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.

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.

Mechanisms underlying the selectivity of meta-diamides between insect resistance to dieldrin (RDL) and human γ-aminobutyric acid (GABA) and glycine receptors.

BACKGROUND: Meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] show high insecticide activity by acting as antagonists to the insect resistance to dieldrin (RDL) γ-aminobutyric acid (GABA) receptors. In contrast, low-level antagonist activities of meta-diamides have been demonstrated against the human GABA type A receptor (GABAA R) α1ß2γ2S, mammalian GABAA R α1ß3γ2S, and the human glycine receptor (GlyR) α1ß. Glycine residue 336 in the membrane-spanning region M3 of the Drosophila RDL GABA receptor is essential for its high sensitivity to meta-diamide 7, [3-benzamido-N-(2-bromo-4-(perfluoropropan-2-yl)-6-(trifluoromethyl)phenyl)-2-fluorobenzamide]. RESULTS: We examined the effects of an equivalent mutation (M288G) in spontaneously opened human GABAA R ß3 homomers using membrane potential assay. Picrotoxin and fipronil blocked spontaneously opened human GABAA Rs ß3 and ß3-M286G in a concentration-dependent manner. In contrast, meta-diamide 7 did not block spontaneously opened GABAA R ß3 homomers, although meta-diamide 7 blocked spontaneously opened GABAA R ß3-M286G homomers. In addition, inhibitory potency of meta-diamide 7 for GABA-induced membrane potential change in cells expressing GABAA R α1ß3-M286G was much higher than that in cells expressing GABAA R α1ß3. In the same way, the equivalent mutation (A288G) in GlyR α1 increased the inhibitory potency of meta-diamide 7 for GlyRs α1 and α1ß. CONCLUSION: Studies substituting an equivalent mutation (M288G) in spontaneously opening human GABAA R ß3 homomers and human GABAA Rs α1ß3 heteromers suggest that M286 in human GABAA R ß3 is important for the low sensitivity to meta-diamide 7. In this study, we summarize the mechanisms underlying the selectivity of meta-diamides between insect RDL and human GABA and glycine receptors. © 2020 Society of Chemical Industry.

Important amino acids for function of the insect Rdl GABA receptor.

BACKGROUND: Insect Rdl GABA receptor is an important insecticide target. To design a novel insecticide, studies on the structures of homologous pentameric ligand-gated ion channels provide information about important amino acids that are necessary for the function of insect Rdl GABA receptors. RESULTS: L9'A, T12'A, T13'A, T13'S, M15'S, and M15'N mutations in the Drosophila Rdl GABA receptor subunit caused the protein to spontaneously adopt the open state conformation. In contrast, the S16'A, S16'T, S17'A, and S17'H mutant homomers showed the same levels of agonist and antagonist sensitivity as the wild-type receptor. The G336M mutation in the Drosophila Rdl GABA receptor abolished the agonist activities of ivermectin and milbemectin, but the F339M mutation did not. Additionally, the F339M mutation caused spontaneous opening of the receptor. In the Drosophila Rdl model, the hydrophobic girdle plays an important role in stabilization of the closed state. Mutations which decrease hydrophobic interactions resulted in spontaneous opening, supporting the importance of the hydrophobic girdle for keeping the channel closed. Through a mutational study of transmembrane 3 (TM3) cytoplasmic domain and Rdl GABA receptor modeling, hydrophobic interactions between TM3 and TM4 and intersubunit interaction were demonstrated to be important for channel gating. Alternatively, the intrasubunit interaction between TM2 and TM3 domains were less important for channel gating in case of Drosophila Rdl GABA receptor. CONCLUSION: This study demonstrates important amino acids critical to the function of the Drosophila Rdl GABA receptor based on the mutational studies and Drosophila Rdl GABA receptor modeling approach. © 2020 Society of Chemical Industry.

Insecticides, biologics and nematicides: Updates to IRAC's mode of action classification - a tool for resistance management.

Insecticide resistance has been and continues to be a significant problem for invertebrate pest control. As such, effective insecticide resistance management (IRM) is critical to maintain the efficacy of current and future insecticides. A technical group within CropLife International, the Insecticide Resistance Action Committee (IRAC) was established 35 years ago (1984) as an international association of crop protection companies that today spans the globe. IRAC's focus is on preserving the long-term utility of insect, mite, and most recently nematode control products through effective resistance management to promote sustainable agriculture and improved public health. A central task of IRAC has been the continual development and documentation of the Mode of Action (MoA) Classification scheme, which serves as an important tool for implementing IRM strategies focused on compound rotation / alternations. Updates to the IRAC MoA Classification scheme provide the latest information on the MoA of current and new insecticides and acaricides, and now includes information on biologics and nematicides. Details for these new changes and additions are reviewed herein.

