Nontarget impacts of neonicotinoids on nectar-inhabiting microbes.

Plant-systemic neonicotinoid (NN) insecticides can exert non-target impacts on organisms like beneficial insects and soil microbes. NNs can affect plant microbiomes, but we know little about their effects on microbial communities that mediate plant-insect interactions, including nectar-inhabiting microbes (NIMs). Here we employed two approaches to assess the impacts of NN exposure on several NIM taxa. First, we assayed the in vitro effects of six NN compounds on NIM growth using plate assays. Second, we inoculated a standardised NIM community into the nectar of NN-treated canola (Brassica napus) and assessed microbial survival and growth after 24 h. With few exceptions, in vitro NN exposure tended to decrease bacterial growth metrics. However, the magnitude of the decrease and the NN concentrations at which effects were observed varied substantially across bacteria. Yeasts showed no consistent in vitro response to NNs. In nectar, we saw no effects of NN treatment on NIM community metrics. Rather, NIM abundance and diversity responded to inherent plant qualities like nectar volume. In conclusion, we found no evidence that NIMs respond to field-relevant NN levels in nectar within 24 h, but our study suggests that context, specifically assay methods, time and plant traits, is important in assaying the effects of NNs on microbial communities.

Anopheles gambiae larvae's ability to grow and emerge in water containing lethal concentrations of clothianidin, acetamiprid, or imidacloprid is consistent with cross-resistance to neonicotinoids.

BACKGROUND: For decades, various agrochemicals have been successfully repurposed for mosquito control. However, preexisting resistance caused in larval and adult populations by unintentional pesticide exposure or other cross-resistance mechanisms poses a challenge to the efficacy of this strategy. A better understanding of larval adaptation to the lethal and sublethal effects of residual pesticides in aquatic habitats would provide vital information for assessing the efficacy of repurposed agrochemicals against mosquitoes. METHODS: We reared field-collected mosquito larvae in water containing a concentration of agrochemical causing 100% mortality in susceptible mosquitoes after 24 h (lethal concentration). Using this experimental setup, we tested the effect of lethal concentrations of a pyrrole (chlorfenapyr, 0.10 mg/l), a pyrethroid (deltamethrin, 1.5 mg/l), and three neonicotinoids including imidacloprid (0.075 mg/l), acetamiprid (0.15 mg/l), and clothianidin (0.035 mg/l) on mortality rates, growth, and survival in third-instar larvae of the two sibling species Anopheles gambiae and Anopheles coluzzii collected from Yaoundé, Cameroon. RESULTS: We found that An. gambiae and An. coluzzii larvae were susceptible to chlorfenapyr and were killed within 24 h by a nominal concentration of 0.10 mg/l. Consistent with strong resistance, deltamethrin induced low mortality in both species. Lethal concentrations of acetamiprid, imidacloprid, and clothianidin strongly inhibited survival, growth, and emergence in An. coluzzii larvae. By contrast, depending on the active ingredient and the population tested, 5-60% of immature stages of An. gambiae were able to grow and emerge in water containing a lethal concentration of neonicotinoids, suggesting cross-resistance to this class of insecticides. CONCLUSIONS: These findings corroborate susceptibility profiles observed in adults and suggest that unintentional pesticide exposure or other cross-resistance processes could contribute to the development of resistance to neonicotinoids in some Anopheles populations.

The Amino Acid Transporter PtCAT7 and Ammonium Nutrition Enhance the Uptake of Thiamethoxam in Citrus Rootstock Seedlings.

