br Materials and methods br Results br Discussion In this
Materials and methods
Discussion In this study, we first showed that fluxametamide, a novel isoxazoline insecticide, inhibits agonist responses in two types of LGCCs, the M. domestica GABACl and GluCl, with higher potency in the former channel versus the latter one (Fig. 3). In addition to GABACls, which are established targets for conventional antagonist insecticides, GluCls are also important targets for selective insecticides because the activation of GluCls by macrocyclic lactones such as abamectin leads to insecticidal activity. Although no insecticide that specifically inhibits GluCls has been identified to date, GluCl antagonists should be safe insecticides with little or low toxicity against vertebrates owing to the protostome-specific expression of GluCls . Meanwhile, given that there are related ion channels in vertebrates, this idea needs to be tested by further studies. Future study should also evaluate whether the inhibition of GluCls by fluxametamide contributes to its insecticidal activity. Fluxametamide potently inhibited GABA-induced currents in the T. urticae wild-type GABACl and the L. striatellus A2′N mutant GABACl, both of which have a “resistant-type” amino PF-05212384 clinical for conventional antagonists, i.e., an amino acid other than Ala, at the 2′-position in TM2  (Fig. 4, Fig. 5). Consequently, this insecticide exhibited high acaricidal/insecticidal activities against T. urticae and not only the susceptible but also the fipronil-resistant populations of L. striatellus (Table 1, Table 2). We previously reported that the A2′N mutation of M. domestica GABACls causes insensitivity to fipronil but does not affect high sensitivity to fluralaner . These findings suggest that isoxazolines such as fluralaner and fluxametamide bind to a site different from the site for IRAC Group 2 insecticides such as fipronil. The plateaued [3H]EBOB displacement by fluxametamide (Fig. 2) and fluralaner  at high concentrations may be a piece of evidence for an allosteric effect caused by binding to a site apart from the EBOB site . Further study is needed to confirm the interaction between the two chemistries. Because several insect pests including planthoppers have developed resistance to fiproles [13,38], the development of insecticides with a novel mode of action is required to circumvent this resistance. Recently, the meta-diamide insecticides such as broflanilide were reported to act as selective antagonists by interacting with the transmembrane subunit interface (TSI) of insect GABACls [19,39]. Similar to the isoxazolines, these insecticides exhibited antagonistic activity against both wild-type and 2′-mutant GABACls. However, the two chemistries have distinct pharmacological profiles. First, unlike the isoxazolines, the meta-diamide insecticides did not inhibit glutamate responses in GluCls . Second, the specific binding of a tritiated meta-diamide to housefly-head membranes was enhanced by channel blocker-type antagonists such as fiproles , whereas the binding of [3H]fluralaner was slightly inhibited by fipronil and neither enhanced nor inhibited by α-endosulfan . These findings suggest that the meta-diamides and the isoxazolines have differential effects on insects. The avermectins, a class of insecticidal, acaricidal, and nematicidal macrolides that includes ivermectin, were shown to bind to the TSI of GluCls . In this context, we have recently found that the substitution of an amino acid (Leu) in the TSI of GluCls with an amino acid (Phe) at the equivalent position of GABACls results in an increase in the antagonistic potency of fluralaner, whereas the same substitution abolished GluCl activation by ivermectin . This finding suggests that fluxametamide, which shares a common skeleton with fluralaner, might also bind to the TSI of LGCCs but through a different interaction than the macrolides. Investigation into the mechanism that causes the difference between the actions of isoxazolines and macrolides might lead to identification of novel pest control chemistries.