Differential metabolism of neonicotinoids by brown planthopper, Nilaparvata lugens, CYP6ER1 variants.

Imidacloprid is very effective in controlling Nilaparvata lugens Stål, which severely damages rice plants. Following heavy imidacloprid use, imidacloprid-resistant N. lugens, which showed cross-resistance to other neonicotinoids, appeared. We used the baculovirus/Sf9 expression system to express CYP6ER1 variants carrying A375del + A376G (del3) mutations, either with or without T318S mutation, which confer imidacloprid resistance in N. lugens. These CYP6ER1 variants metabolized imidacloprid but did not metabolize dinotefuran. Moreover, Drosophila expressing a CYP6ER1 variant carrying T318S + del3 mutations were resistant to imidacloprid, with a resistance ratio of 288.7, whereas the resistance ratio to dinotefuran was 3.6. These findings indicate that N. lugens has a low level of resistance to dinotefuran, and the increase of resistance is slow. We also studied the metabolism of other neonicotinoids, as well as sulfoxaflor and flupyradifurone, by CYP6ER1 variants carrying del3 mutations, either with or without the T318S mutation. Sulfoxaflor, was not metabolized by either CYP6ER1-del3 or CYP6ER1-T318Sdel3 variants. However, these variants did metabolize flupyradifurone. This study sheds light on the substrate selectivity of CYP6ER1 variants.

Differential metabolism of neonicotinoids by Myzus persicae CYP6CY3 stably expressed in Drosophila S2 cells.

The peach-potato aphid, Myzus persicae, is a serious crop pest that has developed imidacloprid resistance, mainly through overexpression of CYP6CY3. Here, we established a metabolic assay using Drosophila S2 cells that stably expressed CYP6CY3. We found that CYP6CY3 showed metabolic activity against imidacloprid, as well as acetamiprid, clothianidin, and thiacloprid, but had no activity against dinotefuran. Our study suggested that stable gene expression in Drosophila S2 cells is useful for examining which insecticide is metabolized by P450 monooxygenases.

Differential metabolism of imidacloprid and dinotefuran by Bemisia tabaci CYP6CM1 variants.

Imidacloprid has been used to control one of most serious pests, Bemisia tabaci. However, B. tabaci has developed imidacloprid resistance mainly by over-expressing CYP6CM1. It was reported that imidacloprid-resistant B. tabaci showed no or low level of cross-resistance against dinotefuran. Here, we expressed CYP6CM1 variants using Sf9/baculovirus and/or Drosophila S2 cells and showed that CYP6CM1 variants metabolized imidacloprid but not dinotefuran. In addition, we demonstrated that imidacloprid and pymetrozine competed for a CYP6CM1 variant more efficiently than dinotefuran, using a luminescent substrate competition assay. These results suggest that lack of metabolic activity of CYP6CM1 variants against dinotefuran caused no or low level of cross-resistance.

Mechanisms of resistance to insecticides targeting RDL GABA receptors in planthoppers.

Lindane and cyclodienes, such as dieldrin and α-endosulfan, represent the first generation of noncompetitive antagonists (NCAs) against the insect RDL GABA receptor. It has been reported that A2'S and A2'G mutations in the membrane-spanning region M2 of the RDL GABA receptor confer resistance to lindane and cyclodienes. Fipronil is a second-generation NCA, but A2'S and A2'G mutations provide a low level of cross-resistance to fipronil. Moreover, a novel A2'N mutation that confers fipronil resistance was found in M2 of the RDL GABA receptors from fipronil-resistant planthoppers in the heterozygous state. Thus, problems are being caused by a worldwide appearance of cyclodiene-resistant pests. And attention must be paid to development of fipronil-resistant planthoppers, such as Sogatella furcifera and Laodelphax striatellus in Japan.

Broflanilide: A meta-diamide insecticide with a novel mode of action.

Broflanilide is a meta-diamide [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamide] that exhibits high larvicidal activity against Spodoptera litura. It has been suggested that broflanilide is metabolized to desmethyl-broflanilide and that it acts as a noncompetitive resistant-to-dieldrin (RDL) γ-aminobutyric acid (GABA) receptor antagonist. The binding site of desmethyl-broflanilide was demonstrated to be distinct from that of conventional noncompetitive antagonists such as fipronil. It has been proposed that the site of action for desmethyl-broflanilide is close to G336 in the M3 region of the Drosophila RDL GABA receptor. However, although the site of action for desmethyl-broflanilide appears to overlap with that of macrocyclic lactones, different modes of actions have been demonstrated for desmethyl-broflanilide and the macrocyclic lactones. The mechanisms underlying the high selectivity of meta-diamides are also discussed in this review. Broflanilide is expected to become a prominent insecticide because it is effective against pests with resistance to cyclodienes and fipronil.

