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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.

Ion channels on cell membrane are molecular targets of more than half peptide neurotoxins from spiders. From Pardosa pseudoannulata, a predatory spider on a range of insect pests, we characterized a peptide neurotoxin PPTX-04 with an insecticidal activity. PPTX-04 showed high toxicity to Nilaparvata lugens, a main prey of P. pseudoannulata, and the toxicity was not affected by the resistance to etofenprox (IUPAC chemical name:1-ethoxy-4-[2-methyl-1-[(3-phenoxyphenyl)methoxy]propan-2-yl]benzene, purity: 99%). On N. lugens voltage-gated sodium channel NlNa v 1 expressed in Xenopus oocytes, PPTX-04 prolonged the channel opening and induced tail currents, which is similar to pyrethroid insecticides. However, PPTX-04 potency on NlNa v 1 was not affected by mutations conferring pyrethroid resistance in insects, which revealed that PPTX-04 and pyrethroids should act on different receptors in NlNa v 1. In contrast, two mutations at the extracellular site 4 significantly reduced PPTX-04 potency, which indicated that PPTX-04 would act on a potential receptor containing the site 4 in NlNa v 1. The result from the molecular docking supported the conclusion that the binding pocket of PPTX-04 in NlNa v 1 should contain the site 4. In summary, PPTX-04 had high insecticidal activity through acting on a distinct receptor site in insect Na v , and was a potential resource to control insect pests and manage resistance to pyrethroids., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Cytochrome P450 monooxygenases (P450s) are recognized as a major contributor to metabolic resistance in insects to most insecticides, through gene overexpressions and protein mutations. MicroRNA (miRNA), an important post-transcriptional regulator, has been reported to promote insecticide resistance by mediating the expression of detoxification enzyme genes., Results: In the present study, we reported that a novel microRNA PC-5p-3991_515 was involved in the post-transcriptional regulation of CYP417A2 and mediated the triflumezopyrim susceptibility in the small brown planthopper (SBPH), Laodelphax striatellus (Fallén). The tissue expression profiles showed that CYP417A2 was highly expressed in fat body. CYP417A2 was significantly up-regulated at 12, 36, 60, 84 and 108 h after the triflumezopyrim treatment. RNA interference (RNAi) against CYP417A2 significantly increased triflumezopyrim susceptibility in SBPH. According to the prediction by miRanda and TargetScan software, three miRNAs were indicated to bind to CYP417A2. However, when oversupply of agomir, only two miRNAs, PC-3p-625_4405 and PC-5p-3991_515, significantly increased the susceptibility to triflumezopyrim and decreased CYP417A2 levels. Furthermore, PC-5p-3991_515 was confirmed to be involved in the post-transcriptional regulation of CYP417A2 by dual luciferase reporter assay. Meanwhile, PC-5p-3991_515 was co-localized with CYP417A2 in the midgut in situ hybridization., Conclusion: Our findings revealed that the novel microRNA, PC-5p-3991_515, post-transcriptionally regulated CYP417A2 expression, which then mediated the triflumezopyrim susceptibility in SBPH. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Four P450s were reported to be important for imidacloprid resistance in Nilaparvata lugens , a major insect pest on rice, which was confirmed in this study in an imidacloprid-resistant strain (ImiR). Here we found that only two ( CYP4CE1 and CYP6ER1 ) from these four P450 genes were overexpressed in a nitenpyram-resistant strain (NitR) when compared to a susceptible strain (SUS). CYP4CE1 RNAi reduced nitenpyram and imidacloprid resistance in NitR and ImiR strains, with a greater reduction in nitenpyram resistance. The transcription factor FoxO mediated nitenpyram resistance in NitR and ImiR strains, but it was not differentially expressed among strains. The potential reason for the differential regulation of FoxO on CYP4CE1 expression was mainly from sequence differences in the CYP4CE1 promoter between susceptible and resistant insects. In six FoxO response elements predicted in the CYP4CE1 promoter, the single-nucleotide polymorphisms were frequently detected in over 50% of NitR and ImiR individuals. The luciferase reporter assays showed that two mutations, -650T/G and -2205T/A in two response elements at the positions of -648 and -2200 bp, mainly contributed to the enhanced regulation on CYP4CE1 expression by FoxO in resistant insects. The frequency was over 69% for both -650T/G and -2205T/A detected in NitR and ImiR individuals but less than 20% in SUS insects. In conclusion, CYP4CE1 overexpression importantly contributed to nitenpyram resistance in N. lugens , and two mutations in the CYP4CE1 promoter of resistant insects led to an enhanced regulation on CYP4CE1 expression by FoxO.

Background: Excessive use of chemical insecticides raises concerns about insecticide resistance, urging the development of novel insecticides. Peptide neurotoxins from spider venom are an incredibly rich source of ion channel modulators with potent insecticidal activity. A neurotoxin U1-Atypitoxin-Cs1a from the spider Calommata signata was annotated previously. It was of interest to investigate its insecticidal activity and potential molecular targets., Results: Cs1a was heterologously expressed, purified and pharmacologically characterized here. The recombinant neurotoxin inhibited high-voltage-activated calcium channel currents with an median inhibitory concentration (IC 50 ) value of 0.182 ± 0.026 μm on cockroach DUM neurons and thus was designated as ω-Atypitoxin-Cs1a. The recombinant Cs1a was toxic to three insect pests of agricultural importance, Nilaparvata lugens, Spodoptera frugiperda and Plutella xylostella with median lethal concentration (LD 50 ) values of 0.121, 0.172 and 0.356 nmol g -1 , respectively, at 24 h postinjection. Cs1a was equivalently toxic to both insecticide-susceptible and -resistant insects. Cs1a exhibited low toxicity to Danio rerio with an LD 50 of 2.316 nmol g -1 ., Conclusion: Our results suggest that ω-Atypitoxin-Cs1a is a potent Ca V channel inhibitor and an attractive candidate reagent for pest control and resistance management. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Photopharmacology involving azobenzene or dithienylethene has changed established methods of studying receptor functions, allowing for increasing the spatiotemporal resolution. There are no photopharmacological tools available for the invertebrate nicotinic acetylcholine receptor (nAChR). Here, we report a photochromic ligand, dithienylethene-imidacloprid (DitIMI), using a dithienylethene photoswitch embedded in the nAChR agonist imidacloprid. It was found that DitIMI displayed good photochromism and fluorescence switching behaviors upon irradiation with UV/vis light in aqueous solution. We demonstrated that open -DitIMI has low spontaneous in vitro and in vivo activity but can be photoisomerized to a highly active closed -form. Surprisingly, the photoswitchable DitIMI showed a large difference in insecticidal activity between the open and closed forms, in which the insecticidal activity against Aphis craccivora of the ring-closed isomers for DitIMI was 355 times that of the corresponding ring-open isomers. This photoisomerization can further be translated to photomodulation of neuron membrane potential and behavioral responses of living mosquito larvae and American cockroaches. The photomanipulation of nACh neurotransmission opens new avenues to understanding inhibitory circuits in intact animals.

