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With the aim of identifying novel neonicotinoid insecticides with low bee toxicity, a series of compounds bearing thiazolidine moiety, which has been shown to be low bee toxic, were rationally designed through substructure splicing strategy and evaluated insecticidal activities. The optimal compounds A24 and A29 exhibited LC 50 values of 30.01 and 17.08 mg/L against Aphis craccivora , respectively. Electrophysiological studies performed on Xenopus oocytes indicated that compound A29 acted on insect nAChR, with EC 50 value of 50.11 μM. Docking binding mode analysis demonstrated that A29 bound to Lymnaea stagnalis acetylcholine binding protein through H-bonds with the residues of D_Arg55, D_Leu102, and D_Val114. Quantum mechanics calculation showed that A29 had a higher highest occupied molecular orbit (HOMO) energy and lower vertical ionization potential (IP) value compared to the high bee toxic imidacloprid, showing potentially low bee toxicity. Bee toxicity predictive model also indicated that A29 was nontoxic to honeybees. Our present work identified an innovative insecticidal scaffold and might facilitate the further exploration of low bee toxic neonicotinoid insecticides.

As one of the dominant natural enemies for insect pests, the pond wolf spider, Pardosa pseudoannulata, plays important roles in pest control. Insecticide applications threaten P. pseudoannulata and consequently weaken its control effects. The roles of P450 monooxygenases in insecticide detoxifications have been richly reported in insects, but there are few reported in spiders. In this study, 120 transcripts encoding P. pseudoannulata P450s were identified based on whole genome sequencing. Compared to P450s of Aedes aegypti and Nilaparvata lugens, several novel P450 families were found, such as CYP3310. KEGG analysis of the CYP3310 family indicated that the family might be involved in the synthesis and metabolism of polyunsaturated fatty acids and hydrocarbons. The potential P450s involved in insecticide metabolism were obtained according to the high FPKM values in fat bodies based on transcriptome sequencing. However, none of the selected P450 genes was significantly upregulated by the treatments of deltamethrin or imidacloprid. The present study provides genomic and transcriptomic information of spider P450s, especially for their roles in the synthesis and metabolism of endogenous and exogenous compounds, such as polyunsaturated fatty acids, hydrocarbons and insecticides., (Copyright © 2021. Published by Elsevier Inc.)

The importance of glutathione S-transferases (GSTs) in imidacloprid resistance in Nilaparvata lugens , a major rice pest, and other insects was often excluded, mostly due to the slight effects of diethyl maleate (DEM) on synergizing imidacloprid in resistant populations. Here, we found that the synergistic effects of DEM were time-dependent. At 24 or 48 h, the time often selected to record mortalities in imidacloprid bioassay, DEM really did not cause an obvious increase in imidacloprid toxicity. However, significant effects were observed after 72 h. The results revealed that GSTs, as phase II detoxification enzymes to metabolize secondary products generated from phase I detoxification enzymes, were also important in imidacloprid resistance in N. lugens , but might have occurred a little later than that of P450s and CarEs as phase I enzymes. The constitutive overexpression in the imidacloprid-resistant strain G25 and expression induction by imidacloprid in the susceptible strain S25 indicated that four GST genes, NlGSTs1 , NlGSTs2 , NlGSTe1 , and NlGSTm1 , were important in imidacloprid resistance, which was confirmed by RNAi test. The higher expression levels and more expression induction by imidacloprid in the midgut and fat body compared to the whole insect supported the important roles of these four GSTs, which was also supported by the more overexpression times in the midgut and fat body versus the whole insect between G25 and S25 strains. Taking the data together, the study ascertained the roles of GSTs in imidacloprid resistance in N. lugens .

