Insecticide Target Research Articles

Characterization of the ryanodine receptor gene in Encarsia formosa (Gahan) and its expression profile in response to diamide insecticides

Ryanodine receptors (RyRs) are the targets of diamide insecticides, which have been identified and characterized in a dozen insect pests of Lepidoptera, Hemiptera, Diptera and Coleoptera, but limited attention has been paid to the RyR in parasitoid natural enemies. Without this knowledge, it will hinder our effective and efficient application using both parasitoid natural enemies and diamide insecticides simultaneously in the integrated pest management (IPM). In this study, the full-length cDNA of RyR was cloned from Encarsia formosa (EfRyR), a parasitic wasp used worldwide for the biological control of whitefly. Its expression profile was examined in various tissues of E. formosa adults. The toxicities of four diamide insecticides to E. formosa were measured, and then the expression profile of EfRyR after 12 h and 24 h exposure to the LC50 dosages of diamide insecticides was investigated. The results showed that the full-length cDNA of EfRyR was 16, 778 bp including a 15, 345 bp open reading frame, and two alternative splice (AS) sites. Comparing to its expression in the abdomen, EfRyR was highly expressed in the head (11.9-fold) and the thorax (3.7-fold). The toxicities of four dimide insecticides against E. formosa from low to high were chlorantraniliprole (LC50 = 367.84 mg L−1), cyantraniliprole (221.72 mg L−1), cyclaniliprole (51.77 mg L−1), and tetrachlorantraniliprole (8.35 mg L−1). The expressions of EfRyR and its variants with AS were significantly increased after E. formosa adults were exposed to different diamide insecticides. This study improves our understanding of the RyR in parasitoid wasps and provides useful information on IPM by using E. formosa.

Loss of the Dβ1 nicotinic acetylcholine receptor subunit disrupts bursicon-driven wing expansion and diminishes adult viability in Drosophila melanogaster.

Cholinergic signaling dominates the insect central nervous system, contributing to numerous fundamental pathways and behavioral circuits. However, we are only just beginning to uncover the diverse roles different cholinergic receptors may play. Historically, insect nicotinic acetylcholine receptors have received attention due to several subunits being key insecticide targets. More recently, there has been a focus on teasing apart the roles of these receptors, and their constituent subunits, in native signaling pathways. In this study, we use CRISPR-Cas9 genome editing to generate germline and somatic deletions of the Dβ1 nicotinic acetylcholine receptor subunit and investigate the consequences of loss of function in Drosophila melanogaster. Severe impacts on movement, male courtship, longevity, and wing expansion were found. Loss of Dβ1 was also associated with a reduction in transcript levels for the wing expansion hormone bursicon. Neuron-specific somatic deletion of Dβ1 in bursicon-producing neurons (CCAP-GAL4) was sufficient to disrupt wing expansion. Furthermore, CCAP-GAL4-specific expression of Dβ1 in a germline deletion background was sufficient to rescue the wing phenotype, pinpointing CCAP neurons as the neuronal subset requiring Dβ1 for the wing expansion pathway. Dβ1 is a known target of multiple commercially important insecticides, and the fitness costs exposed here explain why field-isolated target-site resistance has only been reported for amino acid replacements and not loss of function. This work reveals the importance of Dβ1-containing nicotinic acetylcholine receptors in CCAP neurons for robust bursicon-driven wing expansion.

Insecticide resistance and target site mutations (G119S ace-1 and L1014F kdr) in Anopheles gambiae s.l. in Enugu state, Southeastern Nigeria

