Knock-in mutagenesis in Drosophila Rdl underscores the critical role of the conserved M3 glycine in mediating the actions of broflanilide and isocycloseram on GABA receptors.

γ-Aminobutyric acid receptors (GABARs) are crucial targets for pest control chemicals, including meta-diamide and isoxazoline insecticides, which act as negative allosteric modulators of insect GABARs. Previous cell-based assays have indicated that amino acid residues in the transmembrane cavity between adjacent subunits of Drosophila RDL GABAR (i.e., Ile276, Leu280, and Gly335) are involved in mediating the action of meta-diamides. In this study, to confirm this result at the organismal level, we employed CRISPR/Cas9-mediated genome editing, generated six transgenic Drosophila strains carrying substitutions in these amino acid residues, and investigated their sensitivity to broflanilide and isocycloseram. Flies homozygous for the I276F mutation did not exhibit any change in sensitivity to the tested insecticides compared to the control flies. Conversely, I276C homozygosity was lethal, and heterozygous flies exhibited ∼2-fold lower sensitivity to broflanilide than the control flies. Flies homozygous for the L280C mutation survived into adulthood but exhibited infertility. Both heterozygous and homozygous L280C flies exhibited ∼3- and âˆ¼20-fold lower sensitivities to broflanilide and isocycloseram, respectively, than the control flies. The reduction in sensitivity to isocycloseram in L280C flies diminished to ∼3-fold when treated with piperonyl butoxide. Flies homozygous for the G335A mutation reached the adult stage. However, they were sterile, had small bodies, and exhibited reduced locomotion, indicating the critical role of Gly335 in RDL function. These flies exhibited markedly increased tolerance to topically applied broflanilide and isocycloseram, demonstrating that the conserved Gly335 is the target of the insecticidal actions of broflanilide and isocycloseram. Considering the significant fitness costs, the Gly335 mutation may not pose a serious risk for the development of resistance in field populations of insect pests. However, more careful studies using insect pests are needed to investigate whether our perspective applies to resistance development under field conditions.

Mechanistic insights into the selectivity of bicyclophosphorothionate antagonists for housefly versus rat GABA receptors.

BACKGROUND: γ-Aminobutyric acid (GABA) receptors (GABARs) are validated targets of insecticides. Bicyclophosphorothionates are a group of insecticidal compounds that act as noncompetitive antagonists of GABARs. We previously reported that the analogs exhibit various degrees of selectivity for housefly versus rat GABARs, depending on substitutions at the 3- and 4-positions. We here sought to elucidate the unsolved mechanisms of the receptor selectivity using quantitative structure-activity relationship (QSAR), molecular docking, and molecular dynamics approaches. RESULTS: Three-dimensional (3D)-QSAR studies using Topomer comparative molecular field analysis quantitatively demonstrated how the introduction of a small alkyl group at the 3-position of bicyclophosphorothionates contributes to the housefly versus rat GABAR selectivity. To investigate the molecular mechanisms of the selective action, bicyclophosphorothionates were docked into housefly Resistance to dieldrin (RDL) GABAR and rat α1ß2γ2 GABAR homology models built using the published 3D-structures of human GABARs as templates. The results of molecular docking and molecular dynamics simulations revealed that the 2'Ala and 6'Thr residues of the RDL subunit within the channel are the key amino acids for binding to the housefly GABARs, whereas the 2'Ser residue of γ2 subunit plays a crucial role in binding to rat GABARs. CONCLUSION: We revealed the molecular mechanisms underlying the selective antagonistic action of bicyclophosphorothionates on housefly versus rat GABARs. The information presented should help design and develop novel, safe GABAR-targeting insecticides. © 2023 Society of Chemical Industry.

A glutamate-gated chloride channel as the mite-specific target-site of dicofol and other diphenylcarbinol acaricides.

