Synthesis and activity-detection of photoswitchable ligands with fipronil to insect.

BACKGROUND: Ionotropic γ-aminobutyric acid (GABA) receptor (GABAR) in an insect is the major inhibitory receptor and is one of the most important targets for insecticides. Due to the high spatiotemporal resolution of GABAR, the photopharmacological ligands acting on it in vertebrates but not insect have been developed. RESULTS: In this study, two types of photochromic ligands (PCLs) including DTFIPs (DTFIP1 and DTFIP2) and ABFIPs (p-, m-, and o-ABFIP) were synthesized by incorporating photoswitch azobenzene or dithienylethene into fipronil (FIP), which is the antagonist of insect GABAR. Their photomodulation was measured by mosquito larval behavior, and their potential action mechanism was explored by the two-electrode voltage clamp (TEVC) technique in vitro. DTFIP1 and m-ABFIP exhibited the most significant difference of insecticidal activity by about 90- and 5-fold to mosquito larvae between non-irradiated and irradiated formation, respectively, and allowed for optical control of mosquito swimming activity. TEVC assay results indicated that m-ABFIP and DTFIP1 enable optical control over the homomeric LsRDL-type GABAR, which is achieved by regulating the chloride channel of resistance to dieldrin (RDL)-type GABAR by photoisomerization. CONCLUSION: Our results suggested that PCLs synthesized from fipronil provide an alternative and precise tool for studying insect ionotropic GABARs and GABA-dependent behavior. © 2022 Society of Chemical Industry.

Sublethal doses of broflanilide prevents molting in the fall armyworm, Spodoptera frugiperda via altering molting hormone biosynthesis.

Broflanilide is a novel insecticide with a unique mode of action on the insect GABA receptor and is registered worldwide for the control of agricultural pests. It shows high efficacy in controlling the fall armyworm (FAW) Spodoptera frugiperda, which is a destructive pest to various crops. FAW was exposed to sublethal concentrations of broflanilide to determine its impact on insect development. Sublethal doses (LD10 and LD30) caused failure of ecdysis, reduced body length of larvae, malformation of pupae, and vestigial wing formation in adults. Also, broflanilide at LD30 significantly reduced the amount of molting hormone (MH). After exposure to LD10 or LD30 broflanilide, expression of five Halloween genes, which participate in MH biosynthesis, were found to be altered. Specifically, the transcript levels of SfrCYP307A1 (Spook), SfrCYP314A1 (Shade) and SfrCYP315A1 (Shadow) in 3rd day larvae were significantly decreased as well as SfrCYP302A1 (Disembodied) and SfrCYP306A1 (Phantom) in 5th day pupae. In contrast, the transcript levels of SfrCYP302A1 in 3rd day larvae, SfrCYP307A1 and SfrCYP314A1 in 5th day pupae, and SfrCYP306A1, SfrCYP307A1 and SfrCYP315A1 in 0.5th day adults were significantly increased. Our results demonstrate that broflanilide caused the failure of ecdysis in FAW possibly by influencing the intake of cholesterol through inhibition of feeding and also via altering expression of genes important for MH biosynthesis.

Functional Characteristics of the Lepidopteran Ionotropic GABA Receptor 8916 Subunit Interacting with the LCCH3 or the RDL Subunit.