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 I max of cycloxaprid by 37.0% and increased EC 50 values by 1.9-fold, while the I max of imidacloprid was reduced by 72.0%, and EC 50 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 EC 50 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.

Cytochrome P450 enzymes metabolize various xenobiotics in insects. Compared to numerous P450s associated with insecticide detoxification and resistance, fewer have been identified to bioactivate proinsecticides in insects. Here we reported that two P450s, CYP4C62 and CYP6BD12, in Nilaparvata lugens could bioactivate chlorpyrifos, an organophosphorus insecticide, into its active ingredient chlorpyrifos-oxon in vivo and in vitro . RNAi knockdown of these two genes significantly reduced the sensitivity to chlorpyrifos and the formation of chlorpyrifos-oxon in N. lugens . Chlorpyrifos-oxon was generated when chlorpyrifos was incubated with the crude P450 enzyme prepared from N. lugens or recombinant CYP4C62 and CYP6BD12 enzymes. The expression reduction of CYP4C62 and CYP6BD12 and alternative splicing in CYP4C62 reduced the oxidation of chlorpyrifos into chlorpyrifos-oxon, which contributed importantly to chlorpyrifos resistance in N. lugens . This study revealed a novel mechanism of insecticide resistance due to the bioactivation reduction, which would be common for all currently used proinsecticides.

CYP6ER1 overexpression is a prevalent mechanism for neonicotinoid resistance in Nilaparvata lugens . Except for imidacloprid, the metabolism of other neonicotinoids by CYP6ER1 lacked direct evidence. In this study, a CYP6ER1 knockout strain ( CYP6ER1 -/- ) was constructed using the CRISPR/Cas9 strategy. The CYP6ER1 -/- strain showed much higher susceptibility to imidacloprid and thiacloprid with an SI (sensitivity index, LC 50 of WT/LC 50 of CYP6ER1 -/- ) of over 100, which was 10-30 for four neonicotinoids (acetamiprid, nitenpyram, clothianidin, and dinotefuran) and less than 5 for flupyradifurone and sulfoxaflor. Recombinant CYP6ER1 showed the highest activity to metabolize imidacloprid and thiacloprid and moderate activity for the other four neonicotinoids. Main metabolite identification and oxidation site prediction revealed that CYP6ER1 activities were insecticide structure-dependent. The most potential oxidation site of imidacloprid and thiacloprid was located in the five-membered heterocycle with hydroxylation activity. For the other four neonicotinoids, the potential site was within the ring opening of a five-membered heterocycle, indicating N -desmethyl activity.

