G3'MTMD3 in the insect GABA receptor subunit, RDL, confers resistance to broflanilide and fluralaner.

Meta-diamides (e.g. broflanilide) and isoxazolines (e.g. fluralaner) are novel insecticides that target the resistant to dieldrin (RDL) subunit of insect γ-aminobutyric acid receptors (GABARs). In this study, we used in silico analysis to identify residues that are critical for the interaction between RDL and these insecticides. Substitution of glycine at the third position (G3') in the third transmembrane domain (TMD3) with methionine (G3'M TMD3), which is present in vertebrate GABARs, had the strongest effect on fluralaner binding. This was confirmed by expression of RDL from the rice stem borer, Chilo suppressalis (CsRDL) in oocytes of the African clawed frog, Xenopus laevis, where the G3'MTMD3 mutation almost abolished the antagonistic action of fluralaner. Subsequently, G3'MTMD3 was introduced into the Rdl gene of the fruit fly, Drosophila melanogaster, using the CRISPR/Cas9 system. Larvae of heterozygous lines bearing G3'MTMD3 did not show significant resistance to avermectin, fipronil, broflanilide, and fluralaner. However, larvae homozygous for G3'MTMD3 were highly resistant to broflanilide and fluralaner whilst still being sensitive to fipronil and avermectin. Also, homozygous lines showed severely impaired locomotivity and did not survive to the pupal stage, indicating a significant fitness cost associated with G3'MTMD3. Moreover, the M3'GTMD3 mutation in the mouse Mus musculus α1ß2 GABAR increased sensitivity to fluralaner. Taken together, these results provide convincing in vitro and in vivo evidence for both broflanilide and fluralaner acting on the same amino acid site, as well as insights into potential mechanisms leading to target-site resistance to these insecticides. In addition, our findings could guide further modification of isoxazolines to achieve higher selectivity for the control of insect pests with minimal effects on mammals.

Sublethal effect and detoxifying metabolism of metaflumizone and indoxacarb on the fall armyworm, Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera, Noctuidae), is a highly polyphagous invasive pest that damages various crops. Pesticide control is the most common and effective strategy to control FAW. In this study, we evaluated the toxicity of metaflumizone and indoxacarb against third-instar FAW larvae using the insecticide-incorporated artificial diet method under laboratory conditions. Both metaflumizone and indoxacarb exhibited substantial toxicity against FAW, with LC50 values of 2.43 and 14.66 mg/L at 72 h, respectively. The sublethal effects of metaflumizone and indoxacarb on parental and F1 generation FAW were investigated by exposing third-instar larvae to LC10 and LC30 concentrations of these insecticides. Sublethal exposure to these two insecticides significantly shortened adult longevity, extended pupal developmental times and led to reduced pupal weight, pupation rates, and adult fecundity in the treated parental generation and F1 generation at LC10 or LC30 concentrations, in comparison to the control group. The larval developmental times were shortened in the parental generation but prolonged in the F1 generation, after being treated with sublethal concentrations of metaflumizone. Furthermore, larvae exposed to LC10 or LC30 concentrations of indoxacarb exhibited elevated activity levels of cytochrome P450 monooxygenase and glutathione S-transferase, which coincides with the observed synergistic effect of piperonyl butoxide and diethyl maleate. In conclusion, the high toxicity and negative impact of metaflumizone and indoxacarb on FAW provided significant implications for the rational utilization of insecticides against this pest.

Identification and characterization of odorant receptors in Plutella xylostella antenna response to 2,3-dimethyl-6-(1-hydroxy)-pyrazine.

Diamondback moth (Plutella xylostella), a worldwide migratory pest that is developing strong resistance to various chemical insecticides. It has been determined that four natural pyrazines isolated from Allium tuberosum showed significant repellent activity to P. xylostella, but the molecular target still unknown. In the present study, a novel synthetic route for 2,3-dimethyl-6-(1-hydroxy)-pyrazine which has the most significant repellent activity with a purity of 90.60% was established. Simultaneously, the bioassay result declared that the repellent grade was IV at a dosage of 0.01 mg which was the same as to the published data. Transcriptomics analysis detected 1643 upregulated and 3837 downregulated genes in P. xylostella antennae following this pyrazine exposure. Then, 2142 differentially expressed genes were annotated using Gene Ontology and 2757 genes were annotated by Kyoto Encyclopedia of Genes and Genomes. Moreover, this procedure identified 84 odour perception-related genes, 58 odorant receptor (OR) genes including 57 conventional ORs and the odorant receptor co-receptor (Orco, atypical odorant receptor) gene, and 26 odorant-binding protein (OBP) genes. Based on quantitative real time PCR (RT-qPCR) and differential expression results, 9 OR genes including the Orco were cloned and characterised. In summary, this study provides important basis for the utilization of pyrazines as the main active ingredients or lead compounds to developing new botanical pesticides, which will reduce application of chemical pesticides and postpone the development of resistance.