Dicofol has been widely used to control phytophagous mites. Although dicofol is chemically related to DDT, its mode of action has remained elusive. Here, we mapped dicofol resistance in the spider mite Tetranychus urticae to two genomic regions. Each region harbored a glutamate-gated chloride channel (GluCl) gene that contained a mutation-G314D or G326E-known to confer resistance against the unrelated acaricide abamectin. Using electrophysiology assays we showed that dicofol and other diphenylcarbinol acaricides-bromopropylate and chlorobenzilate-induce persistent currents in Xenopus oocytes expressing wild-type T. urticae GluCl3 receptors and potentiate glutamate responses. In contrast, the G326E substitution abolished the agonistic activity of all three compounds. Assays with the wild-type Drosophila GluClα revealed that this receptor was unresponsive to dicofol. Homology modeling combined with ligand-docking confirmed the specificity of electrophysiology assays. Altogether, this work elucidates the mode of action of diphenylcarbinols as mite-specific agonists of GluCl.

External amino acid residues of insect GABA receptor channels dictate the action of the isoxazoline ectoparasiticide fluralaner.

BACKGROUND: Fluralaner is the first isoxazoline ectoparasiticide developed to protect companion animals from fleas and ticks. Fluralaner primarily inhibits arthropod γ-aminobutyric acid receptors (GABARs), which are ligand-gated ion channels comprising five subunits arranged around the channel pore. We previously reported that the action site of fluralaner resides at the M1-M3 transmembrane interface between adjacent GABAR subunits. To investigate whether fluralaner interacts with the second transmembrane segment (M2) located deep in the interface, we generated four housefly RDL GABAR mutants with non-conservative amino acid substitutions in the M2 region. RESULTS: Electrophysiological analysis of GABARs expressed in Xenopus oocytes indicated that S313A and S314A mutants exhibited fluralaner sensitivities similar to that of the wild type. M312S mutant exhibited approximately seven-fold lower sensitivity than that of the wild type. Notably, the N316L mutant was almost insensitive to fluralaner. CONCLUSION: The findings of this study indicate that the conserved external amino acid residues of insect GABAR channels play a critical role in the antagonistic effect of fluralaner. © 2023 Society of Chemical Industry.

Controlled expression of nicotinic acetylcholine receptor-encoding genes in insects uncovers distinct mechanisms of action of the neonicotinoid insecticide dinotefuran.

Dinotefuran, a neonicotinoid, is a unique insecticide owing to its structure and action. We took two approaches that employed insects with controlled expression of nicotinic acetylcholine receptor (nAChR)-encoding genes to gain insight into the uniqueness of dinotefuran. First, we examined the insecticidal activity of dinotefuran and imidacloprid against brown planthoppers (Nilaparvata lugens), in which the expression of eight (of 13) individual subunit-encoding genes was specifically reduced using RNA interference. Knockdown of the tested gene, except one, resulted in a decrease in sensitivity to imidacloprid, whereas the sensitivity of N. lugens to dinotefuran decreased only when two of the eight genes were knocked down. These findings imply that a major dinotefuran-targeted nAChR subtype may contain specific subunits although imidacloprid acts on a broad range of receptor subtypes. Next, we examined the effects of knockout of Drosophila α1 subunit-encoding gene (Dα1) on the insecticidal effects of dinotefuran and imidacloprid. Dα1-deficient flies (Dα1KO) demonstrated the same sensitivity to dinotefuran as control flies, but a decreased sensitivity to imidacloprid. This difference was attributed to a reduction in imidacloprid-binding sites in Dα1KO flies, whereas the binding of dinotefuran remained unchanged. RNA sequencing analysis indicated that Dα2 expression was specifically enhanced in Dα1KO flies. These findings suggest that changes in Dα1 and Dα2 expression contribute to the differences in the insecticidal activity of dinotefuran and imidacloprid in Dα1KO flies. Overall, our findings suggest that dinotefuran acts on distinct nAChR subtypes.

Structural insights into the interaction between gabazine (SR-95531) and Laodelphax striatellus GABA receptors.