Acetylcholinesterase (AChE) is an essential neurotransmitter hydrolase in nervous systems of animals and its number varies among species. So far, five AChEs have been identified in the natural enemy Pardosa pseudoannulata. Here we found that Ppace1, Ppace2 and Ppace5 were highly expressed in the spider brain, among which the mRNA level of Ppace5, but not Ppace1 and Ppace2, could be up-regulated by organophosphorus insecticides at their sublethal concentrations. In spider brain, the treatment by organophosphorus insecticides at the sublethal concentrations could increase total AChE activity, although high concentrations inhibited the activity. The activity that increased from the sublethal concentration pretreatment could compensate for the activity inhibition due to subsequent application of organophosphorus insecticides at lethal concentrations, and consequently reduce the mortality of spiders. PpAChE1 and PpAChE2 were highly sensitive to organophosphorus insecticides, and their activities would be strongly inhibited by the insecticides. In contrast, PpAChE5 displayed relative insensitivity towards organophosphorus insecticides, but with the highest catalytic efficiency for ACh. That meant the up-regulation of Ppace5 under insecticide exposure was important for maintaining AChE activity in spider brain, when PpAChE1 and PpAChE2 were inhibited by organophosphorus insecticides. The study demonstrated that multiple AChEs in the spider brain worked collaboratively, with part members for maintaining AChE activity and other members responding to organophosphorus inhibition, to provide protection from organophosphorus insecticides. In fields, high concentration insecticides are often applied when ineffective controls of insect pests occur due to relative-low concentration of insecticides in last round application. This application pattern of organophosphorus insecticides provides more chances for P. pseudoannulata to survive and controlling insect pests as a natural enemy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Insect glutathione S-transferases (GSTs) participate in detoxifying insecticides and plant metabolites in two different ways, metabolizing toxic components and remedying oxidative stress. Here in Nilaparvata lugens, a major insect pest on rice, the roles of cytosolic GSTs in resistance to insecticides and to plant defences were evaluated. The over-expression in four resistant strains indicated that NlGSTs1 and NlGSTs2 were essential to resistances to four test insecticides and H 2 O 2 through an antioxidation mechanism. RNAi verified the antioxidation function of NlGSTs1 and NlGSTs2 in the resistances as a common mechanism, regardless of the structural differences among insecticides and H 2 O 2 . NlGSTs1 and NlGSTs2 also provided protection for N. lugens against rice defense by the same mechanism, reducing H 2 O 2 levels when N. lugens were fed on the resistant rice variety Mudogo. The antioxidation activity of recombinant NlGSTs1 and NlGSTs2 is higher than their direct detoxification, which supported the ability of these two GSTs to remedy oxidative stress. For oxidative stress remediation as a common mechanism of NlGSTs1 and NlGSTs2 in both insecticide resistance and host adaptability, the development of insecticide resistance might enhance the ability of insects to remedy oxidative stress from feeding on resistant rice variety and thus to lower the resistance level of rice variety to N. lugens. The results call for careful assessment on N. lugens control when both insecticides and resistant rice variety are applied., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Insecticides harm the beneficial organisms, such as predatory spiders, through direct killing or regulation of the development and reproduction. In this study, the bioassay showed that the treatment of juvenile hormone (JH) analogue fenoxycarb delayed the moulting of Pardosa pseudoannulata, a dominant predatory spider in paddy fields. In order to figure out the regulatory mechanism of fenoxycarb on the spider development, we systematically analyzed JH biosynthesis in P. pseudoannulata. All genes involved in JH biosynthesis pathway were retrieved from the genome of P. pseudoannulata, except for CYP15A1. The absence of CYP15A1 was in agreement with the identification of methyl farnesoate (MF) rather than JH III in the spider. The delayed moulting and decreased expression of JH biosynthesis-related genes in the MF-applied spiderlings supported that MF was an active JH. Fenoxycarb treatment significantly upregulated the transcriptional level of JH biosynthesis-related genes and consequently delayed the spiderling moulting. In the spider development, ecdysteroid played the opposite role, in contrast to MF, to accelerate the development, as our previous study. Here we found that the treatment of ecdysteroid analogue tebufenozide accelerated P. pseudoannulata spiderling moulting, which resulted from the expressional suppression of ecdysteroid biosynthesis-related genes. In total, the JH and ecdysteroid analogues affected the development of P. pseudoannulata by the expressional regulation of biosynthesis-related genes, which would be helpful for the evaluation of hormone analogue insecticides in environmental safety, and useful for the protection and application of P. pseudoannulate and related spider species., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Pyrethroid insecticides prolong the opening of insect sodium channels by binding to two predicted pyrethroid receptor sites (PyR), PyR1 and PyR2. Many naturally-occurring sodium channel mutations that confer pyrethroid resistance (known as knockdown resistance, kdr) are located at PyR1. Recent studies identified two new mutations, V253F and T267A, at PyR2, which co-exist with two well-known mutations F1534C or M918T, at PyR1, in pyrethroid-resistant populations of Aedes aegypti and Nilaparvata lugens, respectively. However, the role of the V253F and T267A mutations in pyrethroid resistance has not been functionally examined. Here we report functional characterization of the V253F and T267A mutations in the Ae. aegypti sodium channel AaNa v 2-1 and the N. lugens sodium channel NlNa v 1 expressed in Xenopus oocytes. Both mutations alone reduced channel sensitivity to pyrethroids, including etofenprox. We docked etofenprox in a homology model of the pore module of the NlNa v 1 channel based on the crystal structure of an open prokaryotic sodium channel NavMs. In the low-energy binding pose etofenprox formed contacts with V253, T267 and a previously identified L1014 within PyR2. Combining of V253F or T267A with F1534C or M918T results in a higher level of pyrethroid insensitivity. Furthermore, both V253F and T267A mutations altered channel gating properties. However, V253F- and T267A-induced gating modifications was not observed in the double mutant channels. Our findings highlight the first example in which naturally-found combinational mutations in PyR1 and PyR2 not only confer higher level pyrethroid insensitivity, but also reduce potential fitness tradeoff in pyrethroid-resistant mosquitoes caused by kdr mutation-induced sodium channel gating modifications., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

As one of the most important detoxification enzymes in insects, Glutathione S-transferases (GSTs) play key roles in insecticide resistance via direct metabolism and protection against oxidative stress induced by insecticide exposure. Insect GSTs are often considered as the phase II detoxification enzymes, they have potential function to metabolize fipronil as well as its fipronil's metabolites. In the fipronil-resistant Nilaparvata lugens strain G28, GSTs' inhibitor DEM (diethyl maleate) showed the optimal synergistic effects (5.73-fold), indicating the essential roles of GSTs in the resistance to fipronil in this insect species. Four GST genes, NlGSTs1, NlGSTs2, NlGSTe1 and NlGSTd1, were found over-expressed in G28 when compared to its relative susceptible counterpart strain S28. The roles of these four GSTs in fipronil resistance were confirmed via RNAi. The four GST genes were highly over-expressed in the midgut and/or fat body with detoxification action, which might provide more chances for insects to metabolize fipronil and its metabolites. Additionally, the higher induction levels in the GST gene expression by insecticides in the midgut and/or fat body compared to the whole insect also supported the significant roles of the four GSTs in the detoxification. Above all, the results provided evidences to understand the functions of GSTs in fipronil resistance in N. lugens, and gave a reference for other insects in fipronil resistance., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Insecticide synergists are sought-after due to their potential in improving the pesticide control efficacy with a reduced dose of an active ingredient. We previously reported that a cis-configuration neonicotinoid (IPPA08) exhibited specific synergistic activity toward neonicotinoid insecticides. In this study, we synthesized a series of structural analogues of IPPA08 by converting the pyridyl moiety of IPPA08 into phenyl groups, via facile double-Mannich condensation reactions between nitromethylene compounds and glutaraldehyde. All of the oxabridged neonicotinoid compounds were found to increase the toxicity of imidacloprid against Aphis craccivora . Notably, compound 25 at 0.75 mg/L lowered the LC 50 value of imidacloprid against A. craccivora by 6.54-fold, while a 3.50-fold reduction of the LC 50 value was observed for IPPA08. The results of bee toxicity test showed that compound 25 display selectivity in its effects on imidacloprid toxicity against the honey bee ( Apis mellifera L.). In summary, replacing the pyridyl ring with a phenyl ring was a viable approach to obtain a novel synergist with oxabridged moiety for neonicotinoid insecticides.