Knockdown of the glutamate-gated chloride channel gene decreases emamectin benzoate susceptibility in the fall armyworm, Spodoptera frugiperda.

Emamectin benzoate (EB), a derivative of avermectin, is the primary insecticide used to control the fall armyworm (FAW) in China. However, the specific molecular targets of EB against FAW remain unclear. In this study, we cloned the glutamate-gated chloride channel (GluCl) gene, which is known to be a primary molecular target for avermectin. We first investigated the transcript levels of SfGluCl in FAW and found that the expression level of SfGluCl in the head and nerve cord was significantly higher than that in other tissues. Furthermore, we found that the expression level of SfGluCl was significantly higher in eggs than that in other developmental stages, including larvae, pupae, and adults. Additionally, we identified three variable splice forms of SfGluCl in exons 3 and 9 and found that their splice frequencies remained unaffected by treatment with the LC50 of EB. RNAi mediated knockdown of SfGluCl showed a significant reduction of 42% and 65% after 48 and 72 h of dsRNA feeding, respectively. Importantly, knockdown of SfGluCl sifgnificantly reduced LC50 and LC90 EB treatment induced mortality of FAW larvae by 15% and 44%, respectively, compared to the control group feeding by dsEGFP. In contrast, there were no significant changes in the mortality of FAW larvae treated with the control insecticides chlorantraniliprole and spinetoram. Finally, molecular docking simulations revealed that EB bound to the large amino-terminal extracellular domain of SfGluCl by forming five hydrogen bonds, two alkyl hydrophobic interactions and one salt bridge. These findings strongly suggest that GluCl may serve as one of the molecular targets of EB in FAW, shedding light on the mode of action of this important insecticide.

Identification and characterization of multiple dsRNases from a lepidopteran insect, the tobacco cutworm, Spodoptera litura (Lepidoptera: Noctuidae).

RNA interference (RNAi) efficiency varies among insects. RNAi is highly efficient and systemic in coleopteran insects but quite variable and inefficient in lepidopteran insects. Degradation of double-stranded RNA (dsRNA) by double-stranded ribonucleases (dsRNases) is thought to contribute to the variability in RNAi efficiency observed among insects. One or two dsRNases involved in dsRNA digestion have been identified in a few insects. To understand the contribution of dsRNases to reduced RNAi efficiency in lepidopteran insects, we searched the transcriptome of Spodoptera litura and identified six genes coding for DNA/RNA non-specific endonucleases. Phylogenetic analysis revealed the evolutionary expansion of dsRNase genes in insects. The mRNA levels of three midgut-specific dsRNases increased during the larval stage, and the highest dsRNA-degrading activity was detected in third-instar larvae. Proteins produced via the expression of three midgut-specific dsRNases, and the widely expressed dsRNase3, in a baculovirus system showed dsRNase activity for four out of five dsRNases tested. In addition, the increase in dsRNA-degrading activity and upregulation of dsRNase1 and 2 in larvae fed on cabbage leaves suggests that the diet of S. litura can influence dsRNase expression, dsRNA stability, and thus probably RNAi efficiency. This is the first report that multiple dsRNases function together in an RNAi-recalcitrant insect. The data included in this paper suggest that multiple dsRNases coded by the S. litura genome might contribute to the lower and variable RNAi efficiency reported in this and other lepidopteran insects.

Identification of the ionotropic GABA receptor-like subunits from the striped stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae).