Background: Neonicotinoids, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) used to control Nilaparvata lugens, a major rice insect pest. High imidacloprid resistance has been reported in N. lugens both in the laboratory and in the field. Cycloxaprid (CYC), an oxa-bridged cis-nitromethylene neonicotinoid, showed high insecticidal activity against N. lugens and low cross-resistance in imidacloprid-resistant strains and field populations., Results: Binding studies demonstrated that imidacloprid has two binding sites with different affinities (K d = 3.18 ± 0.43 pm and 1.78 ± 0.19 nm) in N. lugens nAChRs. CYC was poor at displacing [ 3 H]imidacloprid at its high-affinity binding site (K i = 159.38 ± 20.43 nm), but quite efficient at the low-affinity binding site (K i = 1.27 ± 0.35 nm). These data showed that CYC had overlapping binding sites with imidacloprid only at its low-affinity binding site. Therefore, the low displacement ability of CYC against imidacloprid binding at its high-affinity site could partially explain the low cross-resistance of CYC in imidacloprid-resistant populations., Conclusion: The high insecticidal activity, low cross-resistance and different binding properties on insect nAChRs of CYC show that it is a potential insecticide for the control of N. lugens and related insect pests, especially ones with high resistance to neonicotinoids. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Imidacloprid is a key insecticide used for controlling sucking insect pests, including the small brown planthopper (Laodelphax striatellus, Fallén) (Hemiptera: Delphacidae), an important agricultural pest of rice. A strain of L. striatellus (YN-ILR) developed 21-fold resistance when selected with imidacloprid on a susceptible YN strain. An in vitro study on piperonyl butoxide synergism indicated that enhanced detoxification mediated by cytochrome P450s contributed to imidacloprid resistance to some extent, and multiple P450 genes showed altered expression in the imidacloprid-resistant YN-ILR strain compared with the susceptible YN strain (CYP425B1-CYP6BD10 had 1.51- to 11.45-fold higher expression, CYP4CE2-CYP4DD1V2 had 0.12- to 0.57-fold lower expression). While there were no mutations in target nicotinic acetylcholine receptor (nAChR) genes, subunits of Lsα1, Lsβ1, and Lsβ3 in the YN-ILR strain showed 3.86-, 4.39-, and 2.59-fold higher expression and Lsa8 displayed 0.38-fold lower expression than the YN strain. Moreover, 21-fold moderate imidacloprid resistance in individuals of L. striatellus did not produce a fitness cost. The findings suggest that L. striatellus has the capacity to develop resistance to imidacloprid through P450 detoxification and potential target nAChR expression changes, and moderate imidacloprid resistance was not associated with a fitness cost.

Background: Fitness costs associated with insecticide resistance in pest insects have mainly been studied under optimal laboratory conditions. However, resistant insects face more stressors than just insecticides in the field, and how the resistant population reacts to these stressors is of practical importance for the control of pest insects such as the brown planthopper Nilaparvata lugens. The aim of the present study was to explore the impact of population density on the competitiveness of resistant and susceptible individuals., Results: Two isogenic N. lugens populations, a highly imidacloprid-resistant population (HZ-R) with a resistance ratio (RR) of 227.10 and a relatively susceptible population (HZ-S) with an RR of 2.99, were created from a field-resistant population (HZ; RR 62.51). The high resistance levels of HZ-R and HZ were mainly attributable to the overexpression of multiple cytochrome P450 (CYP) genes such as CYP6ER1, CYP6AY1, CYP6CW1 and CYP4CE1 compared with HZ-S, this being supported by piperonyl butoxide synergism. HZ-R was observed to be more resistant to thiacloprid and etofenprox compared with HZ and HZ-S. Most interestingly, in high population density treatments, HZ-S individuals were much more competitive than HZ-R individuals., Conclusion: Imidacloprid-resistant individuals of N. lugens are less competitive than their susceptible counterparts under density pressure. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Acetylcholine binding proteins (AChBPs) are homologs of extracellular domains of nicotinic acetylcholine receptors (nAChRs) and serve as models for studies on nAChRs. Particularly, studies on invertebrate nAChRs that are limited due to difficulties in their heterologous expression have benefitted from the discovery of AChBPs. Thus far, AChBPs have been characterized only in aquatic mollusks, which have shown low sensitivity to neonicotinoids, the insecticides targeting insect nAChRs. However, AChBPs were also found in spiders based on the sequence and tissue expression analysis. Here, we report five AChBP subunits in Pardosa pseudoannulata, a predator enemy against rice insect pests. Spider AChBP subunits shared higher sequence similarities with nAChR subunits of both insects and mammals compared with mollusk AChBP subunits. The AChBP1 subunit of P. pseudoannulata (Pp-AChBP) was then expressed in Sf9 cells. The Ls-AChBP from Lymnaea stagnalis was also expressed for comparison. In both AChBPs, one ligand site per subunit was present at each interface between two adjacent subunits. Neonicotinoids had higher affinities (7.9-18.4 times based on K d or K i values) for Pp-AChBP than for Ls-AChBP, although epibatidine and α-bungarotoxin showed higher affinities for Ls-AChBP. These results indicate that spider AChBP could be used as an alternative model to study the interaction between insect nAChRs and neonicotinoids., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Nicotinic acetylcholine receptor (nAChR) subunits are encoded by a large multigene family and generate a large number of pentameric receptors with various properties. At present, nematode species, such as Caenorhabditis elegans, have the largest number of nAChR subunits. In this study, two nAChR subunits (Bxy-Unc-38 and Bxy-Unc-29) were cloned from Bursaphelenchus xylophilus, a fatal nematode pest on pine trees causing pine wilt disease. When Bxy-Unc-38 and Bxy-Unc-29 were co-expressed in Xenopus oocytes, constructed functional nAChRs showed agonist responses to acetylcholine and imidacloprid, a neonicotinoid insecticide. When complementary RNAs (cRNAs) of Bxy-Unc-38 and Bxy-Unc-29 were injected at different ratios, the assembled nAChRs showed different pharmacological subtypes, especially in terms of the sensitivity to imidacloprid and another two neonicotinoids. At cRNA ratios 1:1 and 1:5 (Bxy-Unc-38: Bxy-Unc-29), nAChRs showed low sensitivity to test neonicotinoids, which were partial agonists on the receptors. In contrast, at cRNA ratio 5:1, the three test neonicotinoids were full agonists and showed much higher potency compared to that on the receptors with cRNA ratio 1:1 and 1:5. For example, EC 50 values of the three neonicotinoids on the receptors with cRNA ratio 1:5 were 170-222 times of those of receptors with cRNA ratio 5:1. The results showed that the subunit stoichiometry of Bxy-Unc-38/Bxy-Unc-29 receptor dramatically affected the agonist potency of neonicotinoids, and even altered the action property. Due to the high sensitivity of the constructed nAChRs at cRNA ratio 5:1, the construct would serve as an important model to study the interaction between invertebrate nAChRs and neonicotinoids., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Due to great diversity of nicotinic acetylcholine receptor (nAChR) subtypes in insects, one β subunit may be contained in numerous nAChR subtypes. In the locust Locusta migratoria, a model insect species with agricultural importance, the third β subunits (Locβ3) was identified in this study, which reveals at least three β subunits in this insect species. Imidacloprid was found to bind nAChRs in L. migratoria central nervous system at two sites with different affinities, with K d values of 0.16 and 10.31nM. The specific antisera (L1-1, L2-1 and L3-1) were raised against fusion proteins at the large cytoplasmic loop of Locβ1, Locβ2 and Locβ3 respectively. Specific immunodepletion of Locβ1 with antiserum L1-1 resulted in the selective loss of the low affinity binding site for imidacloprid, whereas the immunodepletion of Locβ3 with L3-1 caused the selective loss of the high affinity site. Dual immunodepletion with L1-1 and L3-1 could completely abolish imidacloprid binding. In contrast, the immunodepletion of