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 .

Enhanced detoxification and target mutations that weaken insecticide binding ability are major mechanisms of insecticide resistance. Among these, over-expression or site mutations of carboxylesterase (CarE), cytochrome P450s (CYP450), and glutathione-S-transferase (GST) were the main form responsible for insecticide detoxification; however, transcript-level analysis of the relationship of detoxification gene mutations with chlorpyrifos (an organophosphorus insecticide) resistance is scarce thus far. In this study, multiple sites exhibiting polymorphisms within three detoxification genes were firstly examined via sequencing among different chlorpyrifos-resistant and susceptible individuals of Laodelphax striatellus. For example, the mutation frequencies of A374V in LsCarE16 were 83, 33, and 3%, S277A in LsCarE24 were 88, 28, and 3%, E36K in LsCYP426A1 were 100, 65, and 0% for chlorpyrifos-resistant, resistant decay, and susceptible individuals, respectively. Analysis also found expression levels of GSTd1, GSTt1, GSTs2, CYP4DE1U1, and CYP425B1 are coordinated with chlorpyrifos resistance levels; moreover, we found the deficiencies of 43S and 44A as well as two point mutations of E60D and Q61H at N-terminal region of the OP potential target acetylcholinesterase (AChE) in high resistant but not in low-chlorpyrifos resistant individuals. The results above all demonstrated the dynamic evolutionary process of insecticide resistance and revealed some resistance factors that only played roles at certain resistance level; high insecticide resistance in this example is the result of synergistic impact from multiple resistance factors., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.)

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.)

Monitoring resistance and investigating insecticide resistance mechanisms are necessary for controlling the small brown planthopper, Laodelphax striatellus. The susceptibility to four common insecticides of L. striatellus collected from Jiangsu, Anhui, Zhejiang and Jilin provinces of China in 2015 was monitored. The results showed that all field populations remained susceptible to chlorpyrifos and thiamethoxam with resistance ratios (RRs) of 2.3- to 9.5 and 1.6- to 3.3, respectively, while the insects had developed moderate pymetrozine resistance with RRs of 18.7 to 34.5. Resistance against buprofezin had developed to an alarmingly high level in three southeastern provinces of China with RRs of 108.8 to 156.1, but in Jilin it had an RR of only 26.6. Moreover, in line with both the buprofezin and pymetrozine resistance levels, we found LsCYP6CW1 to be over-expressed in all field L. striatellus populations, which indicated that it might be important for cross-resistance between buprofezin and pymetrozine. RNA interference (RNAi) ingestion resulted in the effective suppression of LsCYP6CW1 expression, and significantly increased susceptibility to both buprofezin and pymetrozine compared with the control, which further confirmed that overexpression of LsCYP6CW1 was involved in the cross-resistance to buprofezin and pymetrozine in field L. Striatellus populations.

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.)

Etofenprox, a non-ester pyrethroid insecticide, will be registered to control rice pests such as the brown planthopper (BPH, Nilaparvata lugens Stål) in mainland China. Insecticide resistance is always a problem to the effective control of N. lugens by chemical insecticides. An etofenprox resistance selection of N. lugens was performed in order to understand the related mechanisms. Through successive selection by etofenprox for 16 generations in the laboratory, an etofenprox-resistant strain (G16) with the resistance ratio (RR) of 422.3-fold was obtained. The resistance was partly synergised (2.68-fold) with the metabolic inhibitor PBO, suggesting a role for P450 monooxygenases. In this study, 11 P450 genes were significantly up-regulated in G16, among which eight genes was above 2.0-fold higher than that in US16, a population with the same origin of G16 but without contacting any insecticide in the laboratory. The expression level of four genes (CYP6AY1, CYP6FU1 and CYP408A1 from Clade 3, and CYP425A1 from Clade 4) were above 4.0-fold when compared to US16. RNA interference (RNAi) was performed to evaluate the importance of the selected P450s in etofenprox resistance. When CYP6FU1, CYP425A1 or CYP6AY1 was interfered, the susceptibility was significantly recovered in both G16 and US16, while the knockdown of CYP408A1 or CYP353D1 did not cause significant changes in etofenprox susceptibility. We supposed that CYP6FU1 was the most important P450 member for etofenprox resistance because of the highest expression level and the most noticeable effects on resistance ratios following RNAi., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Insecticide synergists are key components to increase the control efficacy and reduce active ingredient use. Here, we describe a novel insecticide synergist with activity specific for insecticidal neonicotinoids. The synergist IPPA08, a cis configuration neonicotinoid compound with a unique oxabridged substructure, could increase the toxicity of most neonicotinoid insecticides belonging to the Insecticide Resistance Action Committee (IRAC) 4A subgroup against a range of insect species, although IPPA08 itself was almost inactive to insects at synergistic concentrations. Unfortunately, similar effects were observed on the honey bee (Apis mellifera) and the brown planthopper (Nilaparvata lugens), resistant to imidacloprid. IPPA08 did not show any effects on toxicity of insecticides with different targets, which made us define it as a neonicotinoid-specific synergist. Unlike most insecticide synergists, by inhibition of activities of detoxification enzymes, IPPA08 showed no effects on enzyme activities. The results revealed that IPPA08 worked as a synergist through a distinct way. Although the modulating insect nicotinic acetylcholine receptors (nAChRs, targets of neonicotinoid insecticides) were supposed as a possible mode of action for IPPA08 as a neonicotinoid-specific synergist, direct evidence is needed in further studies. In insect pest control, IPPA08 acts as a target synergist to increase neonicotinoid toxicity and reduce the amount of neonicotinoid used. Combinations of IPPA08 and insecticidal neonicotinoids may be developed into new insecticide formulations. In summary, combining an active ingredient with a "custom" synergist appears to be a very promising approach for the development of effective new insecticide products.