Ionotropic γ-aminobutyric acid (GABA) receptors (GABARs) mediate rapid inhibitory neurotransmission in both vertebrates and invertebrates, and are important molecular targets of insecticides. However, components of insect GABARs remain elusive. In addition to CsRDL1 and CsRDL2, the complementary DNAs (cDNAs) of another two GABA receptor-like subunits, CsLCCH3 and Cs8916, were identified from the rice striped stem borer, Chilo suppressalis Walker in the present study. Both CsLCCH3 and Cs8916 subunits shared common structural features, such as a highly-conserved Cys-loop structure, six distinct regions involved in ligand binding (loops A-F), and four transmembrane domains (TM 1-4). Transcript analysis demonstrated that the relative mRNA expression levels of both CsLCCH3 and Cs8916 subunits were the highest in the ventral nerve cord. Regarding developmental stage, transcript levels of both subunits were highest in eggs. Injections of double-stranded RNAs (dsRNAs), including dsRDL1, dsRDL2, dsLCCH3, or ds8916, significantly reduced mRNA abundance after 24 and 48 h. However, no observable effects on the development of C. suppressalis were observed. Injection of dsRDL1 or dsRDL2 did significantly reduce the mortality of C. suppressalis treated with fluralaner. Our results indicated that CsRDLs mediated the susceptibility of C. suppressalis to fluralaner, whereas CsLCCH3 and CsL8916 did not. The current investigation enhances our knowledge of Lepidopteran GABARs and offers a molecular basis for the development of novel insecticides to control C. suppressalis.

Toxicity and sublethal effects of fluralaner on Spodoptera litura Fabricius (Lepidoptera: Noctuidae).

The increasing occurrence of resistance to chemical insecticides in insect pest populations is a serious threat to the integrity of current pest management strategies, and exploring new alternative chemistries is one important way to overcome this obstacle. Fluralaner, as a novel isoxazoline insecticide, has broad spectrum activity against a variety of insect pests, but little data is available about its effect on Lepidopterans. The effects of fluralaner on Spodoptera litura Fabricius, a widespread and polyphagous pest, were evaluated in the present study. Our results showed younger larvae were more susceptible to fluralaner treatment, but feeding and topical applications were similarly effective in 3rd instar larvae. Synergism assays indicated that piperonyl butoxide (PBO) could increase the toxicity of fluralaner to S. litura to a certain degree and P450 may be involved in the detoxification of fluralaner in vivo. Sublethal developmental effects included reduced larval body weight, decreased pupation and emergence, and notched wings in adults, accompanied by changes in the transcript levels of chitinase 5 (CHT5) and juvenile hormone acid methyltransferase (Jhamt), genes vital for insect development. Above results manifested that fluralaner is highly toxic to S. litura larvae via either topical or oral application and provide an indication of how this insecticide is metabolized in vivo. Further, our results provided a foundation for further development of fluralaner as a new tool in insect pest management.

Transcript Level and Sequence Matching Are Key Determinants of Off-Target Effects in RNAi.

Double-stranded RNA (dsRNA) pesticides, those based on RNA interference (RNAi) technology utilizing dsRNA, have shown potential for pest control. However, the off-target effects of dsRNA pose limitations to the widespread application of RNAi and raise concerns regarding potential side effects on other beneficial organisms. The precise impact and underlying factors of these off-target effects are still not well understood. Here, we found that the transcript level and sequence matching jointly regulate off-target effects of dsRNA. The much lower expressed target genes were knocked down to a lesser extent than genes with higher expression levels, and the critical sequence identity of off-target effects is approximately 80%. Moreover, off-target effects could be triggered by a contiguous matching sequence length exceeding 15 nt as well as nearly perfectly matching sequences with one or two base mismatches exceeding 19 nt. Increasing the dosage of dsRNA leads to more severe off-target effects. However, the length of mismatched dsRNA, the choice of different RNAi targets, and the location of target sites within the same gene do not affect the severity of off-target effects. These parameters can be used to guide the design of possibly selective sequences for RNAi, optimize the specificity and efficiency of dsRNA, and facilitate practical applications of RNAi for pest control.

Ecofriendly polysaccharide-based alginate/pluronic F127 semi-IPN hydrogel with magnetic collectability for precise release of pesticides and sustained pest control.