Minireview: Mode of action of meta-diamide insecticides.

Meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] are a distinct class of RDL GABA receptor noncompetitive antagonists showing high insecticidal activity against Spodoptera litura. The mode of action of the meta-diamides was demonstrated to be distinct from that of conventional noncompetitive antagonists (NCAs) such as fipronil, picrotoxin, lindane, dieldrin, and α-endosulfan. It was suggested that meta-diamides act at or near G336 in the M3 region of the Drosophila RDL GABA receptor. Although the site of action of the meta-diamides appears to overlap with that of macrocyclic lactones including avermectins and milbemycins, differential effects of mutations on the actions of the meta-diamides and the macrocyclic lactones were observed. Molecular modeling studies revealed that the meta-diamides may bind to an inter-subunit pocket near G336 in the Drosophila RDL GABA receptor better when in the closed state, which is distinct from the NCA-binding site, which is in a channel formed by M2s. In contrast, the macrocyclic lactones were suggested to bind to an inter-subunit pocket near G336 in the Drosophila RDL GABA receptor when in the open state. Furthermore, mechanisms underlying the high selectivity of meta-diamides are discussed. This minireview highlights the unique features of novel meta-diamide insecticides and demonstrates why meta-diamides are anticipated to become prominent insecticides that are effective against pests resistant to cyclodienes and fipronil.

Comparison between the modes of action of novel meta-diamide and macrocyclic lactone insecticides on the RDL GABA receptor.

Macrocyclic lactones, avermectins, and milbemycins are widely used to control arthropods, nematodes, and endo- and ectoparasites in livestock and pets. Their main targets are glutamate-gated chloride channels. Furthermore, macrocyclic lactones reportedly interact with insect RDL γ-aminobutyric acid (GABA) receptors, but their modes of action on insect RDL GABA receptors remain unknown. In this study, we attempted to better understand the modes of action of macrocyclic lactones on RDL GABA receptors. We observed that ivermectin and milbemectin behaved as allosteric agonists of the Drosophila RDL GABA receptor. G336A, G336S, and G336T mutations had profound effects on the activities of ivermectin and milbemectin, and a G336M mutation abolished the allosteric agonist and antagonist activities of these macrocyclic lactones. These results suggest that G336 in TM3 of the Drosophila RDL GABA receptor is important for the binding of macrocyclic lactones. Recently, it has been suggested that a novel RDL GABA receptor antagonist, 3-benzamido-N-(2-bromo-4-perfluoroisopropyl-6-(trifluoromethyl)phenyl)-2-fluorobenzamide (meta-diamide 7), binds to the transmembrane intersubunit pocket near G336 in the Drosophila RDL GABA receptor. Thus, we compared the effects of mutations around G336 and A302 mutations in TM2 on the activities of macrocyclic lactone and meta-diamide 7. The effects of L281C, V340Q, V340N, A302S, and A302N mutations on the activity of meta-diamide 7 differed from those on ivermectin and milbemectin. Molecular modeling studies showed that macrocyclic lactones docked in the intersubunit pocket near G336 in the Drosophila RDL GABA receptor in the open state. In contrast, meta-diamide 7 docked into the Drosophila RDL GABA receptor in the closed state. This suggests that the modes of action of macrocyclic lactone binding to the wild-type Drosophila RDL GABA receptor differ from those of meta-diamide binding.

Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors.

γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [(3)H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [(3)H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [(3)H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [(3)H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [(3)H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of [(3)H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides in insect GABARs.

Meta-diamide insecticides acting on distinct sites of RDL GABA receptor from those for conventional noncompetitive antagonists.

The RDL GABA receptor is an attractive target of insecticides. Here we demonstrate that meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] are a distinct class of RDL GABA receptor antagonists showing high insecticidal activity against Spodoptera litura. We also suggest that the mode of action of the meta-diamides is distinct from that of conventional noncompetitive antagonists (NCAs), such as fipronil, picrotoxin, lindane, dieldrin, and α-endosulfan. Using a membrane potential assay, we examined the effects of the meta-diamide 3-benzamido-N-(2-bromo-4-(perfluoropropan-2-yl)-6-(trifluoromethyl)phenyl)-2-fluorobenzamide (meta-diamide 7) and NCAs on mutant Drosophila RDL GABA receptors expressed in Drosophila Mel-2 cells. NCAs had little or no inhibitory activity against at least one of the three mutant receptors (A2'S, A2'G, and A2'N), which were reported to confer resistance to NCAs. In contrast, meta-diamide 7 inhibited all three A2' mutant receptors, at levels comparable to its activity with the wild-type receptor. Furthermore, the A2'S·T6'V mutation almost abolished the inhibitory effects of all NCAs. However, meta-diamide 7 inhibited the A2'S・T6'S mutant receptor at the same level as its activity with the wild-type receptor. In contrast, a G336M mutation in the third transmembrane domain of the RDL GABA receptor abolished the inhibitory activities of meta-diamide 7, although the G336M mutation had little effect on the inhibitory activities of conventional NCAs. Molecular modeling studies also suggested that the binding site of meta-diamides was different from those of NCAs. Meta-diamide insecticides are expected to be prominent insecticides effective against A2' mutant RDL GABA receptors with a different mode of action.