Locβ2 had no significant effect on the specific [ 3 H]imidacloprid binding. Taken together, these data indicated that Locβ1 and Locβ3 were respectively contained in the low- and high-affinity binding sites for imidacloprid in L. migratoria, which is different to the previous finding in Nilaparvata lugens that Nlβ1 was in two binding sites for imidacloprid. The involvement of two β subunits separately in two binding sites may decrease the risk of imidacloprid resistance due to putative point mutations in β subunits in L. migratoria., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cycloxaprid, an oxabridged cis-nitromethylene neonicotinoid, showed high insecticidal activity in Hemipteran insect pests. In this study, the action of cycloxaprid was characterized by recombinant receptor Nlα1/rβ2 and cockroach neurons. On Nlα1/β2 in Xenopus oocytes, cycloxaprid acted as a full agonist. The imidacloprid resistance-associated mutation Y151S reduced the Imax of cycloxaprid by 37.0% and increased EC50 values by 1.9-fold, while the Imax of imidacloprid was reduced by 72.0%, and EC50 values increased by 2.3-fold. On cockroach neurons, the maximum currents elicited by cycloxaprid were only 55% of that of acetylcholine, a full agonist, but with close EC50 values of that of trans-neonicotinoids. In addition, cycloxaprid inhibited acetylcholine-evoked currents on insect neurons in a concentration-dependent manner when co-applied with acetylcholine. Cycloxaprid at low concentrations significantly inhibited the activation of nAChRs by acetylcholine, and its inhibition potency at 1 µM was higher than its activation potency on insect neurons. Two action potencies, activation, and inhibition, by cycloxaprid on insect neurons provided an explanation for its high toxicity to insect pests. In summary, as a cis-nitromethylene neonicotinoid, cycloxaprid showed high potency on both recombinant nAChR Nlα1/β2 and cockroach neurons, which guaranteed its high control effects on a variety of insect pests. [ABSTRACT FROM AUTHOR]

Cys-loop ligand-gated channels mediate neurotransmission in insects and are receptors for many insecticides. Some insecticides acting on cysLGIC also have lethal effects on non-targeting organisms, but the mechanism of this negative effect is unclear due to information absence. The identification and analysis of cysLGIC family in Pardosa pseudoannulata , a pond wolf spider, can deepen the understanding of insecticides for natural enemy safety. Thirty-four cysLGIC genes were identified in P. pseudoannulata genome, including nicotinic acetylcholine receptors, γ-aminobutyric acid gated chloride channels, glutamate-gated chloride channels, histamine-gated chloride channels, and pH-sensitive chloride channels. The expansion of GABACls and HisCls accounts for the large number of cysLGICs in P. pseudoannulata, and the alternative splicing events in nAChR and RDL subunits enriched the diversity of the superfamily. Most cysLGIC genes show the highest expression in brain and lowest expression in the early-egg sac stage. Variable residues (R81, V83, R135, N137, F190, and W197) in P. pseudoannulata nAChR β subunits and critical differences in α6 subunit TM4 region compared with insects would apply for the insensitivity to neonicotinoids and spinosyn. In contrast, avermectin and dieldrin may be lethal to P. pseudoannulata due to the similar drugs binding sites in GluCls compared with insects. These findings will provide a valuable clue for natural enemy protection and environmentally friendly insecticide development. [Display omitted] • 34 cysLGIC genes were detected in P. pseudoannulata genome, including nAChRs, GABACls, GluCls, HisCls and pHCls. • Few numbers of nAChR and GABA subunits in P. pseudoannulata have alternative splicing events. • Most of the cysLGIC genes show the highest expression in P. pseudoannulata central neuron system. • The variable residues in nAChR α6 and β1 subunits could account for resistance P. pseudoannulata to the two insecticides. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Cycloxaprid is a new oxabridged cis‐configuration neonicotinoid insecticide, the resistance development potential and underlying resistance mechanism of which were investigated in the small brown planthopper, Laodelphax striatellus (Fallén), an important agricultural pest of rice. RESULTS: A cycloxaprid‐resistant strain (YN‐CPD) only achieved 10‐fold higher resistance, in contrast to 106‐fold higher resistance to buprofezin and 332‐fold higher resistance to chlorpyrifos achieved after exposure to similar selection pressure, and the cycloxaprid selected line showed no cross‐resistance to the buprofezin and chlorpyrifos‐selected resistance strains. Moreover, we identified 10 nicotinic acetylcholine receptor (nAChR) subunits from the transcriptome of L. striatellus, and six segments had open reading frames (ORFs). While we did not find mutations in the nAChR genes of L. striatellus, subunits Lsα1 and Lsβ1 exhibited, respectively, 9.60‐fold and 3.36‐fold higher expression in the resistant strain, while Lsα8 exhibited 0.44‐fold lower expression. Suppression of Lsα1 through ingestion of dsLsα1 led to an increase in susceptibility to cycloxaprid. CONCLUSION: The findings indicate that resistance to cycloxaprid develops slowly compared with resistance to other chemicals and without cross‐resistance to chlorpyrifos or buprofezin; over‐expressed Lsα1 is associated with low cycloxaprid resistance levels, but the importance of over‐expressed Lsβ1 and reduced expression of Lsα8 could not be excluded. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Methane emitted by insects is considered to be an important source of atmospheric methane. Here we report the stimulation of methane emission in the cockroach Periplaneta americana and termite Coptotermes chaohuensis , insects with abundant methanogens, by neonicotinoids, insecticides widely used to control insect pests. Cycloxaprid (CYC) and imidacloprid (IMI) caused foregut expansion in P. americana , and increased the methane emission. Antibiotics mostly eliminated the effects. In P. americana guts, hydrogen levels increased and pH values decreased, which could be significantly explained by the gut bacterium community change. The proportion of several bacterium genera increased in guts following CYC treatment, and two genera from four could generate hydrogen. Hydrogen is a central intermediate in methanogenesis. All increased methanogens in both foregut and hindgut used hydrogen as electron donor to produce methane. Besides, the up-regulation of mcrA , encoding the enzyme for the final step of methanogenesis suggested the enhanced methane production ability in present methanogens. In the termite, hydrogen levels in gut and methane emission also significantly increased after neonicotinoid treatment, which was similar to the results in P. americana. In summary, neonicotinoids changed bacterium community in P. americana gut to generate more hydrogen, which then stimulated gut methanogens to produce and emit more methane. The finding raised a new concern over neonicotinoid applications, and might be a potential environmental risk associated with atmospheric methane. [Display omitted] F0B7 Stimulation of methane production in insects by neonicotinoid insecticides was first reported. F0B7 Neonicotinoid treatment changed bacterium community in gut to produce more hydrogen. F0B7 Methanogens in gut use higher hydrogen concentrations to generate and emit more methane. F0B7 It is a potential environmental risk concern with atmospheric methane of using neonicotinoids. Neonicotinoids stimulate H 2 -limited methane emission in methanogenic insects through the regulation of gut bacterium community, which raised a potential environmental risk. [ABSTRACT FROM AUTHOR]

Neonicotinoids are insect-selective nicotinic acetylcholine receptors (nAChRs) agonists that are used extensively for plant protection and animal health care. Some chaperone proteins, such as 14-3-3 proteins, importantly modulate nAChRs to display the physiological and pharmacological properties. Here we found that there is a 14-3-3 binding motif RSP S TH within the cytoplasmic loop of most insect α8 subunits. In the motif, a potential phosphorylated serine residue, serine 337, was a putative protein kinase A (PKA) substrate. Using Locusta migratoria α8 subunit as a representative, here we demonstrated that Loc14‐3-3ε interacted with the unique phosphoserine (α8S337) of Locα8 subunit to regulate agonist efficacy on hybrid Locα8/β2 nAChRs in Xenopus