Controlled-release systems are crucial for efficient pesticide utilization and environmental protection in agricultural production. The utilization of polysaccharide-based materials derived from biopolymers as carriers for controlling pesticide release holds significant potential. In this work, a reversible near infrared-responsive polysaccharide-based hydrogel (RNPH) was fabricated by employing a semi-interpenetrating polymer network (alginate-FeIII/pluronic F127) as a carrier to encapsulate Fe3O4@polydopamine (FP) and emamectin benzoate (EB)-loaded hollow mesoporous silica. The incorporation of FP into the RNPH introduced a photothermal effect, enabling the precise release of EB through reversible shrinkage of the hydrogel upon NIR irradiation. Additionally, the presence of magnetic Fe3O4 in the system facilitated the rapid removal of remaining RNPH from the environment using a magnet, reducing EB residue. Importantly, RNPH exhibited exceptional controlled-release performance and could be reused for at least 4 cycles. Furthermore, the anti-photolysis ability of EB protected by RNPH was enhanced by 4.8 times compared to EB alone. Moreover, RNPH significantly improved the adhesion of EB to foliar surfaces, thereby reducing the loss of EB while ensuring crop safety. Therefore, the polysaccharide-based hydrogel holds promise as a versatile carrier for the precise release of EB, offering valuable applications in enhancing pesticide bioavailability and promoting environmental safety.

Control efficacy and joint toxicity of metaflumizone mixed with chlorantraniliprole or indoxacarb against the fall armyworm, Spodoptera frugiperda.

BACKGROUND: The fall armyworm (FAW), Spodoptera frugiperda is the main destructive pest of grain crops, and has led to substantial economic losses worldwide. Chemical pesticides are the most effective way to manage FAW. Here, a laboratory test using an artificial diet-incorporated assay was conducted to determine the toxicity of five insecticides and the joint effect of the binary combination insecticides to FAW larvae. A field plot test using foliar spray was carried out to assess the control efficacy of metaflumizone mixed with chlorantraniliprole or indoxacarb against FAW. RESULTS: The bioassay results showed that metaflumizone had a stronger insecticidal effect than indoxacarb toward FAW larvae. Furthermore, the mixture of metaflumizone and chlorantraniliprole in a volume ratio of 3:7 had the strongest synergistic effect against FAW, with a co-toxicity coefficient (CTC) of 317.18. The best synergistic effect for mixtures of metaflumizone and indoxacarb was observed at a 1:9 volume ratio, with a CTC of 185.98. However, there was an antagonistic effect of metaflumizone mixed with emamectin benzoate and with lufenuron, because the co-toxic factor was less than -20 at volume ratios of 8:2 and 9:1, respectively. According to the results of the field trial, metaflumizone mixed with chlorantraniliprole or indoxacarb at a 50% reduction of the application rate can effectively control FAW with efficacy ranging from 77.73% to 94.65% 1-7 days postapplication. CONCLUSION: Overall, our findings suggest that metaflumizone and its binary combination insecticides can be utilized in FAW integrated pest management programs. © 2022 Society of Chemical Industry.

Identification and pharmacological characterization of histamine-gated chloride channels in the fall armyworm, Spodoptera frugiperda.

Histamine-gated chloride channels (HACls) mediate fast inhibitory neurotransmission in invertebrate nervous systems and have important roles in light reception, color processing, temperature preference and light-dark cycle. The fall armyworm, Spodoptera frugiperda is a main destructive pest of grain and row crops. However, the pharmacological characterization of HACls in S. frugiperda remain unknown. In this study, we identified two cDNAs encoding SfHACl1 and SfHACl2 in S. frugiperda. They had similar expression patterns and were most abundantly expressed in the head of larvae and at the egg stage. Electrophysiological analysis with the two-electrode voltage clamp method showed that histamine (HA) and γ-aminobutyric acid (GABA) activated inward currents when SfHACls were singly or collectively expressed with different ratios in Xenopus laevis oocytes. These channels were ≥2000-fold more sensitive to HA than to GABA. They were anion-selective channels, which were highly dependent on changes in external chloride concentrations, but insensitive to changes in external sodium concentrations. The insecticides abamectin (ABM) and emamectin benzoate (EB) also activated these channels with the EC50 to SfHACl1 lower than that to SfHACl2. And the EC50s of ABM and EB to the co-expressed channels gradually increased with increase in the injection ratio of SfHACl2 cRNA. Homology models and docking simulations revealed that HA bound to the large amino-terminal extracellular domain of SfHACl1 and SfHACl2 by forming 4 and 2 hydrogen bonds, respectively. The docking simulations of ABM and EB had similar binding sites in the transmembrane regions. Overall, these findings indicated that HACls act as targets for macrolide, and this study provides theoretical guidance for further derivatization of abamectin insecticides.