Cross-resistance between insecticides, especially from different groups, can be extremely unpredictable, and it has been a serious concern in pest control. Pymetrozine has been widely used to control Nilaparvata lugens with the suspension of imidacloprid for the resistance, and N. lugens has showed obvious pymetrozine resistance in recent years. To investigate the possible cross-resistance between imidacloprid and pymetrozine is very important to avoid the adverse effects on resistance development and pest control. Bioassays of two field populations in five consecutive years showed that imidacloprid resistance decreased greatly, while pymetrozine resistance increased significantly. The synergist piperonyl butoxide (PBO) could synergize both imidacloprid and pymetrozine in all field populations, which indicated the importance of P450s in the resistance to two insecticides. Imidacloprid resistance was reported to be associated with two P450s, CYP6AY1 and CYP6ER1, which could metabolize imidacloprid efficiently. However, the recombinant proteins of these two P450s did not show any enzymatic activity to metabolize pymetrozine. The pymetrozine susceptibility did not change when CYP6AY1 and CYP6ER1 mRNA levels were reduced by RNA interference (RNAi), although which could obviously decrease imidacloprid resistance. In vivo and in vitro studies provided evidences to demonstrate that there was no cross-resistance between imidacloprid and pymetrozine in N. lugens, which was different from the findings in Bemisia tabaci., (Copyright © 2015. Published by Elsevier Inc.)

Two P450 monooxygenase genes, CYP6AY1 and CYP6ER1, were reported to contribute importantly to imidacloprid resistance in the brown planthopper, Nilaparvata lugens. Although recombinant CYP6AY1 could metabolize imidacloprid efficiently, the expression levels of CYP6ER1 gene were higher in most resistant populations. In the present study, three field populations were collected from different countries, and the bioassay, RNAi and imidacloprid metabolism were performed to evaluate the importance of two P450s in imidacloprid resistance. All three populations, DOT (Dongtai) from China, CNA (Chainat) from Thailand and HCM (Ho Chi Minh) from Vietnam, showed high resistance to imidacloprid (57.0-, 102.9- and 89.0-fold). CYP6AY1 and CYP6ER1 were both over expressed in three populations, with highest ratio of 13.2-fold for CYP6ER1 in HCM population. Synergism test and RNAi analysis confirmed the roles of both P450 genes in imidacloprid resistance. However, CYP6AY1 was indicated more important in CNA population, and CYP6AY1 and CYP6ER1 were equal in HCM population, although the expression level of CYP6ER1 (13.2-fold) was much higher than that of CYP6AY1 (4.11-fold) in HCM population. Although the recombinant proteins of both P450 genes could metabolize imidacloprid efficiently, the catalytic activity of CYP6AY1 (Kcat=3.627 pmol/min/pmol P450) was significantly higher than that of CYP6ER1 (Kcat=2.785 pmol/min/pmol P450). It was supposed that both P450 proteins were important for imidacloprid resistance, in which CYP6AY1 metabolized imidacloprid more efficiently and CYP6ER1 gene could be regulated by imidacloprid to a higher level., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The pond wolf spider (Pardosa pseudoannulata) is an important predatory enemy against several insect pests and showed relative different sensitivities to organophosphate and carbamate insecticides compared to insect pests. In our previous studies, two acetylcholinesterases were identified in P. pseudoannulata and played important roles in insecticide sensitivities. In order to understand the contributions of the two acetylcholinesterases to insecticide sensitivities, we firstly employed the RNAi technology in the spider. For a suitable microinjection RNAi method, the injection site, injection volume and interference time were optimized, which then demonstrated that the injection RNAi method was applicable in this spider. With the new RNAi method, it was revealed that both Pp-AChE1 and Pp-AChE2, encoded by genes Ppace1 and Ppace2, were the targets of organophosphate insecticides, but Pp-AChE1 would be more important. In contrast, the carbamate acted selectively on Pp-AChE1. The results showed that Pp-AChE1 was the major catalytic enzyme in P. pseudoannulata and the major target of organophosphate and carbamate insecticides. In a word, an RNAi method was established in the pond wolf spider, which further validated the importance of two acetylcholinesterases in insecticide sensitivities in this spider., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The insect central nervous system (CNS) is the target for many insecticides, and changes in transcript levels could be expected after insecticide applications. In this study, differentially expressed genes in the locust (Locusta migratoria manilensis) CNS in response to imidacloprid treatments at low dose (LD, 10% mortality) and high dose (HD, 80% mortality) were identified. Two nicotine acetylcholine receptor (nAChR) subunits genes and 18 interacting protein genes were regulated at LD, and only one nAChR subunit gene and 11 interacting proteins were regulated at HD. Among the 110 annotated P450 unigenes, 43 unigenes were regulated at LD and 34 unigenes were regulated at HD. Most of the differentially expressed P450 unigenes were mapped to CYP4, in which most unigenes were upregulated at LD, but downregulated at HD. Totally, the numbers and regulation levels of the regulated genes were more at LD than that at HD. Seventeen unigenes were selected to test their expression changes following insecticide treatments by qRT-PCR, in which the changes in more than half of the selected genes were verified. The results revealed the variation in the response of locusts to different insecticide pressure, such as different doses., (© The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.)