oocytes. Co-expression of Loc14-3-3ε caused a dramatic rise of maximal inward currents (I max) of Locα8/β2 for acetylcholine and imidacloprid to 2.9-fold and 3.1-fold of that of Locα8/β2 alone. The S337A substitution of Locα8 reduced the I max rise when Locα8S337A/β2 and Loc14-3-3ε were co-expressed. The increased agonist currents by exogenous Loc14-3-3ε on Locα8/β2 could be almost abolished by either PKA inhibitor KT5720 or 14-3-3 inhibitor difopein. The findings revealed that serine 337 within motif RSP S TH was important for the interaction between insect nAChRs and 14-3-3ε, and inhibiting the interaction would change the pharmacological property of insect nAChRs to agonist such as neonicotinoids which may provide insights to develop new targets for insecticide design. [Display omitted] • 14–3-3ε was a modulator of hybrid nAChRs Locα8/β2. • PKA-driven phosphorylation was involved in the interaction of 14–3-3ε with Locα8. • Phosphoserine 337 within Locα8 RSPSTH was important for the interaction. [ABSTRACT FROM AUTHOR]

Our previous study showed that IPPA08, a cis -configuration neonicotinoid compound with unique oxabridged substructure, acted as a specific synergist to neonicotinoid insecticides targeting nicotinic acetylcholine receptors (nAChRs). Heteropentamer nAChRs have diverse characteristics and can form canonical and noncanonical subunit interfaces. While canonical interfaces have been exploited as targets of many drugs, noncanonical interfaces have received less attention. In this study, the mechanism of IPPA08 synergism was evaluated on hybrid nAChRs consisting of three α1 subunits from the brown planthopper and two rat β1 subunits (Nlα1/rβ2) expressed in Xenopus oocytes. IPPA08 alone evoked inward currents, but only at very high concentrations, greater than 1 mM. However, at concentrations below 200 μM, IPPA08 slowed the decay of inward currents evoked by imidacloprid, but not by acetylcholine, and also increased the sensitivity of Nlα1/rβ2 to imidacloprid. Both modulations by IPPA08 were concentration-dependent in the same concentration range of 10–150 μM. Experimentally induced mutations in canonical (α+/β−) and noncanonical (β+/α−) interfaces of Nlα1/rβ2 receptors were also examined to evaluate the presence of possible binding sites for IPPA08 on the receptors. Our results showed that mutations in the canonical interfaces affected only the potency of IPPA08 as an agonist, while mutations in the noncanonical interfaces affected only the synergistic action of IPPA08. Based on these results, we propose that at low concentrations IPPA08 can act as a positive allosteric modulator of noncanonical interfaces, and likely slow the decay of currents through stabilizing the open-channel state caused by the action of imidacloprid on canonical interfaces. [ABSTRACT FROM AUTHOR]

The overexpression of P450 monooxygenase genes is a main mechanism for the resistance to imidacloprid, a representative neonicotinoid insecticide, in Nilaparvata lugens (brown planthopper, BPH). However, only two P450 genes (CYP6AY1 and CYP6ER1), among fifty-four P450 genes identified from BPH genome database, have been reported to play important roles in imidacloprid resistance until now. In this study, after the confirmation of important roles of P450s in imidacloprid resistance by the synergism analysis, the expression induction by imidacloprid was determined for all P450 genes. In the susceptible (Sus) strain, eight P450 genes in Clade4, eight in Clade3 and two in Clade2 were up-regulated by imidacloprid, among which three genes (CYP6CS1, CYP6CW1 and CYP6ER1, all in Clade3) were increased to above 4.0-fold and eight genes to above 2.0-fold. In contrast, no P450 genes were induced in Mito clade. Eight genes induced to above 2.0-fold were selected to determine their expression and induced levels in Huzhou population, in which piperonyl butoxide showed the biggest effects on imidacloprid toxicity among eight field populations. The expression levels of seven P450 genes were higher in Huzhou population than that in Sus strain, with the biggest differences for CYP6CS1 (9.8-fold), CYP6ER1 (7.7-fold) and CYP6AY1 (5.1-fold). The induction levels for all tested genes were bigger in Sus strain than that in Huzhou population except CYP425B1. Screening the induction of P450 genes by imidacloprid in the genome-scale will provide an overall view on the possible metabolic factors in the resistance to neonicotinoid insecticides. The further work, such as the functional study of recombinant proteins, will be performed to validate the roles of these P450s in imidacloprid resistance. [ABSTRACT FROM AUTHOR]