γ-Aminobutyric acid receptors (GABARs) mediate fast inhibitory neurotransmission and are targets for insecticides. GABARs are composed of five subunits, the composition of which dictates the pharmacological characteristics of GABARs. Both competitive and noncompetitive GABAR antagonists can be used as insecticides. Gabazine is a potent competitive antagonist of mammalian α1ß2γ2 GABARs; however, it is less potent against insect GABARs. To explore how gabazine interacts with GABARs, we examined whether the sensitivity of the small brown planthopper (Laodelphax striatellus) RDL GABAR (LsRDLR) to gabazine is increased when its amino acid residues are substituted with α1ß2γ2 GABAR residues. In the results, two of the generated mutants showed enhanced gabazine sensitivity. Docking simulations of gabazine using LsRDLR homology models and an α1ß2γ2 GABAR cryo-EM structure revealed that the accommodation of gabazine into the "aromatic box" in the orthosteric site lowered the binding energy. This information may help in designing GABAR-targeting insecticides with novel modes of action.

5-(4-Pyridinyl)-3-isothiazolols as Competitive Antagonists of Insect GABA Receptors: Design, Synthesis, and a New Mechanism Leading to Insecticidal Effects.

Ionotropic γ-aminobutyric acid (GABA) receptors (iGABARs) are validated targets of drugs and insecticides. Our previous studies showed that the competitive antagonists of insect iGABARs exhibit insecticidal activities and that the 3-isothiazolol scaffold is used as a lead for developing novel iGABAR antagonists. Here, we designed a novel series of 4-aryl-5-(4-pyridinyl)-3-isothiazolol (4-API) analogs that have various aromatic substituents at the 4-position. Two-electrode voltage clamp experiments showed that all synthesized 4-APIs exhibited antagonistic activity against Musca domestica and Spodoptera litura iGABARs (RDL) expressed in oocytes of Xenopus laevis at 100 µM. Of the 4-APIs, the 4-(1,1'-biphenylyl) analog was the most potent antagonist with IC50s of 7.1 and 9.9 µM against M. domestica and S. litura RDL receptors, respectively. This analog also showed a certain insecticidal activity against S. litura larvae, with >75% mortality at 100 µg/g diet. Molecular docking studies with a M. domestica iGABAR model indicated that the π-π stacking interactions formed between the pyridinyl ring and Y252 and between the 4-substituted aromatic group and Y107 might be important for antagonism by the 4-(1,1'-biphenylyl) analog. Our studies provide important information for designing novel iGABAR antagonists and suggest that the 4-APIs acting on iGABARs are promising insecticide leads for further studies.

Novel insecticidal 1,6-dihydro-6-iminopyridazine derivatives as competitive antagonists of insect RDL GABA receptors.

BACKGROUND: The ionotropic γ-aminobutyric acid (GABA) receptor (iGABAR) is an important target for insecticides and parasiticides. Our previous studies showed that competitive antagonists (CAs) of insect iGABARs have the potential to be used for developing novel insecticides and that the structural modification of gabazine (a representative CA of mammalian iGABARs) could lead to the identification of novel CAs of insect iGABARs. RESULTS: In the present study, a novel series of 1,3-di- and 1,3,5-trisubstituted 1,6-dihydro-6-iminopyridazines (DIPs) was designed using a versatile strategy and synthesized using facile methods. Electrophysiological studies showed that several target DIPs (30 µM) exhibited excellent antagonistic activities against common cutworm and housefly iGABARs consisting of RDL subunits. The IC50 values of 3-(4-methoxyphenyl), 3-(4-trifluoromethoxyphenyl), 3-(4-biphenylylphenyl), 3-(2-naphthyl), 3-(3,4-methylenedioxyphenyl), and 3,5-(4-methoxyphenyl) analogs ranged from 2.2 to 24.8 µM. Additionally, several 1,3-disubstituted DIPs, especially 3-(4-trifluoromethoxyphenyl) and 3-(3,4-methylenedioxyphenyl) analogs, exhibited moderate insecticidal activity against common cutworm larvae, with >60% mortality at a concentration of 100 mg kg-1 . Molecular docking studies showed that the oxygen atom on the three-substituted aromatic ring could form a hydrogen bond with Arg254, which may enhance the activity of these DIPs against housefly iGABARs. CONCLUSION: This systematic study indicated that the presence of a carboxyl side chain shorter by one methylene than that of gabazine at the 1-position of the pyridazine ring is effective for maintaining the stable binding of these DIPs in insect iGABARs. Our study provides important information for the design of novel insect iGABAR CAs. © 2022 Society of Chemical Industry.

State-dependent inhibition of GABA receptor channels by the ectoparasiticide fluralaner.