The pond wolf spider Pardosa pseudoannulata, an important natural predatory enemy of rice planthoppers, is found widely distributed in paddy fields. However, data on the genes involved in insecticide action, detoxification, and response are very limited for P. pseudoannulata, which inhibits the development and appropriate use of selective insecticides to control insect pests on rice. We used transcriptome construction from adult spider cephalothoraxes to analyze and manually identify genes enconding metabolic enzymes and target receptors related to insecticide action and detoxification, including 90 cytochrome P450s, 14 glutathione S-transferases (GSTs), 17 acetylcholinesterases (AChEs), 17 nicotinic acetylcholine receptors (nAChRs), and 17 gamma-aminobutyric acid (GABA) receptors, as well as 12 glutamate-gated chloride channel (GluCl) unigenes. Sequence alignment and phylogenetic analysis revealed the different subclassifications of P450s and GSTs, some important sequence diversities in nAChRs and GABA receptors, polymorphism in AChEs, and high similarities in GluCls. For P450s in P. pseudoannulata, the number of unigenes belonging to the CYP2 clade was much higher than that in CYP3 and CYP4 clades. The results differed from insects in which most P450 genes were in CYP3 and CYP4 clades. For GSTs, most unigenes belonged to the delta and sigma classes, and no epsilon GST class gene was found, which differed from the findings for insects and acarina. Our results will be useful for studies on insecticide action, selectivity, and detoxification in the spider and other related animals, and the sequence differences in target genes between the spider and insects will provide important information for the design of selective insecticides.

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 EC50 value in Clα8(N191F)/β2 of 15.21μM and 5.74μM 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., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

As one of the most important predatory enemies, the miridbug, Cyrtorhinus lividipennis, plays an important role in rice planthoppers control, such as Nilaparvata lugens (brown planthopper). In order to compare insecticide selectivity between C. lividipennis and N. lugens, the contact acute toxicities of six insecticides (diazoxon, paraoxon, carbaryl, fenobucarb, fipronil and ethofenprox) were monitored. The results showed that all tested insecticides were more toxic to C. lividipennis than to N. lugens and fipronil had the biggest difference. The RDL subunit (Cl-RDL) was cloned from C. lividipennis and a RDL isoform (Cl-RDL-In) was also found with 31 amino acids insertion in RDL intracellular region. In order to understand the role of the insertion on insecticide sensitivities, three subunits (Nl-RDL, Cl-RDL and Cl-RDL-In) were constructed to obtain the functional receptors in Xenopus oocytes and the fipronil sensitivities were detected by the voltage-clamp technique. Nl-RDL (IC50=32.36 ± 4.07 µM) was more insensitive to fipronil than Cl-RDL (IC50=6.47 ± 1.12 µM). The insertion in Cl-RDL significantly reduced fipronil sensitivity with IC50 value in Cl-RDL-In of 16.83 ± 2.30 µM. Interestingly, after the elution of fipronil, the current response of Cl-RDL-In appeared obvious recovery, which were not observed in Cl-RDL and Nl-RDL. It might imply that the insertion played a special role in fipronil sensitivity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Pardosa pseudoannulata is an important predatory enemy against insect pests, such as rice planthoppers and leafhoppers. In order to understand the insecticide selectivity between P. pseudoannulata and insect pests, two acetylcholinesterase genes, Pp-ace1 and Pp-ace2, were cloned from this natural enemy. The putative proteins encoded by Pp-ace1 and Pp-ace2 showed high similarities to insect AChE1 (63% to Liposcelis entomophila AChE1) and AChE2 (36% to Culex quinquefasciatus AChE2) with specific functional motifs, which indicated that two genes might encode AChE1 and AChE2 proteins respectively. The recombinant proteins by expressing Pp-ace1 and Pp-ace2 genes in insect sf9 cells showed high AChE activities. The kinetic parameters, Vmax and Km, of two recombinant AChE proteins were significantly different. The sensitivities to six insecticides were determined in two recombinant AChEs. Pp-AChE1 was more sensitive to all tested insecticides than Pp-AChE2, such as fenobucarb (54 times in Ki ratios), isoprocarb (31 times), carbaryl (13 times) and omethoate (6 times). These results indicated that Pp-AChE1 might be the major synaptic enzyme in the spider. By sequence comparison of P. pseudoannulata and insect AChEs, the key amino acid differences at or close to the functional sites were found. The locations of some key amino acid differences were consistent with the point mutation sites in insect AChEs that were associated with insecticide resistance, such as Phe331 in Pp-AChE2 corresponding to Ser331Phe mutation in Myzus persicae and Aphis gossypii AChE2, which might play important roles in insecticide selectivity between P. pseudoannulata and insect pests. Of course, the direct evidences are needed through further studies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Imidacloprid is a key insecticide extensively used for control of Nilaparvata lugens, and its resistance had been reported both in the laboratory selected strains and field populations. A target site mutation Y151S in two nicotinic acetylcholine receptor subunits and enhanced oxidative detoxification have been identified in the laboratory resistant strain, contributing importantly to imidacloprid resistance in N. lugens. To date, however, imidacloprid resistance in field population is primarily attributable to enhanced oxidative detoxification by over-expressed P450 monooxygenases. A resistant strain (Res), originally collected from a field population and continuously selected in laboratory with imidacloprid for more than 40 generations, had 180.8-fold resistance to imidacloprid, compared to a susceptible strain (Sus). Expression of different putative P450 genes at mRNA levels was detected and compared between Res and Sus strains, and six genes were found expressed significantly higher in Res strain than in Sus strain. CYP6AY1 was found to be the most different expressed P450 gene and its mRNA level in Res strain was 17.9 times of that in Sus strain. By expressing in E. coli cells, CYP6AY1 was found to metabolize imidacloprid efficiently with initial velocity calculated of 0.851 ± 0.073 pmol/min/pmol P450. When CYP6AY1 mRNA levels in Res strain was reduced by RNA interference, imidacloprid susceptibility was recovered. In four field populations with different resistance levels, high levels of CYP6AY1 transcript were also found. In vitro and in vivo studies provided evidences that the over-expression of CYP6AY1 was one of the key factors contributing to imidacloprid resistance in the laboratory selected strain Res, which might also be the important mechanism for imidacloprid resistance in field populations, when the target site mutation was not prevalent at present., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of the insect nicotinic acetylcholine receptors (nAChRs) with -NO(2) or -CN group in trans-configuration. Previously we reported the excellent insecticidal activity of a series of nitroconjugated neonicotinoids with -NO(2) 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 (pEC(50) = 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 pIC(50) 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 EC(50), 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 © 2011 Elsevier Ltd. All rights reserved.)