Thiamethoxam (THX), when applied to the soil, can be taken up by citrus roots and subsequently transported to the leaves, providing effective protection of plants against the Asian citrus psyllid (Diaphorina citri Kuwayama). In this study, the field experiments showed that the coapplication of THX and nitrogen fertilizer (AN) did not affect THX uptake in six-year-old citrus plants. However, their coapplication promoted THX uptake in three-year-old Potassium trifoliate rootstocks and relieved the inhibition of AN at a higher level on plant growth characteristics, including biomass and growth of root and stem. RNA-seq analysis found that THX induced upregulation of a cationic amino acid transporter (PtCAT7) in citrus leaves. PtCAT7 facilitated THX uptake in the yeast strain to inhibit its growth, and the PtCAT7 protein was localized on the plasma membrane. Our results demonstrate that THX and N fertilizer can be coapplied and PtCAT7 may be involved in THX uptake in citrus.

Adult mosquitoes of the sibling species Anopheles gambiae and Anopheles coluzzii exhibit contrasting patterns of susceptibility to four neonicotinoid insecticides along an urban-to-rural gradient in Yaoundé, Cameroon.

BACKGROUND: Neonicotinoids are potential alternatives for controlling pyrethroid-resistant mosquitoes, but their efficacy against malaria vector populations of sub-Saharan Africa has yet to be investigated. The aim of the present study was to test the efficacy of four neonicotinoids against adult populations of the sibling species Anopheles gambiae and Anopheles coluzzii sampled along an urban-to-rural gradient. METHODS: The lethal toxicity of three active ingredients for adults of two susceptible Anopheles strains was assessed using concentration-response assays, and their discriminating concentrations were calculated. The discriminating concentrations were then used to test the susceptibility of An. gambiae and An. coluzzii mosquitoes collected from urban, suburban and rural areas of Yaoundé, Cameroon, to acetamiprid, imidacloprid, clothianidin and thiamethoxam. RESULTS: Lethal concentrations of neonicotinoids were relatively high suggesting that this class of insecticides has low toxicity against Anopheles mosquitoes. Reduced susceptibility to the four neonicotinoids tested was detected in An. gambiae populations collected from rural and suburban areas. By contrast, adults of An. coluzzii that occurred in urbanized settings were susceptible to neonicotinoids except acetamiprid for which 80% mortality was obtained within 72 h of insecticide exposure. The cytochrome inhibitor, piperonyl butoxide (PBO), significantly enhanced the activity of clothianidin and acetamiprid against An. gambiae mosquitoes. CONCLUSIONS: These findings corroborate susceptibility profiles observed in larvae and highlight a significant variation in tolerance to neonicotinoids between An. gambiae and An. coluzzii populations from Yaoundé. Further studies are needed to disentangle the role of exposure to agricultural pesticides and of cross-resistance mechanisms in the development of neonicotinoid resistance in some Anopheles species.

Mutation V65I in the ß1 Subunit of the Nicotinic Acetylcholine Receptor Confers Neonicotinoid and Sulfoxaflor Resistance in Insects.

Neonicotinoids have been widely used to control pests with remarkable effectiveness. Excessive insecticides have led to serious insect resistance. Mutations of the nicotinic acetylcholine receptor (nAChR) are one of the reasons for neonicotinoid resistance conferred in various agricultural pests. Two mutations, V65I and V104I, were found in the nAChR ß1 subunit of two neonicotinoid-resistant aphid populations. However, the specific functions of the two mutations remain unclear. In this study, we cloned and identified four nAChR subunits (α1, α2, α8, and ß1) of thrips and found them to be highly homologous to the nAChR subunits of other insects. Subsequently, we successfully expressed two subtypes nAChR (α1/α2/α8/ß1 and α1/α8/ß1) by coinjecting three cofactors for the first time in thrips, and α1/α8/ß1 showed abundant current rapidly. Acetylcholine, neonicotinoids, and sulfoxaflor exhibited different activation capacities for the two subtypes of nAChRs. Finally, V65I was found to significantly reduce the binding ability of nAChR to neonicotinoids and sulfoxaflor through electrophysiology and computer simulations. V104I caused a decrease in agonist affinity (pEC50) but an increase in the efficacy (Imax) of nAChR against neonicotinoids and reduced the binding ability of nAChR to sulfoxaflor. This study provides theoretical and technical support for studying the molecular mechanisms of neonicotinoid resistance in pests.