High sensitivity to neonicotinoid insecticides have been reported in the miridbug Cyrtorhinus lividipennis , an important predatory enemy of rice planthoppers, such as Nilaparvata lugens (brown planthopper). In the present study, the sensitivity of neonicotinoid insecticides between C. lividipennis and N. lugens were detected and compared. The results showed that neonicotinoid insecticides were much more toxic to the miridbug than to the brown planthopper. A nicotinic acetylcholine receptor subunit was cloned from the miridbug and denoted as α8 subunit (Clα8) according to sequence similarities and important functional motifs. Key amino acid differences were found in specific loops from α8 subunits between C . lividipennis (Clα8) and N . lugens (Nlα8). In order to understand the roles of key amino acid differences in insecticide sensitivities, the different amino acid residues in specific loops of Nlα8 were introduced into the corresponding sites in Clα8 to construct several subunit mutants. Clα8 or subunit mutants were co-expressed with rat β2 to obtain the functional receptors in Xenopus oocytes. The single mutation N191F in loop B reduced imidacloprid sensitivity, with EC 50 value in Clα8 N191F /β2 of 15.21 μM and 5.74 μM in Clα8/β2. Interestingly, although the single mutation E240T in loop C did not cause the significant change in imidacloprid sensitivity, it could enhance the effects of N191F and cause more decrease in imidacloprid sensitivity. The results indicated that E240T might contribute to neonicotinoid sensitivity in an indirect way. [ABSTRACT FROM AUTHOR]

Chemical insecticides are still primary methods to control rice planthoppers in China, which not only cause environmental pollution, insecticide residue and insecticide resistance, but also have negative effects on natural enemies, such as Pardosa pseudoannulata (the pond wolf spider), an important predatory enemy of rice planthoppers. Neonicotinoids insecticides, such as imidacloprid and thiacloprid, are insect-selective nAChRs agonists that are used extensively in the areas of crop protection and animal health, but have hypotoxicity to P . pseudoannulata. In the present study, two nAChR α subunits, Ppα1 or Ppα8, were found to be successfully expressed with rβ2 in Xenopus oocytes, but with much different sensitivity to imidacloprid and thiacloprid on two recombinant receptors Ppα1/rβ2 and Ppα8/rβ2. Key amino acid differences were found in and between the important loops for ligand binding. In order to well understand the relationship between the amino acid differences and neonicotinoid sensitivities, different segments in Ppα8 or Ppα1 with key amino acid differences were introduced into the corresponding regions of Ppα1 or Ppα8 to construct chimeras and then co-expressed with rβ2 subunit in Xenopus oocytes. The results from chimeras of both Ppα8 and Ppα1 showed that segments Δ5, Δ6, and Δ7 contributed to neonicotinoid sensitivities directly between two receptors. Although the segment Δ4 including all loop B region had no direct influences on neonicotinoid sensitivities, it could more remarkably influence neonicotinoid sensitivities when co-introductions with Δ5, Δ6 or Δ7. So, key amino acid differences in these four segments were important to neonicotinoid sensitivities, but the difference in Δ4 was likely ignored because of its indirect effects. [ABSTRACT FROM AUTHOR]