A study of insecticide resistant status and detection of G119S and L1014F mutation genes in Anopheles gambiae sensu lato. in Enugu State was conducted between May, 2019 and June, 2020. The aim was to investigate the resistance status of An. gambiae s.l. to four classes of insecticides used in vector control in the area and to identify the target site resistant genes present in resistant mosquitoes. Three randomly selected communities; Awkunanaw (Enugu East), Obukpa (Enugu North) and Awgu (Enugu West senatorial districts) were used as sampling sites. Anopheles gambiae s.l. larvae were collected by dip method and reared to adult for the study. WHO Tube Bioassay was used to determine resistance status. Polymerase Chain Reaction (PCR) was employed for detection of resistance mechanisms. The average mortality of An. gambiae s.l. tested with DDT was 28.3%, deltamethrin 63.53%, bendiocarb 88.33%, while malathion had the highest average mortality rate of 100%. The Acetycholinesterase Resistance Gene (GII9S Ace-1) mutation was detected at low frequencies in 75(100%) randomly selected An. gambiae s.l. from the three senatorial districts while the Knockdown Resistance Gene (LI0I4F kdr) mutation was not detected. Implementation of insecticide resistance management strategies in the state to control the spread of resistance is recommended.

Permethrin resistance in Aedes aegypti: Genomic variants that confer knockdown resistance, recovery, and death.

Pyrethroids are one of the few classes of insecticides available to control Aedes aegypti, the major vector of dengue, chikungunya, and Zika viruses. Unfortunately, evolving mechanisms of pyrethroid resistance in mosquito populations threaten our ability to control disease outbreaks. Two common pyrethroid resistance mechanisms occur in Ae. aegypti: 1) knockdown resistance, which involves amino acid substitutions at the pyrethroid target site—the voltage-gated sodium channel (VGSC)—and 2) enhanced metabolism by detoxification enzymes. When a heterogeneous population of mosquitoes is exposed to pyrethroids, different responses occur. During exposure, a proportion of mosquitoes exhibit immediate knockdown, whereas others are not knocked-down and are designated knockdown resistant (kdr). When these individuals are removed from the source of insecticide, the knocked-down mosquitoes can either remain in this status and lead to dead or recover within a few hours. The proportion of these phenotypic responses is dependent on the pyrethroid concentration and the genetic background of the population tested. In this study, we sequenced and performed pairwise genome comparisons between kdr, recovered, and dead phenotypes in a pyrethroid-resistant colony from Tapachula, Mexico. We identified single-nucleotide polymorphisms (SNPs) associated with each phenotype and identified genes that are likely associated with the mechanisms of pyrethroid resistance, including detoxification, the cuticle, and insecticide target sites. We identified high association between kdr and mutations at VGSC and moderate association with additional insecticide target site, detoxification, and cuticle protein coding genes. Recovery was associated with cuticle proteins, the voltage-dependent calcium channel, and a different group of detoxification genes. We provide a list of detoxification genes under directional selection in this field-resistant population. Their functional roles in pyrethroid metabolism and their potential uses as genomic markers of resistance require validation.

Structure-dependent receptor subtype selectivity and G protein subtype preference of heterocyclic agonists in heterologously expressed silkworm octopamine receptors

(R)-Octopamine (OA), a major invertebrate biogenic amine, plays an important role in a wide variety of physiological processes as a neurohormone, neuromodulator, and neurotransmitter in insects. OA receptors (OARs) are class A G protein-coupled receptors that specifically bind OA to activate downstream signaling cascades by coupling to G proteins and presumably other regulatory proteins. These receptors are broadly classified as α- and β-adrenergic-like OARs (α- and β-ALOARs). OARs are considered important targets of insecticides and acaricides. In the present study, we examined the actions of an array of 13 heterocyclic OAR agonists with the moieties that correspond to the phenyl group and the basic nitrogen atom of OA on α- and β-ALOARs from the silkworm (Bombyx mori) and the signaling pathways activated through these actions. The results indicated that these compounds display structure-dependent receptor subtype selectivity and G protein subtype preference, underscoring the need to determine which subtype and signaling pathway mediates toxicologically relevant effects for the efficient discovery of novel pest control chemicals. The results of insecticidal assays using B. mori larvae suggested that the activation of signal transduction pathways via α-ALOARs might be mainly responsible for the toxicological effects of the heterocycles.