Synthesis and Insecticidal Evaluation of 3,5-Dicyanopyridines Against Cotton Aphids via Microwave-Assisted Multicomponent Reactions.

Certain 2-amino-6-alkoxy-4-arylpyridine-3,5-dicyanide 1a-e were prepared via a straightforward process using microwave technology rather than conventional methods. This involved reaction of arylidenemalononitrile thru propanedinitrile in the occurrence of sodium alkoxide under MW. While, their positional isomer 4-amino-6-alkoxy-2-arylpyridine-3,5-dicyanide 3a-j have been separated from the reaction of aryl aldehydes with 2-aminoprop-1-ene-1,1,3-tricarbonitrile 2 in the presence of sodium alkoxide using microwave technic. Furthermore, the insecticidal properties of all synthesized compounds were observed with respect to Cotton aphid nymphs and adults. Neonicotinoid pesticides are indicated as the most effective pesticides toward aphids and many other pests. Many insecticides are discovered as novelties. As a result, several pyridine compounds were chemical method synthesized to serve as equivalents of neonicotinoids, a broad class of insecticides. With LC50 value of 0.03 mg/L, components 3g exhibit the highest insecticidal bioactivity. This work discusses how to find new chemicals that could be used as insecticidal agents in the future.

Dual mutations in the whitefly nicotinic acetylcholine receptor ß1 subunit confer target-site resistance to multiple neonicotinoid insecticides.

Neonicotinoid insecticides, which target insect nicotinic acetylcholine receptors (nAChRs), have been widely and intensively used to control the whitefly, Bemisia tabaci, a highly damaging, globally distributed, crop pest. This has inevitably led to the emergence of populations with resistance to neonicotinoids. However, to date, there have been no reports of target-site resistance involving mutation of B. tabaci nAChR genes. Here we characterize the nAChR subunit gene family of B. tabaci and identify dual mutations (A58T&R79E) in one of these genes (BTß1) that confer resistance to multiple neonicotinoids. Transgenic D. melanogaster, where the native nAChR Dß1 was replaced with BTß1A58T&R79E, were significantly more resistant to neonicotinoids than flies where Dß1 were replaced with the wildtype BTß1 sequence, demonstrating the causal role of the mutations in resistance. The two mutations identified in this study replace two amino acids that are highly conserved in >200 insect species. Three-dimensional modelling suggests a molecular mechanism for this resistance, whereby A58T forms a hydrogen bond with the R79E side chain, which positions its negatively-charged carboxylate group to electrostatically repulse a neonicotinoid at the orthosteric site. Together these findings describe the first case of target-site resistance to neonicotinoids in B. tabaci and provide insight into the molecular determinants of neonicotinoid binding and selectivity.

The role of the Bemisia tabaci and Trialeurodes vaporariorum cytochrome-P450 clade CYP6DPx in resistance to nicotine and neonicotinoids.

The alkaloid, nicotine, produced by tobacco and other Solanaceae as an anti-herbivore defence chemical is one of the most toxic natural insecticides in nature. However, some insects, such as the whitefly species, Trialeurodes vaporariorum and Bemisia tabaci show strong tolerance to this allelochemical and can utilise tobacco as a host. Here, we used biological, molecular and functional approaches to investigate the role of cytochrome P450 enzymes in nicotine tolerance in T. vaporariorum and B. tabaci. Insecticide bioassays revealed that feeding on tobacco resulted in strong induced tolerance to nicotine in both species. Transcriptome profiling of both species reared on tobacco and bean hosts revealed profound differences in the transcriptional response these host plants. Interrogation of the expression of P450 genes in the host-adapted lines revealed that P450 genes belonging to the CYP6DP subfamily are strongly upregulated in lines reared on tobacco. Functional characterisation of these P450s revealed that CYP6DP1 and CYP6DP2 of T. vaporariorum and CYP6DP3 of B. tabaci confer resistance to nicotine in vivo. These three genes, in addition to the B. tabaci P450 CYP6DP5, were also found to confer resistance to the neonicotinoid imidacloprid. Our data provide new insight into the molecular basis of nicotine resistance in insects and illustrates how divergence in the evolution of P450 genes in this subfamily in whiteflies may have impacted the extent to which different species can tolerate a potent natural insecticide.