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.

Expression and functional analysis of cytochrome P450 genes in the integument of the oriental armyworm, Mythimna separata (Walker).

BACKGROUND: Mythimna separata is a devastating agricultural pest that has recently developed insecticide resistance. Integument-specific cytochrome P450s were reported to participate in cuticle formation and could be potential targets for pesticide synthesis. RESULTS: The transcriptome of integuments of M. separata larvae was constructed, generating a total of 38 058 unigenes with an average length of 1243 bp. These unigenes are enriched in functional categories such as lipid transport and metabolism, and secondary metabolites biosynthesis, transport and catabolism. Amongst unigenes, cytochrome P450s were identified and 66 unique P450s with complete open reading frames were named. These P450s were divided into 17 families and 32 subfamilies, containing conserved motifs such as helix C, helix I, helix K, and the heme-binding region. RNA-Seq and RT-qPCR analyses showed different expression levels of P450s in integuments of M. separata larvae. Further RT-qPCR analysis of P450s among different tissues showed that five P450s, especially CYP4G199, were specifically highly expressed in integuments. Moreover, knockdown of CYP4G199 disturbed cuticle formation, leading to imperfection in larval cuticle, and prevented pupation of M. separata. CONCLUSION: Transcriptome of larval integuments provided sequence and expression of genes in M. separata. CYP4G199 is specifically highly expressed in larval integuments and is important for cuticle formation in M. separata.

Identification of Olfactory Genes From the Greater Wax Moth by Antennal Transcriptome Analysis.

The olfactory system is used by insects to find hosts, mates, and oviposition sites. Insects have different types of olfactory proteins, including odorant-binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs) to perceive chemical cues from the environment. The greater wax moth, Galleria mellonella, is an important lepidopteran pest of apiculture. However, the molecular mechanism underlying odorant perception in this species is unclear. In this study, we performed transcriptome sequencing of G. mellonella antennae to identify genes involved in olfaction. A total of 42,544 unigenes were obtained by assembling the transcriptome. Functional classification of these unigenes was determined by searching against the Gene Ontology (GO), eukaryotic orthologous groups (KOG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. We identified a total of 102 olfactory-related genes: 21 OBPs, 18 CSPs, 43 ORs, 18 IRs, and 2 SNMPs. Results from BLASTX best hit and phylogenetic analyses showed that most of the genes had a close relationship with orthologs from other Lepidoptera species. A large number of OBPs and CSPs were tandemly arrayed in the genomic scaffolds and formed gene clusters. Reverse transcription-quantitative PCR results showed that GmelOBP19 and GmelOR47 are mainly expressed in male antennae. This work provides a transcriptome resource for olfactory genes in G. mellonella, and the findings pave the way for studying the function of these genes.

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.

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.

Biochemical Comparison of dsRNA Degrading Nucleases in Four Different Insects.

Double stranded RNAs (dsRNA) degrading nuclease is responsible for the rapid degradation of dsRNA molecules, and thus accounts for variations in RNA interference (RNAi) efficacy among insect species. Here, the biochemical properties and tissue-specific activities of dsRNA degrading nucleases in four insects (Spodoptera litura, Locusta migratoria, Periplaneta americana, and Zophobas atratus) from different orders were characterized using a modified assay method. The results revealed that all insect dsRNA degrading nucleases tested showed high activity in alkaline environments at optimal Mg2+ concentrations and elevated temperatures. We also found that enzymes from different insects varied in terms of their optimal reaction conditions and kinetic parameters. Whole body enzyme activity differed dramatically between insect species, although enzymes with higher substrate affinities (lower Km) were usually balanced by a smaller Vmax to maintain a proper level of degradative capacity. Furthermore, enzyme activities varied significantly between the four tested tissues (whole body, gut, hemolymph, and carcass) of the insect species. All the insects tested showed several hundred-fold higher dsRNA degrading activity in their gut than in other tissues. Reaction environment analysis demonstrated that physiological conditions in the prepared gut fluid and serum of different insects were not necessarily optimal for dsRNA degrading nuclease activity. Our data describe the biochemical characteristics and tissue distributions of dsRNA degrading activities in various insects, not only explaining why oral delivery of dsRNA often produces lower RNAi effects than injection of dsRNA, but also suggesting that dsRNA-degrading activities are regulated by physiological conditions. These results allow for a better understanding of the properties of dsRNA degrading nucleases, and will aid in the development of successful RNAi strategies in insects.