Resistance development and limited lepidopteran activities call for the discovery of "super-neonicotinoids" solving these problems. Compounds with the cis-configuration offer an opportunity for further optimization. Fixing the nitro group in the cis-configuration provided a new approach for neonicotinoid molecular design. Introductions of the heterocycle or a bulky group are two synthesis concepts to fix the cis-configuration of the nitro group. The design, synthesis, bioactivity, and preliminary modes of action of five types of cis-neonicotinoids are reviewed. cis- and trans-neonicotinoids have some differences in bioactivities and modes of action. This study focused, especially, on the reaction diversities of nitromethylene analogues of imidacloprid with various aldehydes.

cis-Neonicotinoids are a type of neonicotinoid, in which the nitro or the cyano group are in cis-configuration relative to heteroaromatic moiety, which show excellent activities against a range of insect species. This review covers cis-neonicotinoids with commercialization perspectives, structural optimization (phenylazoneonicotinoids and chlorothiazolyl analogues of Paichongding), modes of action studies, radiao-synthesis of Paichongding and Cycloxaprid, and photostability of neonicotinoids.

A series of tetrahydroimidazo[1,2-a]pyridine derivatives were synthesized by the reaction of 2-vinyl-4,5-dihydroimidazole derivatives with substituted benzylidenemalononitrile via a catalyst-free aza-Diels-Alder reaction. Insecticidal activities of target compounds were tested against pea aphids ( Aphis craccivora ), which showed that activities were strongly influenced by the substituents and their positions. Especially, the introduction of a fluoro group at the 2- position increased activities.

A series of divalent and oxabridged neonicotinoids were synthesized by reactions of nitromethylene analogues of imidacloprid and dialdehydes, and their structures were confirmed by (1)H NMR, (13)C NMR, high-resolution mass spectroscopy, and X-ray diffraction analysis. The bioassays indicated that some of them were endowed with excellent insecticidal activities against cowpea aphid ( Aphis craccivora ), armyworm ( Pseudaletia separata Walker), and brown planthopper ( Nilaparvata lugens ). Divalent neonicotinoid 6 and oxabridged 8a had higher activities than imidacloprid against cowpea aphids and armyworm; furthermore, the activity of 8a was 40.4-fold higher than that of imidacloprid against imidacloprid-resistant brown planthopper.

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. The brown planthopper (Nilaparvata lugens), an insect pest of rice crops throughout Asia, is an important target species for control with neonicotinoid insecticides such as imidacloprid. Studies with nAChRs purified from N. lugens have identified two [(3)H]imidacloprid binding sites with different affinities (K(d) = 3.5 +/- 0.6 pM and 1.5 +/- 0.2 nM). Co-immunoprecipitation studies with native preparations of N. lugens nAChRs, using subunit-selective antisera, have demonstrated the co-assembly of Nlalpha1, Nlalpha2 and Nlbeta1 subunits into one receptor complex and of Nlalpha3, Nlalpha8 and Nlbeta1 into another. Immunodepletion of Nlalpha1 or Nlalpha2 subunits resulted in the selective loss of the lower affinity imidacloprid binding site, whereas immunodepletion of Nlalpha3 or Nlalpha8 caused the selective loss of the high-affinity site. Immunodepletion of Nlbeta1 resulted in a complete absence of specific imidacloprid binding. In contrast, immunodepletion with antibodies selective for other N. lugens nAChR subunits (Nlalpha4, Nlalpha6, Nlalpha7 and Nlbeta2) had no significant effect on imidacloprid binding. Taken together, these data suggest that nAChRs containing Nlalpha1, Nlalpha2 and Nlbeta1 constitute the lower affinity binding site, whereas nAChRs containing Nlalpha3, Nlalpha8 and Nlbeta1 constitute the higher affinity binding site for imidacloprid in N. lugens., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

One nicotinic acetylcholine receptor non-alpha subunit was cloned from the pond wolf spider, Pardosa pseudoannulata, an important predatory enemy of some insect pests with agricultural importance, such as the green peach aphid Myzus persicae. The subunit shows high amino acid identities to insect beta1 subunits (74-78%), and was denoted as Ppbeta1. Although high identities are found between Ppbeta1 and insect beta1 subunits, amino acid differences are found within loops D, E and F, important segments contributing to ligand binding. The effects of amino acid differences within these loops were evaluated by introducing loops of insect or spider beta1 subunits into rat beta2 subunit and co-expressing with insect alpha subunit. The corresponding regions of rat beta2 chimera beta2(Mpbeta1) (beta2 with loops D, E and F from M. persicae beta1 subunit Mpbeta1) were replaced by loops D, E and F of Ppbeta1 singly or together to construct different chimeras. When these chimeras were co-expressed with insect Nlalpha1, it was found that the replacement of loops D, E and F of beta2(Mpbeta1) by that of Ppbeta1 resulted in a right-ward shift of the imidacloprid dose-response curves, reflecting increases in EC(50), compared to Nlalpha1/beta2(Mpbeta1). By contrast, the influences on ACh potency were minimal. The further study showed that R81Q, N137G and F190W differences, within loops D, E and F respectively, contributed mainly to these sensitivity changes. This study contributes to our understanding of the molecular mechanism underlying selectivity of neonicotinoids against insects over spiders.

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. Previously, we have identified a nAChR point mutation (Y151S) associated with insecticide resistance in the brown planthopper Nilaparvata lugens. Although this mutation has been identified in two different N. lugens nAChR subunits (Nlalpha1 and Nlalpha3) because of difficulties in heterologous expression of Nlalpha3; its influence on agonist potency has been examined only in Nlalpha1-containing nAChRs. Here we describe the cloning of a novel nAChR subunit from N. lugens (Nlalpha8), together with evidence for its co-assembly with Nlalpha3 in native and recombinant nAChRs. This has, for the first time, enabled the functional effects of the Nlalpha3(Y151S) mutation to be examined. The Nlalpha3(Y151S) mutation has little effect on agonist potency of acetylcholine but has a dramatic effect on neonicotinoid insecticides (reducing I(max) values and increasing EC(50) values). The apparent affinity of neonicotinoids was higher and the effect of the Y151S mutation on neonicotinoid agonist potency was more profound in Nlalpha3-containing, rather than Nlalpha1-containing nAChR. We conclude that Nlalpha3- and Nlalpha1-containing nAChRs may be representative of two distinct insect nAChR populations.

