School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, United Kingdom.
Rothamsted Research, West Common, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom.
Insect Biochem Mol Biol. 2020 Jul;122:103388. doi: 10.1016/j.ibmb.2020.103388. Epub 2020 May 4.
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 V for activation and steady state inactivation of sodium currents (I) 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 (EC 14.5 nM) than s-kdr (EC 133 nM). We also found that in both strains, I 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 EC 5.84 nM) was more sensitive to deltamethrin than was the inactivating type (wild-type EC 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 I 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 I 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 (EC 42 nM) than the k splice variant (EC 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.
电压门控钠离子通道（VGSCs）是拟除虫菊酯杀虫剂作用的主要靶标，对拟除虫菊酯的抗性归因于 VGSC 基因的突变。昆虫中的 VGSCs 仅由一个基因编码，其结构和功能的多样性来自于转录后修饰，特别是选择性剪接。我们使用来自对拟除虫菊酯敏感（野生型）和抗性（s-kdr）家蝇神经元的全细胞膜片钳技术，表明与野生型相比，钠电流（I）的激活和稳态失活的 V 区在 s-kdr 神经元中明显去极化，并且 10 nM 溴氰菊酯显著超极化了这两种参数在易感而非 s-kdr 家蝇的神经元中。同样，尾电流对溴氰菊酯的敏感性在野生型神经元（EC 14.5 nM）中比 s-kdr 神经元（EC 133 nM）更高。我们还发现，在这两种品系中，I 有两种类型：一种是强烈失活的（占峰值的 6.8%）电流，另一种是更持久的（占峰值的 17.1%）电流。尾电流分析表明，两种品系（野生型 EC 5.84 nM）的持久电流对溴氰菊酯的敏感性均高于失活型（野生型 EC 35.1 nM）。先前已经表明，果蝇 VGSC 中的外显子 l 的存在导致比包含外显子 k 的替代剪接变体更持久的 I，我们使用家蝇头部 cDNA 的 PCR 来确认替代剪接变体 k 和 l 的家蝇同源物的存在。通过检查表达果蝇 para VGSC 的 k 或 l 变体的非洲爪蟾卵母细胞中的 I 来确定它们对溴氰菊酯敏感性的影响。在存在不同浓度溴氰菊酯的情况下，对尾电流进行分析表明，l 剪接变体比 k 剪接变体（EC 866 nM）更敏感（EC 42 nM）。我们得出结论，除了存在点突变外，对拟除虫菊酯的靶标抗性可能涉及剪接变体的差异表达。