The ionotropic γ-aminobutyric acid (iGABA) receptor is commonly considered as a fast inhibitory channel and is an important insecticide target. Since 1990, RDL, LCCH3, and GRD have been successively isolated and found to be potential subunits of the insect iGABA receptor. More recently, one orphan gene named 8916 was found and considered to be another potential iGABA receptor subunit according to its amino acid sequence. However, little information about 8916 has been reported. Here, the 8916 subunit from Chilo suppressalis was studied to determine whether it can form part of a functional iGABA receptor by co-expressing this subunit with CsRDL1 or CsLCCH3 in the Xenopus oocyte system. Cs8916 or CsLCCH3 did not form functional ion channels when expressed alone. However, Cs8916 was able to form heteromeric ion channels when expressed with either CsLCCH3 or CsRDL1. The recombinant heteromeric Cs8916/LCCH3 channel was a cation-selective channel, which was sensitive to GABA or ß-alanine. The current of the Cs8916/LCCH3 channel was inhibited by dieldrin, endosulfan, fipronil, or ethiprole. In contrast, fluralaner, broflanilide, and avermectin showed little effect on the Cs8916/LCCH3 channel (IC50s > 10 000 nM). The Cs8916/RDL1 channel was sensitive to GABA, but was significantly different in EC50 and Imax for GABA to those of homomeric CsRDL1. Fluralaner, fipronil, or dieldrin showed antagonistic actions on Cs8916/RDL1. In conclusion, Cs8916 is a potential iGABA receptor subunit, which can interact with CsLCCH3 to generate a cation-selective channel that is sensitive to several insecticides. Also, as Cs8916/RDL1 has a higher EC50 than homomeric CsRDL1, Cs8916 may affect the physiological functions of CsRDL1 and therefore play a role in fine-tuning GABAergic signaling.

Novel alternative splicing of GABA receptor RDL exon 9 from Laodelphax striatellus modulates agonist potency.

The resistance to dieldrin gene (RDL) encodes the primary subunit of the insect ionotropic γ-aminobutyric acid (GABA) receptor (GABAR), which is the target of phenylpyrazole and isoxazoline insecticides. The splice variants in exons 3 and 6 of RDL, which have been widely explored in many insects, modulate the agonist potency of the homomeric RDL GABAR and potentially play an important role in the development of insects. In the present study, four splice variants of exon 9 were identified in RDL of the small brown planthopper, Laodelphax striatellus (LsRDL), resulting in LsRDL-9a, LsRDL-9a', LsRDL-9b, and LsRDL-9c. LsRDL-9a has one more amino acid (E, glutamic acid) compared with LsRDL-9a', and LsRDL-9b lacked two amino acids and had seven different amino acids compared with LsRDL-9c. Two-electrode voltage-clamp recording on LsRDLs expressed in Xenopus oocytes showed that alternative splicing of exon 9 has significant impact on LsRDL sensitivity to the agonists GABA and ß-alanine, whereas no significant difference was observed in the potencies of the non-competitive antagonists (NCAs) ethiprole and fluralaner on the splice variants. Our results suggest that alternative splicing of RDL exon 9 broadens functional capabilities of the GABAR in L. striatellus by influencing the action of GABA.

Acute toxicity, bioconcentration, elimination, action mode and detoxification metabolism of broflanilide in zebrafish, Danio rerio.

Broflanilide, a novel meta-diamide insecticide, shows high insecticidal activity against agricultural pests and is scheduled to be launched onto the market in 2020. However, little information about its potential toxicological effects on fish has been reported. In this study, broflanilide showed low toxicity to the zebrafish, Danio rerio, with LC50 > 10 mg L-1 at 96 h and also did not inhibit GABA-induced currents of the heteromeric Drα1ß2Sγ2 GABA receptor. Broflanilide showed medium bioconcentration level with a bioconcentration factor at steady state (BCFss) of 10.02 and 69.40 in D. rerio at 2.00 mg L-1 and 0.20 mg L-1, respectively. In the elimination process, the concentration of broflanilide rapidly decreased within two days and slowly dropped below the limit of quantification after ten days. In the 2.00 mg L-1 broflanilide treatment, CYP450 activity was significantly increased up to 3.11-fold during eight days. Glutathione-S- transferase (GST) activity significantly increased by 91.44 % within four days. In conclusion, the acute toxicity of broflanilide was low, but it might induce chronic toxicity, affecting metabolism. To our knowledge, this is the first report of the toxicological effects of broflanilide on an aquatic organism, which has the potential to guide the use of broflanilide in the field.

Lethal and sublethal effects of fluralaner on the two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae).