γ-Aminobutyric acid (GABA) receptors (GABARs) are ligand-gated Cl- channels, which cause an influx of Cl- that inhibits excitation in postsynaptic cells upon activation. GABARs are important targets for drugs and pest control chemicals. We previously reported that the isoxazoline ectoparasiticide fluralaner inhibits GABA-induced currents in housefly (Musca domestica) GABARs by binding to the putative binding site in the transmembrane subunit interface. In the present study, we investigated whether fluralaner inhibits the GABA response in the GABAR activated state, the resting state, or both, using two-electrode voltage clamp electrophysiology protocols. We found that inhibition progresses over time to steady-state levels by repeated short applications of GABA during fluralaner perfusion. The GABA response was not impaired by fluralaner treatment in the GABAR resting state. However, once inhibited, the GABA response was not restored by repeated applications of GABA. These findings suggest that fluralaner might reach the binding site of the activated conformation of GABARs in a stepwise fashion and tightly bind to it.

Structure-dependent receptor subtype selectivity and G protein subtype preference of heterocyclic agonists in heterologously expressed silkworm octopamine receptors.

(R)-Octopamine (OA), a major invertebrate biogenic amine, plays an important role in a wide variety of physiological processes as a neurohormone, neuromodulator, and neurotransmitter in insects. OA receptors (OARs) are class A G protein-coupled receptors that specifically bind OA to activate downstream signaling cascades by coupling to G proteins and presumably other regulatory proteins. These receptors are broadly classified as α- and ß-adrenergic-like OARs (α- and ß-ALOARs). OARs are considered important targets of insecticides and acaricides. In the present study, we examined the actions of an array of 13 heterocyclic OAR agonists with the moieties that correspond to the phenyl group and the basic nitrogen atom of OA on α- and ß-ALOARs from the silkworm (Bombyx mori) and the signaling pathways activated through these actions. The results indicated that these compounds display structure-dependent receptor subtype selectivity and G protein subtype preference, underscoring the need to determine which subtype and signaling pathway mediates toxicologically relevant effects for the efficient discovery of novel pest control chemicals. The results of insecticidal assays using B. mori larvae suggested that the activation of signal transduction pathways via α-ALOARs might be mainly responsible for the toxicological effects of the heterocycles.

Ion channels and G protein-coupled receptors as targets for invertebrate pest control: from past challenges to practical insecticides.

In the late 1970s, we discovered that toxic bicyclic phosphates inhibit the generation of miniature inhibitory junction potentials, implying their antagonism of γ-aminobutyric acid (GABA) receptors (GABARs; GABA-gated chloride channels). This unique mode of action provided a strong incentive for our research on GABARs in later years. Furthermore, minor structural changes conferred insect GABAR selectivity to this class of compounds, convincing us of the possibility of GABARs as targets for insecticides. Forty years later, third-generation insecticides acting as allosteric modulator antagonists at a distinctive site of action in insect GABARs were developed. G protein-coupled receptors (GPCRs) are also promising targets for pest control. We characterized phenolamine receptors functionally and pharmacologically. Of the tested receptors, ß-adrenergic-like octopamine receptors were revealed to be the most sensitive to the acaricide/insecticide amitraz. Given the presence of multiple sites of action, ion channels and GPCRs remain potential targets for invertebrate pest control.

Molecular cloning and pharmacology of Min-UNC-49B, a GABA receptor from the southern root-knot nematode Meloidogyne incognita.