Clothianidin-resistant Anopheles gambiae adult mosquitoes from Yaoundé, Cameroon, display reduced susceptibility to SumiShield® 50WG, a neonicotinoid formulation for indoor residual spraying.

BACKGROUND: Chronic exposure of mosquito larvae to pesticide residues and cross-resistance mechanisms are major drivers of tolerance to insecticides used for vector control. This presents a concern for the efficacy of clothianidin, an agricultural neonicotinoid prequalified for Indoor Residual Spraying (IRS). METHODS: Using standard bioassays, we tested if reduced susceptibility to clothianidin can affect the efficacy of SumiShield® 50WG, one of four new IRS formulations containing clothianidin. We simultaneously monitored susceptibility to clothianidin and to SumiShield 50WG, testing adults of Anopheles gambiae, An. coluzzii and Culex sp sampled from urban, suburban and agricultural areas of Yaoundé, Cameroon. RESULTS: We found that in this geographic area, the level of susceptibility to the active ingredient predicted the efficacy of SumiShield 50WG. This formulation was very potent against populations that reached 100% mortality within 72 h of exposure to a discriminating concentration of clothianidin. By contrast, mortality leveled off at 75.4 ± 3.5% within 7 days of exposure to SumiShield 50WG in An. gambiae adults collected from a farm where the spraying of the two neonicotinoids acetamiprid and imidacloprid for crop protection is likely driving resistance to clothianidin. CONCLUSIONS: Despite the relatively small geographic extend of the study, the findings suggest that cross-resistance may impact the efficacy of some new IRS formulations and that alternative compounds could be prioritized in areas where neonicotinoid resistance is emerging.

CYP4CS5-mediated thiamethoxam and clothianidin resistance is accompanied by fitness cost in the whitefly Bemisia tabaci.

BACKGROUND: Understanding the trade-offs between insecticide resistance and the associated fitness is of particular importance to sustainable pest control. One of the most devastating pest worldwide, the whitefly Bemisia tabaci, has developed resistance to various insecticides, especially the neonicotinoid group. Although neonicotinoid resistance often is conferred by P450s-mediated metabolic resistance, the relationship between such resistance and the associated fitness phenotype remains largely elusive. By gene cloning, quantitative reverse transcription (qRT)-PCR, RNA interference (RNAi), transgenic Drosophila melanogaster, metabolism capacity in vitro and 'two sex-age stage' life table study, this study aims to explore the molecular role of a P450 gene CYP4CS5 in neonicotinoid resistance and to investigate whether such resistance mechanism carries fitness costs in the whitefly. RESULTS: Our bioassay tests showed that a total of 13 field-collected populations of B. tabaci MED biotype displayed low-to-moderate resistance to thiamethoxam and clothianidin. Compared to the laboratory susceptible strain, we then found that an important P450 CYP4CS5 was remarkably upregulated in the field resistant populations. Such overexpression of CYP4CS5 had a good match with the resistance level among the whitefly samples. Further exposure to the two neonicotinoids resulted in an increase in CYP4CS5 expression. These results implicate that overexpression of CYP4CS5 is closely correlated with thiamethoxam and clothianidin resistance. RNAi knockdown of CYP4CS5 increased mortality of the resistant and susceptible populations after treatment with thiamethoxam and clothianidin in bioassay, but obtained an opposite result when using a transgenic line of D. melanogaster expressing CYP4CS5. Metabolic assays in vitro revealed that CYP4CS5 exhibited certain capacity of metabolizing thiamethoxam and clothianidin. These in vivo and in vitro assays indicate an essential role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance in whitefly. Additionally, our life-table analysis demonstrate that the field resistant whitefly exhibited a prolonged development time, shortened longevity and reduced fecundity compared to the susceptible, suggesting an existing fitness cost as a result of the resistance. CONCLUSION: Collectively, in addition to the important role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance, this resistance mechanism is associated with fitness costs in the whitefly. These findings not only contribute to the development of neonicotinoids resistance management strategies, but also provide a new target for sustainable whitefly control. © 2023 Society of Chemical Industry.