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 alpha subunits together with loops D-F present in either non-alpha subunits or homomer-forming alpha subunits. A new non-alpha subunit was cloned from Nilaparvata lugens, a major rice pest in many parts of Asia, showing very high amino acid identity to other insect beta1 subunits, and was denoted as N. lugens beta1 (Nlbeta1). Six A-to-I RNA editing sites were found in Nlbeta1 N-terminal domain, in which only one site was previously reported in Drosophila melanogaster Dbeta1 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.

Background: The brown planthopper (BPH), Nilaparvata lugens Stål, is a major rice pest in many parts of Asia. Neonicotinoids such as imidacloprid and dinotefuran are widely used for control of this pest, and resistance to these insecticides has developed in recent years. This pest has also been widely exposed to triazophos and fenvalerate, although these insecticides are not used for the control of the pest directly. Here, the effects of sublethal doses of these four insecticides on the reproduction and wing formation in BPH were examined., Results: Imidacloprid and dinotefuran reduced the fecundity of BPH to 68.8% and 52.4% in macropterous families, and to 57.9% and 43.1% in brachypterous families, when compared with the untreated controls. By contrast, triazophos and fenvalerate increased fecundity. In both macropterous and brachypterous families, sublethal doses of imidacloprid and dinotefuran showed significant induction of macropterous adults., Conclusion: Imidacloprid and dinotefuran could reduce the fecundity of BPH to a significant extent, demonstrating further activity against this pest in addition to their direct toxicity. The significant induction of macropterous adults by sublethal doses of imidacloprid and dinotefuran is of importance in the management of this pest, particularly in the areas of predicting the size of emigrating populations and the numbers of insects likely to occur in newly colonized areas.

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively in areas of crop protection and animal health to control a variety of insect pest species. Here, we describe studies performed with nAChR subunits Nlalpha1 and Nlalpha2 cloned from the brown planthopper Nilaparvata lugens, a major insect pest of rice crops in many parts of Asia. The influence of Nlalpha1 and Nlalpha2 subunits upon the functional properties of recombinant nAChRs has been examined by expression in Xenopus oocytes. In addition, the influence of a Nlalpha1 mutation (Y151S), which has been linked to neonicotinoid lab generated resistance in N. lugens, has been examined. As in previous studies of insect alpha subunits, functional expression has been achieved by co-expression with the mammalian beta2 subunit. This approach has revealed a significantly higher apparent affinity of imidacloprid for Nlalpha1/beta2 than for Nlalpha2/beta2 nAChRs. In addition, evidence has been obtained for the co-assembly of Nlalpha1 and Nlalpha2 subunits into 'triplet' nAChRs of subunit composition Nlalpha1/Nlalpha2/beta2. Evidence has also been obtained which demonstrates that the resistance-associated Y151S mutation has a significantly reduced effect on neonicotinoid agonist activity when Nlalpha1 is co-assembled with Nlalpha2 than when expressed as the sole alpha subunit in a heteromeric nAChR. These findings may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance in insect field populations.

Background: Rice brown planthopper, Nilaparvata lugens Stål, is a primary insect pest of cultivated rice, and effective control is essential for economical crop production. Resistance to neonicotinoid insecticides, in particular imidacloprid, has been reported as an increasing constraint in recent years. In order to investigate the extent of resistance, 24 samples of N. lugens were collected from China, India, Indonesia, Malaysia, Thailand and Vietnam during 2005 and 2006. Their responses to two diagnostic doses of imidacloprid (corresponding approximately to the LC(95) and 5 x LC(95) of a susceptible strain) were examined., Results: Ten of the 12 samples collected during 2005 were found to be susceptible to imidacloprid, but two late-season samples from India showed reduced mortality at both diagnostic doses. All 13 strains collected in 2006 showed reduced mortality at both doses when compared with the susceptible strain. Dose-response lines showed resistance in one of the most resistant field strains to be approximately 100-fold compared with the susceptible standard., Conclusion: The data demonstrate the development and spread of neonicotinoid resistance in N. lugens in Asia and support reports of reduced field efficacy of imidacloprid., (Copyright (c) 2008 Society of Chemical Industry.)

Neonicotinoid insecticides are potent selective agonists of insect nicotinic acetylcholine receptors (nAChRs). Since their introduction in 1991, resistance to neonicotinoids has been slow to develop, but it is now established in some insect field populations such as the planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia. We have reported recently the identification of a target-site mutation (Y151S) within two nAChR subunits (Nlalpha1 and Nlalpha3) from a laboratory-selected field population of N. lugens. In the present study, we have examined the influence of this mutation upon the functional properties of recombinant nAChRs expressed in Xenopus oocytes (as hybrid nAChRs, co-expressed with a rat beta2 subunit). The agonist potency of several nicotinic agonists has been examined, including all of the neonicotinoid insecticides that are currently licensed for either crop protection or animal health applications (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam). The Y151S mutation was found to have no significant effect on the maximal current (I(max)) observed with the endogenous agonist, acetylcholine. In contrast, a significant reduction in I(max) was observed for all neonicotinoids (the I(max) for mutant nAChRs ranged from 13 to 81% of that observed on wild-type receptors). In addition, nAChRs containing the Y151S mutation caused a significant rightward shift in agonist dose-response curves for all neonicotinoids, but of varying magnitude (shifts in EC(50) values ranged from 1.3 to 3.6-fold). The relationship between neonicotinoid structure and their potency on nAChRs containing the Y151S target-site mutation is discussed.