BACKGROUND: Root-knot nematodes are plant-parasitic nematodes that cause immense damage to a broad range of cultivated crops by forming root galls, resulting in yield losses in crops. To facilitate the development of faster-acting selective nematicides, we cloned three cDNAs encoding UNC-49B proteins from the southern root-knot nematode Meloidogyne incognita and examined their functional and pharmacological properties by two-electrode voltage clamp electrophysiology using a Xenopus oocyte expression system. RESULTS: The three cloned cDNAs encoded Min-UNC-49B, Min-UNC-49B-L and Min-UNC-49B-XL; the last two proteins have longer N-terminal regions than the first protein. When expressed in Xenopus oocytes, these proteins responded to γ-aminobutyric acid (GABA) to activate currents with high-micromolar or low-millimolar half-maximal effective concentration (EC50 ) values, indicating the formation of functional homo-pentameric GABA receptors. Fipronil and picrotoxinin inhibited GABA-induced currents with high-nanomolar and low-micromolar half-maximal inhibitory concentration (IC50 ) values, respectively, in oocytes expressing Min-UNC-49B. The G2'A and T6'M mutations in the second transmembrane domain of Min-UNC-49B enhanced and reduced the sensitivity of Min-UNC-49B to these two antagonists, respectively. Samaderine B and SF-14 inhibited GABA responses in oocytes expressing Min-UNC-49B with low-micromolar and high-nanomolar IC50 values, respectively. Ivermectin, α-terpineol, thymol and methyl eugenol exerted dual effects on Min-UNC-49B by potentiating currents induced by low concentrations of GABA and inhibiting currents induced by high concentrations of GABA. CONCLUSION: We have shown that structurally diverse compounds act at Min-UNC-49B GABA receptors. Our results may serve as a starting point to decipher the molecular function of native GABA receptors of plant-parasitic nematodes, which could aid in the structure-based design of novel nematicides. © 2020 Society of Chemical Industry.

Further characterization of distinct high-affinity binding sites for dinotefuran in the abdominal nerve cord of the American cockroach Periplaneta americana (Blattodea).

Dinotefuran (DTF) is a systemic neonicotinoid insecticide characterized by a tetrahydrofuran ring. In the present study, we examined the characteristics of DTF binding to native nicotinic acetylcholine receptors (nAChRs) expressed in the American cockroach Periplaneta americana using radioligand-binding methods. The Scatchard analysis, using [3H]imidacloprid (IMI), indicated that IMI has a single class of high-affinity binding sites in the P. americana nerve cord. In contrast, the Scatchard analysis using [3H]DTF indicated that DTF has two different classes of binding sites. Both DTF and IMI were found to bind to one of the classes, for which DTF showed low affinity. The other class, for which DTF showed high affinity, was localized in the abdominal nerve cord but not in the thoracic nerve cord. IMI showed low affinity for the high-affinity DTF binding sites. Our data suggest that DTF binds with high affinity to a nAChR subtype distinct from the high-affinity subtype for IMI. This difference might be responsible, at least in part, for the difference in resistance development to DTF and IMI in P. americana.

A point mutation in the ß-adrenergic-like octopamine receptor: possible association with amitraz resistance.

BACKGROUND: Amitraz is a unique formamidine-class acaricide/insecticide that effectively controls ticks, mites, and insect pests. However, the recent emergence of amitraz-resistant cattle ticks is a serious problem that requires an urgent solution. A nonsynonymous single nucleotide polymorphism (A181T) leading to an amino acid substitution (I61F) in the ß-adrenergic-like (ß-AL) octopamine receptor (OAR) of amitraz-resistant southern cattle ticks (Rhipicephalus microplus) (RmßAOR) was proposed to be a cause of the amitraz resistance. However, it remains unclear whether this substitution exerts any functional effect on the action of amitraz. To make this clear, the functional role of this mutation was examined using an orthologous OAR (BmOAR2) from the silkworm (Bombyx mori). RESULTS: Both amitraz and its metabolite N2 -(2,4-dimethylphenyl)-N1 -methyformamidine (DPMF) elevated intracellular cyclic AMP levels as orthosteric OAR agonists in HEK-293 cells stably expressing BmOAR2. The I45F mutant of BmOAR2 (equivalent to I61F in RmßAOR) was generated and tested for its sensitivity to amitraz and DPMF. The assay result showed that the I45F mutation reduces the potency of DPMF to a level similar to that of the endogenous agonist (R)-OA in wild-type BmOAR2. CONCLUSION: The amino acid substitution found in the first transmembrane segment of RmßAOR most likely causes target-site insensitivity to DPMF, which might contribute to the resistance of R. microplus to amitraz. This needs to be further confirmed using RmßAOR. © 2020 Society of Chemical Industry.

Potential of Competitive Antagonists of Insect Ionotropic γ-Aminobutyric Acid Receptors as Insecticides.