Abstract: Neonicotinoid insecticides, such as imidacloprid, are selective agonists of the insect nicotinic acetylcholine receptors (nAChRs) with –NO2 or –CN group in trans-configuration. Previously we reported the excellent insecticidal activity of a series of nitroconjugated neonicotinoids with –NO2 or –CN group in cis-configuration by replacing nitromethylene pharmacophore with a nitroconjugated system. To understand the action mode of these nitroconjugated neonicotinoids, a representative member IPPA152201 was chosen to perform toxicity and pharmacology studies. IPPA152201 showed a comparable toxicity with imidacloprid against Nilaparvata lugens in a susceptible strain and had no significant cross-resistance in an imidacloprid resistant strain. IPPA152201 showed good efficacies on the isolated cockroach neurons (pEC50 = 5.91 ± 0.14) and the evoked responses by IPPA152201 could be blocked by the typical nAChRs antagonists methyllycaconitine citrate (MLA) and dihydro-β-erythroidine (DHβE), with pIC50 of 6.56 ± 0.07 and 6.89 ± 0.12. The efficacy of IPPA152201 on hybrid receptors Nlα1/β2 in Xenopus oocytes and response inhibition by MLA and DHβE were also observed. These data demonstrate that IPPA152201 acts on insect nAChRs as an agonist. In addition, the influence of a Nlα1 mutation (Y151S), which has been linked to the lab-generated neonicotinoid resistance in N. lugens, has been examined. Compared to the wildtype Nlα1/β2, this mutation reduced I max for IPPA152201 to 63.2% and caused a 1.5-fold increase in EC50, which is much smaller than the effects on imidacloprid. The high insecticidal activity and little influence by Y151S mutation make IPPA152201 to be a potential insecticide to manage N. lugens. [Copyright &y& Elsevier]

Abstract: Nicotinic acetylcholine (ACh) receptors (nAChRs) are ligand-gated ion channels which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The nAChR agonist-binding site is formed by loops A–C present in α subunits together with loops D–F present in either non-α subunits or homomer-forming α subunits. A new non-α subunit was cloned from Nilaparvata lugens, a major rice pest in many parts of Asia, showing very high amino acid identity to other insect β1 subunits, and was denoted as N. lugens β1 (Nlβ1). Six A-to-I RNA editing sites were found in Nlβ1 N-terminal domain, in which only one site was previously reported in Drosophila melanogaster Dβ1 and the other five were newly identified. Among the six editing sites, four caused amino acid changes, in which the site 2 (E2) and site 5 (E5) caused an N to D change in loop D (N73D) and loop E (N133D) respectively. E2 frequency was high in Sus (susceptible) strain and E5 frequency was high in Res (resistant) strain. By expressing in Xenopus oocytes, N73D editing was found to reduce the agonist potency of both ACh and imidacloprid, and the influence on ACh was more significant than on imidacloprid. By contrast, N133D editing only affected imidacloprid potency. These results indicated, although E2 and E5 editings both caused an N to D change in important loops, their roles in neonicotinoid insensitivity might be different. [Copyright &y& Elsevier]

Abstract: Nicotinic acetylcholine (ACh) receptors (nAChRs) are ligand-gated ion channels which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The nAChR agonist-binding site is present at the interface of adjacent subunits and is formed by loops A–C present in α subunits together with loops D–F present in either non-α subunits or homomer-forming α subunits. To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of altering insect-specific loops D, E and F in hybrid nAChRs containing insect and mammalian subunits (Nlα1 from the brown planthopper Nilaparvata lugens and β2 from rat). Introduction of the insect-specific loops D, E and F, singly or together, into rat β2 subunit resulted in a leftward shift of the imidacloprid dose–response curves for nAChRs Nlα1–β2 chimeras, reflecting decreases in EC50, compared to wildtype nAChRs Nlα1–β2. By contrast, the influences on ACh potency were minimal or negligible. The effects of loop D could be interpreted by the earlier findings of Shimomura et al. [2006. Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist-binding site. Mol. Pharmacol. 70, 1255–1263.], in which T77R and E79V were shown to be responsible for neonicotinoid selectivity. In the present study, S131Y(R) and D133N in loop E and T191W and P192K in loop F were found to contribute to the neonicotinoid selectivity of insect-specific loops E and F. These results indicated the insect-specific loops D, E and F each play important roles in neonicotinoids selectivity. This study contributes to our understanding of the molecular mechanism underlying selectivity of neonicotinoids against insects over vertebrates. [Copyright &y& Elsevier]