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.

Fiprole insecticide resistance of Laodelphax striatellus: electrophysiological and molecular docking characterization of A2'N RDL GABA receptors.

BACKGROUND: Phenylpyrazole (fiprole) insecticides, including ethiprole, fipronil and flufiprole with excellent activity on rice planthoppers, are very important in Asia but resistance has developed after decades of use. The molecular mechanism of fipronil- but not ethiprole-resistance has been previously studied in rice planthoppers. In our laboratory, a small brown planthopper Laodelphax striatellus strain with ethiprole-resistance was cultured and the molecular mechanisms of ethiprole resistance and of cross-resistance among fiprole insecticides were investigated. RESULTS: Ethiprole-resistant L. striatellus has >5000-fold resistance compared to the susceptible strain, and exhibits around 200-fold cross-resistance with fipronil and flufiprole. RDL genes were isolated from susceptible and ethiprole-resistant L. striatellus and expressed in Xenopus oocytes. Electrophysiological studies showed fiprole insecticides inhibited γ-aminobutyric acid (GABA)-induced current with IC50 = 0.1-1.4 µM to LsRDL-S homomers. In LsRDL-R with A2'N mutation, only 1-13% inhibition was observed on treatment with 10 µM ethiprole, fipronil or flufiprole. Homology models indicate A2'N mutation allows crosslinking hydrogen bonding between Asn sidechains at the 2' position around the channel pore, blocking insecticides from interacting near this position. In contrast, insecticides showed favorable binding near A2' in wild-type L. striatellus. CONCLUSION: Cross-resistance is increasing for fiprole insecticides in L. striatellus and management strategies are necessary to minimize resistance. © 2018 Society of Chemical Industry.

Acute toxicity, bioconcentration, elimination and antioxidant effects of fluralaner in zebrafish, Danio rerio.

Fluralaner is a novel isoxazoline insecticide which shows high insecticidal activity against parasitic, sanitary and agricultural pests, but there is little information about the effect of fluralaner on non-target organisms. This study reports the acute toxicity, bioconcentration, elimination and antioxidant response of fluralaner in zebrafish. All LC50 values of fluralaner to zebrafish were higher than 10 mg L-1 at 24, 48, 72 and 96 h. To study the bioconcentration and elimination, the zebrafish were exposed to sub-lethal concentrations of fluralaner (2.00 and 0.20 mg L-1) for 15 d and then held 6 d in clean water. The results showed medium BCF of fluralaner with values of 12.06 (48 h) and 21.34 (144 h) after exposure to 2.00 and 0.20 mg L-1 fluralaner, respectively. In the elimination process, a concentration of only 0.113 mg kg-1 was found in zebrafish on the 6th day after removal to clean water. After exposure in 2.00 mg L-1 fluralaner, the enzyme activities of SOD, CAT, and GST, GSH-PX, CarE and content of MDA were measured. Only CAT and CarE activities were significantly regulated and the others stayed at a stable level compared to the control group. Meanwhile, transcriptional expression of CYP1C2, CYP1D1, CYP11A were significantly down-regulated at 12 h exposed to 2.00 mg L-1 of fluralaner. Except CYP1D1, others CYPs were up-regulated at different time during exposure periods. Fluralaner and its formulated product (BRAVECTO®) are of low toxicity to zebrafish and are rapidly concentrated in zebrafish and eliminated after exposure in clean water. Antioxidant defense and metabolic systems were involved in the fluralaner-induced toxicity. Among them, the activities of CAT and CarE, and most mRNA expression level of CYPs showed fast response to the sub-lethal concentration of fluralaner, which could be used as a biomarker relevant to the toxicity.