Ionotropic γ-aminobutyric acid (GABA) receptors (GABARs) represent an important insecticide target. Currently used GABAR-targeting insecticides are non-competitive antagonists (NCAs) of these receptors. Recent studies have demonstrated that competitive antagonists (CAs) of GABARs have functions of inhibiting insect GABARs similar to NCAs and that they also exhibit insecticidal activity. CAs have different binding sites and different mechanisms of action compared to those of NCAs. Therefore, GABAR CAs should have the potential to be developed into novel insecticides, which could be used to overcome the developed resistance of insect pests to conventional NCA insecticides. Although research on insect GABAR CAs has lagged behind that on mammalian GABAR CAs, research on the CAs of insect ionotropic GABARs has made great progress in recent years, and several series of heterocyclic compounds, such as 3-isoxazolols and 6-iminopyridazines, have been identified as insect GABAR CAs. In this review, we briefly summarize the design strategies, structures, and biological activities of the novel GABAR CAs that have been found in the past decade. Updated information about GABAR CAs may benefit the design and development of novel GABAR-targeting insecticides.

Effects of intersubunit amino acid substitutions on GABA receptor sensitivity to the ectoparasiticide fluralaner.

The isoxazoline ectoparasiticide fluralaner exerts antiparasitic effects by inhibiting the function of γ-aminobutyric acid (GABA) receptors (GABARs). The present study was conducted to identify the amino acid residues that contribute to the high sensitivity of insect GABARs to fluralaner. We generated housefly (Musca domestica) GABARs with amino acid substitutions in the first through third α-helical transmembrane segments (TM1-TM3) of the RDL subunit using site-directed mutagenesis and examined the effects of the substitutions on the sensitivity of GABARs expressed in Xenopus oocytes to fluralaner using two-electrode voltage clamp electrophysiology. The Q271L substitution in TM1 caused a significant reduction in the sensitivity to fluralaner. Although the I274A and I274F substitutions in TM1 did not affect fluralaner sensitivity, the I274C substitution significantly enhanced the sensitivity to fluralaner. In contrast, the L278C substitution in TM1 reduced fluralaner sensitivity. Substitutions of Gly333 in TM3 led to substantial reductions in the sensitivity to fluralaner. These findings indicate that Gln271, Ile274, Leu278, and Gly333, which are situated in the outer half of the transmembrane subunit interface, are closely related to the antagonism of GABARs by fluralaner.

Spatiotemporally different expression of alternatively spliced GABA receptor subunit transcripts in the housefly Musca domestica.

Insect γ-aminobutyric acid (GABA) receptors are important as major inhibitory neurotransmitter receptors and targets for insecticides. The housefly GABA receptor subunit gene MdRdl is alternatively spliced at exons 3 (a or b) and 6 (c or d) to yield the variants of ac, ad, bc, and bd combinations. In the present study, the expression of the MdRdl transcript in the body parts and in the developmental stages of the housefly Musca domestica was examined by quantitative polymerase chain reaction using specific primers that amplify the combinations of alternative exons. The results indicated that the transcripts of MdRdl, including four combinations, were highly expressed in the adult stage. MdRdlbd was the most abundant in the adult head. The expression pattern did not change in the adult stage over 7 days after eclosion. The expression level of the MdRdl bd transcript in the female head was similar to that of the male head. In contrast, MdRdl bc was the predominant transcript in the pupal head and the adult leg. Because the homomeric Rdl bc GABA receptor has a high affinity for GABA, our results provide grounds for designing agonist or competitive-antagonist insecticides that target the orthosteric site of the GABA receptor containing this Rdl variant.

4-Aryl-5-carbamoyl-3-isoxazolols as competitive antagonists of insect GABA receptors: Synthesis, biological activity, and molecular docking studies.

