locus have also been reported, L1014S and L1014H. Here we have used expression of L1014 modified Drosophila para VGSCs in Xenopus oocytes with two-electrode voltage clamp to characterise all three mutations. The mutations L1014F and L1014H caused significant depolarizing shifts in the half activation voltage (V(50,act)) from -17.3 mV (wild-type) to -13.1 and -13.5 mV respectively, whereas L1014S caused no shift in V(50,act) but its currents decayed significantly faster than wild-type channels. Treatment of the wild-type channel with deltamethrin (≥ 1 nM), permethrin (≥ 30 nM) or DDT (≥ 1 μM) resulted in hyperpolarizing shifts in V(50,act). Deltamethrin, permethrin and DDT also produced "tail currents" with EC₅₀s of 0.043, 0.40 and 65 μM and maximum modifications of 837, 325 and 7% respectively. L1014F provided a high level of resistance against all insecticides for both measured parameters. L1014H most effectively combated deltamethrin induced tail currents while L1014S strongly resisted the large DDT induced shifts in V(50,act). We conclude that L1014H and L1014S may have arisen through heavy exposure to specific pyrethroids and DDT respectively.