Use of double-stranded RNA targeting ß2 divergent nicotinic acetylcholine receptor subunit to control pea aphid Acyrthosiphon pisum at larval and adult stages.

BACKGROUND: In recent years, the use of the RNA interference technology (RNAi) has emerged as one of the new strategies for species-specific control of insect pests. Its specificity depends on the distinctiveness of the target gene sequence for a given species. In this work, we assessed in the pea aphid Acyrthosiphon pisum (A. pisum) the use of a double-stranded RNA (dsRNA) that targets the ß2 divergent nicotinic acetylcholine receptor (nAChR) subunit (dsRNA-ß2), which shares low sequence identity with other subunits, to control populations of this pest at different developmental stages. Because nAChRs are targeted by neonicotinoid insecticides such as imidacloprid, we also assessed the effect of dsRNA-ß2 coupled to this insecticide on aphid survival. Finally, because the effect of a control agent on beneficial insect must be considered before any use of new pest management strategies, the acute toxicity of dsRNA-ß2 combined with imidacloprid was evaluated on honeybee Apis mellifera. RESULTS: In this work, we demonstrated that dsRNA-ß2 alone has an insecticidal effect on aphid larvae and adults. Moreover, dsRNA-ß2 and imidacloprid effects on aphid larvae and adults were additive, meaning that dsRNA-ß2 did not alter the efficacy of imidacloprid on these two developmental stages. Also, no obvious acute toxicity on Apis mellifera was reported. CONCLUSION: Using RNAi that targets ß2 divergent nAChR subunit is effective alone or combined with imidacloprid to control A. pisum at larval and adult stages. Because no obvious Apis mellifera mortality has been reported, this RNAi-based pest management strategy should be considered to control insect pest. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Design, Synthesis, and Synergistic Activities of Eight-Membered Carbon Bridged Neonicotinoid Derivatives.

Insecticide synergists are an effective approach to increase the control efficacy and reduce active ingredient usage. In order to explore neonicotinoid-specific synergists with novel scaffolds and higher potency, a series of eight-membered carbon bridged neonicotinoid derivatives were designed and synthesized in accordance with our previous research. The synergistic effects of the target compounds on neonicotinoids in Aphis craccivora were evaluated, and the structure-activity relationships were summarized. The results indicated that most of the target compounds exhibited significant synergistic effects on imidacloprid in A. craccivora at low concentrations. In particular, compound 1 at a concentration of 1 mg/L reduced the LC50 value of imidacloprid from 0.856 mg/L to 0.170 mg/L. Meanwhile, compound 1 also increased the insecticidal activity of most neonicotinoid insecticides belonging to the Insecticide Resistance Action Committee (IRAC) 4 A subgroup against A. craccivora. The present study might be meaningful for directing the design of neonicotinoid-specific synergists.

Molecular Evolutionary Mechanisms of CYP6ER1vA-Type Variant Associated with Resistance to Neonicotinoid Insecticides in Field Populations of Nilaparvata lugens.