Competitive antagonists (CAs) of ionotropic GABA receptors (GABARs) reportedly exhibit insecticidal activity and have potential for development as novel insecticides for overcoming emerging resistance to traditional GABAR-targeting insecticides. Our previous studies demonstrated that 4,5-disubstituted 3-isoxazolols or 3-isothiazolols are an important class of insect GABAR CAs. In the present study, we synthesized a series of 4-aryl-5-carbamoyl-3-isoxazolols and examined their antagonism of insect GABARs expressed in Xenopus oocytes. Several of these 3-isoxazolols exhibited potent antagonistic activities against housefly and common cutworm GABARs, with IC50 values in the low-micromolar range in both receptors. 4-(3-Amino-4-methylphenyl)-5-carbamoyl-3-isoxazolol (3u) displayed the highest antagonism, with IC50 values of 2.0 and 0.9 µM in housefly and common cutworm GABARs, respectively. Most of the synthesized 3-isoxazolols showed moderate larvicidal activities against common cutworms, with more than 50% mortality at 100 µg/g. These results indicate that 4-monocyclic aryl-5-carbamoyl-3-isoxazolol is a promising scaffold for insect GABAR CA discovery and provide important information for the design and development of GABAR-targeting insecticides with a novel mode of action.

Molecular cloning, spatiotemporal and functional expression of GABA receptor subunits RDL1 and RDL2 of the rice stem borer Chilo suppressalis.

Insect γ-aminobutyric acid (GABA) receptor (GABAR) is one of the major targets of insecticides. In the present study, cDNAs (CsRDL1A and CsRDL2S) encoding the two isoforms of RDL subunits were cloned from the rice stem borer Chilo suppressalis. Transcripts of both genes demonstrated similar expression patterns in different tissues and developmental stages, although CsRDL2S was ∼2-fold more abundant than CsRDL1A throughout all development stages. To investigate the function of channels formed by CsRDL subunits, both genes were expressed in Xenopus laevis oocytes singly or in combination in different ratios. Electrophysiological results using a two-electrode voltage clamp demonstrated that GABA activated currents in oocytes injected with both cRNAs. The EC50 value of GABA in activating currents was smaller in oocytes co-injected with CsRDL1A and CsRDL2S than in oocytes injected singly. The IC50 value of the insecticide fluralaner in inhibiting GABA responses was smaller in oocytes co-injected with different cRNAs than in oocytes injected singly. Co-injection also changed the potency of the insecticide dieldrin in oocytes injected singly. These findings suggested that heteromeric GABARs were formed by the co-injections of CsRDL1A and CsRDL2S in oocytes. Although the presence of Ser at the 2'-position in the second transmembrane segment was responsible for the insensitivity of GABARs to dieldrin, this amino acid did not affect the potencies of the insecticides fipronil and fluralaner. These results lead us to hypothesize that C. suppressalis may adapt to insecticide pressure by regulating the expression levels of CsRDL1A and CsRDL2S and the composition of both subunits in GABARs.

Fluxametamide: A novel isoxazoline insecticide that acts via distinctive antagonism of insect ligand-gated chloride channels.

Fluxametamide is a novel wide-spectrum insecticide that was discovered and synthesized by Nissan Chemical Industries, Ltd. To identify the mode of action of fluxametamide, we first performed [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) binding assays. Fluxametamide potently inhibited the specific binding of [3H]EBOB to housefly-head membranes, suggesting that fluxametamide affects insect γ-aminobutyric acid (GABA)-gated chloride channels (GABACls). Next, the antagonism of housefly GABACls and glutamate-gated chloride channels (GluCls) was examined using the two-electrode voltage clamp (TEVC) method. Fluxametamide inhibited agonist responses in both ion channels expressed in Xenopus oocytes in the nanomolar range, indicating that this insecticide is a ligand-gated chloride channel (LGCC) antagonist. The insecticidal and LGCC antagonist potencies of fluxametamide against fipronil-susceptible and fipronil-resistant strains of small brown planthoppers and two-spotted spider mites, which are insensitive to fipronil, were evaluated. Fluxametamide exhibited similar levels of both activities in these fipronil-susceptible and fipronil-resistant arthropod pests. These data indicate that fluxametamide exerts distinctive antagonism of arthropod GABACls by binding to a site different from those for existing antagonists. In contrast to its profound actions on the arthropod LGCCs, the antagonistic activity of fluxametamide against rat GABACls and human glycine-gated chloride channels was nearly insignificant, suggesting that fluxametamide has high target-site selectivity for arthropods over mammals. Overall, fluxametamide is a new type of LGCC antagonist insecticide with excellent safety for mammals at the target-site level.