BACKGROUND: Two-spotted spider mite (TSSM), Tetranychus urticae Koch is a serious agricultural pest and has developed high levels of resistance to many acaricides. Hence, screening new acaricides is important in the control of TSSM. Fluralaner, a novel isoxazoline, has high activity against agricultural and ectoparasitic pests; however, little information is available about its effects on agricultural mites. Lethal and sublethal effects of fluralaner towards TSSM eggs and female adults were therefore evaluated in this study. RESULTS: Fluralaner exhibited higher activity towards TSSM female adults (LC50 , 0.49 mg L-1 ) compared with commercial acaricides including fenpyroximate, pyridaben and hexythiazox, and also excellent activity towards eggs (LC50 , 0.36 mg L-1 ). The results of a sublethal effect study showed a significant reduction in the duration and fecundity of female adults with increases in the tested concentrations. The net reproductive rate (R0 ) decreased to 67.34 ± 3.96 and 50.29 ± 3.40 offspring per individual in LC10 and LC30 treatments compared with control (108.31 ± 7.61 offspring per individual). The intrinsic rate of increase (r) and finite rate of increase (λ) in the controls (0.30 and 1.36 d-1 ) were significantly higher than in the LC30 treatment (0.25 and 1.29 d-1 ); whereas, they were almost identical to values for the LC10 treatment. No change in mean generation time (T) was observed at sublethal concentrations treatments. CONCLUSION: Our results demonstrated that fluralaner has high acaricidal activity and could be considered a potential acaricide for TSSM management. © 2019 Society of Chemical Industry.

Neurotoxicity and mode of action of fluralaner on honeybee Apis mellifera L.

BACKGROUND: Fluralaner, a novel pesticide that targets the γ-aminobutyric acid (GABA) receptor (GABAR) subunit of resistant to dieldrin (RDL), exhibits strong potential to be an insecticide to control agricultural insect pests. However, the risk and action of fluralaner to economic insects, e.g., honeybee Apis mellifera Linnaeus, remains unclear. RESULTS: In this study, both oral and contact toxicity of fluralaner to honeybee were found to be 0.13 µg adult-1 . Abamectin, dieldrin, ethiprole, α-endosulfan, fipronil and fluralaner strongly inhibited the GABA-induced current in A. mellifera RDL (AmRDL), expressed in Xenopus laevis oocytes, with median inhibitory concentration (IC50 ) values of 7.99, 868.1, 27.10, 412.0, 11.21 and 13.59 nM, respectively. The binding free energy and electrophysiological response of AmRDL and insecticides were opposite. The correlation values between toxicity (to A. mellifera) and binding free energy/electrophysiological inhibition (to AmRDL) were at a moderate level. CONCLUSION: In conclusion, we report for the first time the notable risk of fluralaner to honeybee in vivo and compared the actions of GABAR-targeted insecticides on the AmRDL receptor. © 2019 Society of Chemical Industry.

Interaction of insecticides with heteromeric GABA-gated chloride channels from zebrafish Danio rerio (Hamilton).

The ionotropic GABAA receptor (GABAAR) is the main fast inhibitory post-synaptic receptor and is also an important insecticidal target. Effect of insecticides on fish has attracted intensive attention. However, no systematic study on heteromeric zebrafish GABAAR expressed in oocytes has been reported to date. In this study, the α1 subunit, the ß2S subunit lacking 47 amino acid residues compared with the ß2L subunit, and the γ2 subunit having five transmembrane domains were isolated from zebrafish Danio rerio. The responses of homomeric and heteromeric (α1, ß2S and γ2) channels to agonists and GABAAR-targeted compounds were detected with two-electrode voltage clamp. Dose-dependent responses were observed in heteromeric α1ß2S, ß2Sγ2, and α1ß2Sγ2 GABAR channels with EC50 values at 21.75, 6291, and 33.69 µM for GABA-induced current and 3.28, 155.5, and 3.79 mM for ß-alanine-induced current, respectively. However, no response was induced by benzamidine in all GABAR channels. Abamectin, dieldrin, fluralaner and fipronil could strongly inhibited GABA-induced inward current ≥50% at 10-6 M, while α-endosulfan, flufiprole and ethiprole only inhibited GABA-induced current <50%. This study has clarified the interaction of insecticides with the heteromeric GABAAR channel, which could help us further explore the potential function and toxicological importance of GABAARs from D. rerio.

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.