The evolution of insecticide resistance has threatened the control of Nilaparvata lugens. Research on mechanisms behind neonicotinoid resistance in N. lugens remains incomplete. This study examined P450-mediated resistance to neonicotinoids in a resistant N. lugens strain (XA-2017-3G). The overexpression of CYP6ER1 in the XA-2017-3G strain plays a role in neonicotinoid resistance, as confirmed by RNA interference. Phenotypic analyses of CYP6ER1-mediated resistance in strains, including laboratory-susceptible, field-collected, and imidacloprid-laboratory further-selected strains, revealed that the vA-type/vL-type genotype exhibited greater resistance to neonicotinoids compared to the vA-type/vA-type genotype. The mRNA expression levels of CYP6ER1vA-type were closely correlated with the levels of neonicotinoid resistance in N. lugens strains, in which CYP6ER1vA-type overexpression is in part attributed to increased copy numbers of CYP6ER1. CYP6ER1vA-type-mediated neonicotinoid resistance was further confirmed by a CYP6ER1vA-type transgenic Drosophila melanogaster line. Taken together, our findings strongly suggest that the overexpression of CYP6ER1vA-type, which can be partially attributed to copy number variations, plays a crucial role in N. lugens resistance to neonicotinoids.

Vegetable oil-based surfactants are adjuvants that enhance the efficacy of neonicotinoid insecticides and can bias susceptibility testing in adult mosquitoes.

BACKGROUND: The standard operating procedure for testing the susceptibility of adult mosquitoes to neonicotinoid or butenolide insecticides recommends using a vegetable oil ester (Mero) as a surfactant. However, there is growing evidence that this adjuvant contains surfactants that can enhance insecticide activity, mask resistance and bias the bioassay. METHODOLOGY/PRINCIPAL FINDINGS: Using standard bioassays, we tested the effects of commercial formulations of vegetable oil-based surfactants similar to Mero on the activity of a spectrum of active ingredients including four neonicotinoids (acetamiprid, clothianidin, imidacloprid and thiamethoxam) and two pyrethroids (permethrin and deltamethrin). We found that three different brands of linseed oil soap used as cleaning products drastically enhanced neonicotinoid activity in Anopheles mosquitoes. At 1% (v/v), the surfactant reduced the median lethal concentration, LC50, of clothianidin more than 10-fold both in susceptible and in resistant populations of Anopheles gambiae. At 1% or 0.5% (v/v), linseed oil soap restored the susceptibility of adult mosquitoes fully to clothianidin, thiamethoxam and imidacloprid and partially to acetamiprid. By contrast, adding soap to the active ingredient did not significantly affect the level of resistance to permethrin or deltamethrin suggesting that vegetable oil-based surfactants specifically enhance the potency of some classes of insecticides. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that surfactants are not inert ingredients, and their use in susceptibility testing may jeopardize the ability to detect resistance. Further research is needed to evaluate the potential, the limitations and the challenges of using some surfactants as adjuvants to enhance the potency of some chemicals applied in mosquito control.

Over-expression of UDP-glycosyltransferase UGT353G2 confers resistance to neonicotinoids in whitefly (Bemisia tabaci).

The whitefly, Bemisia tabaci, comes up high metabolic resistance to most neonicotinoids in long-term evolution, which is the key problem of pest control. UGT glycosyltransferase, as a secondary detoxification enzyme, plays an indispensable role in detoxification metabolism. In this study, UGT inhibitors, 5-nitrouracil and sulfinpyrazone, dramatically augmented the toxic damage of neonicotinoids to B. tabaci. A UGT named UGT353G2 was identified in whitefly, which was notably up-regulated in resistant strain (3.92 folds), and could be induced by most neonicotinoids. Additionally, the using of RNA interference (RNAi) suppresses UGT353G2 substantially increased sensitivity to neonicotinoids in resistant strain. Our results support that UGT353G2 may be involved in the neonicotinoids resistance of whitefly. These findings will help further verify the functional role of UGTs in neonicotinoid resistance.

Metal-Organic Framework-Based Insecticide and dsRNA Codelivery System for Insecticide Resistance Management.