Concentration-dependent effects of GABA on insensitivity to fipronil in the A2'S mutant RDL GABA receptor from fipronil-resistant Oulema oryzae (Coleoptera: Chrysomelidae).

The beetle Oulema oryzae Kuwayama (Coleoptera: Chrysomelidae), an important pest of rice, has developed fipronil resistance in Japan. Molecular cloning and sequence analysis of O. oryzae RDL gamma-aminobutyric acid (GABA) receptor subunit (OO-RDL) genes from fipronil-susceptible and -resistant O. oryzae identified the A2'S mutation (index number for the M2 membrane-spanning region). To investigate the effect of the A2'S mutation on fipronil resistance, we stably expressed the wild-type and mutant OO-RDL homomers in Drosophila Mel-2 cells. A membrane potential assay exhibited that the IC50 values of fipronil for inhibition of the response to EC80 GABA of the wild-type and A2'S mutant OO-RDL homomers were 0.09 microM and 0.11 microM, respectively. However, the IC50 values of fipronil for inhibition of the response to EC95 GABA of the wild-type and A2'S mutant OO-RDL homomers were 0.11 microM and approximately equal to 5 microM, respectively. These results suggest that the GABA concentration is an important factor affecting fipronil resistance in O. oryzae carrying the A2'S mutation in OO-RDL.

The A2'N mutation of the RDL gamma-aminobutyric acid receptor conferring fipronil resistance in Laodelphax striatellus (Hemiptera: Delphacidae).

The planthopper Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae) is a serious insect pest of rice, Oryza sativa L., and has developed resistance to fipronil in Japan. Sequence analysis of L. striatellus RDL gamma-aminobutyric acid (GABA) receptor subunit (LS-RDL) genes from a fipronil-resistant population and a fipronil-susceptible strain identified the A2'N mutation (index number for M2 membrane-spanning region), that was previously implicated in fipronil resistance in the planthopper Sogatella furcifera (Horváth) (Hemiptera: Delphacidae). Nineteen of 21 fipronil-resistant L. striatellus individuals were genotyped as heterozygous for the A2'N mutation, suggesting that this mutation is associated with fipronil resistance and that most fipronil-resistant L. striatellus express wild-type and A2'N mutant LS-RDL simultaneously. To confirm the role of the A2'N mutation of LS-RDL, Drosophila Mel-2 cells were transfected with wild-type and A2'N mutant LS-RDL genes, either individually or together. A membrane potential assay showed that fipronil had no inhibitory effect at 10 microM on cells transfected with the A2'N mutant LS-RDL gene with or without the wild-type LS-RDL gene. By contrast, the IC50 value of fipronil for wild-type LS-RDL homomers was 14 nM. These results suggest that the A2'N mutation of the RDL GABA receptor subunit confers fipronil resistance in L. striatellus as well as S. furcifera.

Modulation of androgen receptor transcriptional activity by anti-acne reagents.

BACKGROUND: To study the potential anti-androgenic activity of roxithromycin (RXM), we previously used human dermal fibroblasts transiently transfected with the expression vector of androgen receptor (AR) coactivator ARA55 as the in vitro model reflecting the end-organ hypersensitivity. OBJECTIVE: To examine the potential anti-androgenic activity of anti-acne therapeutic agents, nadifloxacin (NDFX), RXM, all-trans retinoic acid (atRA), and glycolic acid (GA), we carried out the transient transfection assays using the CV-1 cells as a more sensitive assay system. RESULTS: The result showed that 5 microg/ml of RXM suppress 10(-9)M R1881-induced AR transcriptional activity by 21.2%. 50 microg/ml of NDFX can suppress AR transcriptional activity to 29.8%. Furthermore, the assays with treatment of 1, 5, 10, or 50 microg/ml NDFX in the presence of 1 microg/ml RXM showed that 5, 10, or 50 microg/ml NDFX inhibits the AR transactivity by 32.7, 31.1 or 61.0%, respectively, indicating the synergistic effect of NDFX and RXM. Besides 10(-5)M atRA suppressed the R1881-induced luciferase activity by 50%, but GA did not alter AR transactivity. CONCLUSIONS: We demonstrated that anti-acne agents available in the clinical practice can exert anti-androgenic effects in the treatment of acne.