The modes of action of insecticides are classified by the

The modes of action of insecticides are classified by the Insecticide Resistance Action Committee (IRAC) [6]. In this classification, a large proportion of insecticides are categorized as nerve- and muscle-targeting insecticides, which encompass GABACl antagonists (Group 2) and GluCl allosteric modulators (Group 6). GABACl antagonist insecticides include different chemotypes, i.e., 2A cyclodiene organochlorines such as endosulfan and chlordane and 2B phenylpyrazoles (fiproles) such as fipronil and ethiprole. GluCls are also important targets for insecticides and parasiticides because they are found only in invertebrates and are major targets for macrolide parasiticides/insecticides such as avermectins and milbemycins. Among the insecticides that act as GABACl antagonists, some potently inhibit GluCls as well [7], whereas others are more GABACl-specific [5]. LGCC-targeting pest control chemicals are currently in use, but the emergence of pest insects and mites resistant to these agents is a serious problem. An Ala-to-Ser mutation responsible for high resistance to dieldrin (Rdl) (designated as A2’S) was first identified in the second amino 2211 in the second TM (TM2) of the Drosophila melanogaster GABACl subunit [8,9]. The Drosophila strain showed resistance not only to dieldrin but also to picrotoxinin and fipronil [9,10]. Furthermore, when expressed in Xenopus oocytes, the Drosophila simulans GABACl double mutant with an Ala-to-Gly substitution in TM2 and a Thr-to-Met substitution in the third TM (TM3) showed low sensitivity to fipronil [11]. The A2’S mutation was also recently observed in ethiprole-resistant brown planthoppers (Nilaparvata lugens) [12]. Several other mutations of GABACl subunits associated with resistance to fiproles were reported in agricultural pests [13]. An Ala-to-Asn (A2′N) mutation was identified in the small brown planthopper, Laodelphax striatellus [14]. The A2′N mutant of the L. striatellus GABACl showed lower sensitivity to fipronil than the A2’S mutant. In a GABACl subunit (Tu_Rdl1 Lon) from a field-collected population of the two-spotted spider mite, Tetranychus urticae, His is situated at the 2′-position in TM2, and Ala is present at the position equivalent to the Thr-to-Met mutation in TM3 of the abovementioned Drosophila GABACl double mutant [15]. These amino acids are responsible for the lower sensitivity to fipronil [16]. Point mutations in TM3 of GluCl subunits have been identified in abamectin-resistant T. urticae [15]. The mutation of a conserved Gly in TM3 of the T. urticae GluCl3 (Tu_GluCl3 Mar) was recently reported to disrupt the sensitivity of the channel to abamectin and milbemycin [17]. Because of the emergence of resistant insects and mites, the development of new acaricides/insecticides with novel modes of action and higher effectiveness against resistant mites/insects is urgently required. In recent years, two novel chemotypes of GABACl antagonists, isoxazolines [18] and meta-diamides [19], have been reported. An important aspect of these chemistries is that these antagonists exhibit potent blockage of GABACls with mutations conferring insensitivity to fipronil. However, these two chemotypes are uniquely differentiated: the isoxazoline ectoparasiticide fluralaner inhibits both GABACls and GluCls [18], whereas the meta-diamide insecticides do not inhibit GluCls [20]. Fluxametamide (Fig. 1) is a newer isoxazoline insecticide that exhibits high insecticidal activity against a variety of insect species, such as Lepidoptera, Thysanoptera, Acarina, and Diptera [21]. Here, we show that fluxametamide inhibits both housefly (Musca domestica) GABACls and GluCls, as does fluralaner. We also provide evidence that fluxametamide exerts excellent antagonist effects on GABACls insensitive to existing IRAC Group 2 antagonists, i.e., the 2′ mutant of the small brown planthopper (L. striatellus) GABACl and the wild-type two-spotted spider mite (T. urticae) GABACl. Finally, we demonstrate that fluxametamide has no deleterious effect on mammalian LGCCs, namely, the α1β2γ2 GABACl and the α1 GlyCl. These results allow us to conclude that fluxametamide is a safe insecticide acting as a distinctive LGCC antagonist.