Targeted silencing of resistance-associated genes by specific double-stranded RNA (dsRNA) is an attractive strategy for overcoming insecticide resistance in insect pests. However, silencing target genes of insect pests by feeding on dsRNA transported via plants remains challenging. Herein, a codelivery system of insecticide and dsRNA is designed by encapsulating imidacloprid and dsNlCYP6ER1 within zeolitic imidazolate framework-8 (ZIF-8) nanoparticles to improve the susceptibility of Nilaparvata lugens (Stål) to imidacloprid. With an average particle size of 195 nm and a positive surface charge, the derived imidacloprid/dsNlCYP6ER1@ZIF-8 demonstrates good monodispersity. Survival curve results showed that the survival rates of N. lugens treated with imidacloprid and imidacloprid@ZIF-8 were 82 and 62%, respectively, whereas, in the imidacloprid/dsNlCYP6ER1@ZIF-8 treatment group, the survival rate of N. lugens is only 8%. Pot experiments demonstrate that the survival rate in the imidacloprid/dsNlCYP6ER1@ZIF-8 treatment group was much lower than that in the imidacloprid treatment group, decreasing from 54 to 24%. The identification of NlCYP6ER1 expression and the fluorescence tracking of ZIF-8 demonstrate that ZIF-8 can codeliver dsRNA and insecticide to insects via rice. Safety evaluation results showed that the dsNlCYP6ER1@ZIF-8 nanoparticle had desirable biocompatibility and biosafety to silkworm. This dsRNA and insecticide codelivery system may be extended to additional insecticides with potential resistance problems in the future, greatly enhancing the development of pest resistance management.

The interaction of nicotinic acetylcholine receptor subunits Ldα3, Ldα8 and Ldß1 with neonicotinoids in Colorado potato beetle, Leptinotarsa decemlineata.

The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is an extremely destructive notifiable quarantine pest. Over the last two decades, neonicotinoid insecticides, particularly thiamethoxam and imidacloprid, have been used to control it in Xinjiang, and local field populations have developed different levels of resistance in consequence. However, the contributions of nicotinic acetylcholine receptors (nAChRs) to neonicotinoid resistance are currently poorly understood in CPB. Previous studies have shown that nAChRα1, α3, α8 and ß1 are major target subunits for neonicotinoids in some model and important agricultural insects including nAChRα1 subunit of L. decemlineata (Ldα1). In this study, the expression levels of Ldα3, Ldα8 and Ldß1 following 72 h of treatments with median lethal doses of thiamethoxam and imidacloprid were compared using real-time quantitative PCR. These genes were then individually and simultaneously knocked down with Ldα1 by RNA interference (RNAi) using a double-stranded RNA (dsRNA) feeding method for six days to explore their roles in CPB susceptibility to imidacloprid and thiamethoxam. The results showed that the expressions of Ldα3, Ldα8 and Ldß1 were significantly decreased by 36.99-74.89% after thiamethoxam and imidacloprid treatments, compared with the control. The significant downregulation of the target genes resulting from RNAi significantly reduced the mortality of adults exposed to thiamethoxam and imidacloprid by 34.53% -56.44% and 28.78%-43.93%, respectively. Furthermore, the adult survival rates were not affected by every dsRNA-feeding treatment, while the body weight of the test adults significantly deceased after four and six days of individual gene RNAi. This study showed that Ldα3, Ldα8 and Ldß1 are down-regulated by thiamethoxam and imidacloprid and play important roles in the tolerance of CPB to neonicotinoids.

Structural Insights into Neonicotinoids and N-Unsubstituted Metabolites on Human nAChRs by Molecular Docking, Dynamics Simulations, and Calcium Imaging.

Neonicotinoid pesticides were initially designed in order to achieve species selectivity on insect nicotinic acetylcholine receptors (nAChRs). However, concerns arose when agonistic effects were also detected in human cells expressing nAChRs. In the context of next-generation risk assessments (NGRAs), new approach methods (NAMs) should replace animal testing where appropriate. Herein, we present a combination of in silico and in vitro methodologies that are used to investigate the potentially toxic effects of neonicotinoids and nicotinoid metabolites on human neurons. First, an ensemble docking study was conducted on the nAChR isoforms α7 and α3ß4 to assess potential crucial molecular initiating event (MIE) interactions. Representative docking poses were further refined using molecular dynamics (MD) simulations and binding energy calculations using implicit solvent models. Finally, calcium imaging on LUHMES neurons confirmed a key event (KE) downstream of the MIE. This method was also used to confirm the predicted agonistic effect of the metabolite descyano-thiacloprid (DCNT).

Transcriptomic investigation of the molecular mechanisms underlying resistance to the neonicotinoid thiamethoxam and the pyrethroid lambda-cyhalothrin in Euschistus heros (Hemiptera: Pentatomidae).

BACKGROUND: Laboratory-selected resistant strains of Euschistus heros to thiamethoxam (NEO) and lambda-cyhalothrin (PYR) were recently reported in Brazil. However, the mechanisms conferring resistance to these insecticides in E. heros remain unresolved. We utilized comparative transcriptome profiling and single nucleotide polymorphism (SNP) calling of susceptible and resistant strains of E. heros to investigate the molecular mechanism(s) underlying resistance. RESULTS: The E. heros transcriptome was assembled, generating 91 673 transcripts with a mean length of 720 bp and N50 of 1795 bp. Comparative gene expression analysis between the susceptible (SUS) and NEO strains identified 215 significantly differentially expressed (DE) transcripts. DE transcripts associated with the xenobiotic metabolism were all up-regulated in the NEO strain. The comparative analysis of the SUS and PYR strains identified 204 DE transcripts, including an esterase (esterase FE4), a glutathione-S-transferase, an ABC transporter (ABCC1) and aquaporins that were up-regulated in the PYR strain. We identified 9588 and 15 043 nonsynonymous SNPs in the PYR and NEO strains. One of the SNPs (D70N) detected in the NEO strain occurs in a subunit (α5) of the nAChRs, the target site of neonicotinoid insecticides. Nevertheless, this residue position in α5 is not conserved among insects. CONCLUSIONS: Neonicotinoid and pyrethroid resistance in laboratory-selected E. heros is associated with a potential metabolic resistance mechanism by the overexpression of proteins commonly involved in the three phases of xenobiotic metabolism. Together these findings provide insight into the potential basis of resistance in E. heros and will inform the development and implementation of resistance management strategies against this important pest. © 2023 Society of Chemical Industry.

Cytochrome P450 CYP6DB3 was involved in thiamethoxam and imidacloprid resistance in Bemisia tabaci Q (Hemiptera: Aleyrodidae).

High level resistance for a variety of insecticides has emerged in Bemisia tabaci, a globally notorious insect. Neonicotinoid insecticides have been applied widely to control B. tabaci. Whether a differentially expressed gene CYP6DB3 discovered from transcriptome data of B. tabaci is involved in the resistance to neonicotinoid insecticides remains unclear. In the study, CYP6DB3 expression was significantly up-regulated in both thiamethoxam- and imidacloprid-resistant strains relative to the susceptive strains. We also found that CYP6DB3 expression was up-regulated after B. tabaci adults were exposed to thiamethoxam and imidacloprid. Moreover, knocking down CYP6DB3 expression via feeding corresponding dsRNA significantly reduced CYP6DB3 mRNA levels by 34.1%. Silencing CYP6DB3 expression increased the sensitivity of B. tabaci Q adults against both thiamethoxam and imidacloprid. Overexpression of CYP6DB3 gene reduced the toxicity of imidacloprid and thiamethoxam to transgenic D. melanogaster. In addition, metabolic studies showed that CYP6DB3 can metabolize 24.41% imidacloprid in vitro. Collectively, these results strongly support that CYP6DB3 plays an important role in the resistance of B. tabaci Q to imidacloprid and thiamethoxam. This work will facilitate a deeper insight into the part of cytochrome P450s in the evolution of insecticide resistance and provide a theoretical basis for the development of new integrated pest resistance management.