Identification of whitefly (Bemisia tabaci) proteins interacting with Tomato leaf curl Bangalore virus coat protein gene using Y2H system

Tomato leaf curl Bangalore virus (ToLCBV) (Geminiviridae) causes the economically important tomato leaf curl virus disease (ToLCVD), and is transmitted by the whitefly, Bemisia tabaci (Gennadius) (Aleyrodidae: Hemiptera). Successful transmission of the virus by the insect requires safe translocation of the virus through different barriers inside the vector. During the translocation, the viral coat protein will interact with many whitefly proteins, while a few of them are beneficial to virus (e.g. heat shock proteins) but some may be harmful (e.g. whitefly immune proteins). We carried out the yeast two hybrid (Y2H) assays to identify the proteins of the B.tabaci Asia 1 genetic group interacting with ToLCBV coat protein (CP). The Y2H assay initially identified a total of 425 putative interacting whitefly proteins on a low stringent selection media, and they were reduced to 324 when the yeast colonies were grown on a high stringent media, and of which about 274 colonies produced single bands in colony PCR experiments while the remaining colonies produced multiple bands. Further, high selection pressure assays confirmed a total of 102 whitefly proteins interacting with ToLCBV CP and these included the heat shock proteins (HSPs) 70 kDa, GroEL, nucleoproteins, vitellogenins, apolipophorins, 40 s ribosomal proteins, sorbitol dehydrogenase, dipeptidyl peptidase, E3 ubiquitin, annexin, GTP cyclohydrolase, tropomyosin, salivary secreted proteins, succinate dehydrogenase, lachesins, enolase and others. The identified proteins could be potential targets for novel whitefly control strategies such as using RNAi or insecticide target sites for developing future disease and whitefly control strategies.

Ion channels and G protein-coupled receptors as targets for invertebrate pest control: from past challenges to practical insecticides.

In the late 1970s, we discovered that toxic bicyclic phosphates inhibit the generation of miniature inhibitory junction potentials, implying their antagonism of γ-aminobutyric acid (GABA) receptors (GABARs; GABA-gated chloride channels). This unique mode of action provided a strong incentive for our research on GABARs in later years. Furthermore, minor structural changes conferred insect GABAR selectivity to this class of compounds, convincing us of the possibility of GABARs as targets for insecticides. Forty years later, third-generation insecticides acting as allosteric modulator antagonists at a distinctive site of action in insect GABARs were developed. G protein-coupled receptors (GPCRs) are also promising targets for pest control. We characterized phenolamine receptors functionally and pharmacologically. Of the tested receptors, β-adrenergic-like octopamine receptors were revealed to be the most sensitive to the acaricide/insecticide amitraz. Given the presence of multiple sites of action, ion channels and GPCRs remain potential targets for invertebrate pest control.

Trehalase inhibition by validamycin A may be a promising target to design new fungicides and insecticides.

The introduction of insecticides and fungicides in agriculture has improved crop yields and, consequently, the quality of life for many people, especially in what is widely considered as the 'first world'. However, the indiscriminate use of dangerous chemical insecticides has led to pest resistance, human and animal poisoning and environmental pollution. Biochemical and genetic evidence concludes that the non-reducing disaccharide trehalose plays an essential role in the pathobiology of many insects and fungi. Both organisms share identical pathway for trehalose biosynthesis (the TPS/TPP pathway), while a high degree of homology in their trehalose hydrolysis capacity (trehalase activities) has also been demonstrated. In the search for new, effective and environmentally sustainable compounds, a set of trehalase inhibitors has emerged as a potentially interesting antifungal and insecticidal target. In particular, the trehalose analogue, Validamycin A, which has a strong inhibitory effect on several trehalases, has been successfully introduced for the treatment of various diseases caused by insects and fungi. Herein, we review the main features of the specific interaction between Validamycin A and trehalase as well as the expected advantages of the applications based on trehalase inhibition as insecticides and fungicides. © 2021 Society of Chemical Industry.

A Tale of Two Transcriptomic Responses in Agricultural Pests via Host Defenses and Viral Replication.

Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) is a baculovirus that causes systemic infections in many arthropod pests. The specific molecular processes underlying the biocidal activity of AcMNPV on its insect hosts are largely unknown. We describe the transcriptional responses in two major pests, Spodoptera frugiperda (fall armyworm) and Trichoplusia ni (cabbage looper), to determine the host–pathogen responses during systemic infection, concurrently with the viral response to the host. We assembled species-specific transcriptomes of the hemolymph to identify host transcriptional responses during systemic infection and assessed the viral transcript abundance in infected hemolymph from both species. We found transcriptional suppression of chitin metabolism and tracheal development in infected hosts. Synergistic transcriptional support was observed to suggest suppression of immune responses and induction of oxidative stress indicating disease progression in the host. The entire AcMNPV core genome was expressed in the infected host hemolymph with a proportional high abundance detected for viral transcripts associated with replication, structure, and movement. Interestingly, several of the host genes that were targeted by AcMNPV as revealed by our study are also targets of chemical insecticides currently used commercially to control arthropod pests. Our results reveal an extensive overlap between biological processes represented by transcriptional responses in both hosts, as well as convergence on highly abundant viral genes expressed in the two hosts, providing an overview of the host–pathogen transcriptomic landscape during systemic infection.

Characterization and functional analysis of two acetylcholinesterase genes in Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae)

Two acetylcholinesterase genes (Boace1 and Boace2) were cloned from Bradysia odoriphaga, a devastating soil pest that mainly damages Chinese chives. The Boace1 encodes BoAChE1 protein consisting of 696 amino acid residues, while Boace2 encodes BoAChE2 containing 638 amino acids. Phylogenetic analysis showed that Boace1 and Boace2 are appeared to be distinct clusters. The gene expression patterns at different development stages and various body parts tissues were examined, and their biological functions were characterized by RNA interference and analog docking prediction. The results showed that both Boace genes were expressed in all developmental stages and examined tissues. The transcript level of Boace2 was significantly higher than Boace1 in all tested samples, and Boace1 was found most abundant in the head while Boace2 was highly expressed in the fat body of B. odoriphaga. The silencing of Boace1 and Boace2 significantly decreased the AChE activity of 36.6% and 14.8% separately, and increased the susceptibility of B. odoriphaga to phoxim, with 60.8% and 44.7% mortality. Besides, overexpression and gene duplication of Boace1 were found in two field resistant populations, and two major mutations, A319S and G400V, were detected in Boace1. Moreover, the docking results revealed that BoAChE1 had a higher affinity towards organophosphorus than BoAChE2. It is concluded that Boace2 is the most abundant ace type in B. odoriphaga, while both Boace play vital roles. Boace1 might play a major neurological function and more likely be the prime target for insecticides, while Boace2 might play some important unidentified roles.

Molecular Characterization of the β2-like Octopamine Receptor of Helicoverpa armigera.

Helicoverpa armigera is a devastating polyphagous and cosmopolitan crop pest. There are reports of this insect being resistant to a variety of pesticides raising concern worldwide. The Octopamine (OA) binding β2-like receptor (OAR), a GPCR, is widely distributed in the nervous system of the insect and plays essential roles in the physiology and development and thus is an important target for insecticides. Yet, the molecular characterization of the H. armigera OAR (HarmOAR) and rational design of compounds based on this receptor is lacking. As a first step, we performed multiple sequence alignment of all insect OARs, which revealed that the sequences contained all conserved class A GPCR motifs. Phylogenetic studies showed clade-specific variations in the protein sequences primarily arising owing to differences in the ICL3 loop region. Further, a structural model of HarmOAR was built using the inactive human β2AR as a template. 0.9µs atomistic simulations revealed conserved inter helical contacts and water molecules of HarmOAR. The detailed binding of octopamine was studied using molecular docking and 0.3µs atomistic simulations. Twenty-two insecticides active against octopamine receptors of other insects were compiled and docked to HarmOAR followed by rescoring with binding free energies to prioritize them for H. armigera. Our study suggests α-terpineol to be a good candidate as an insecticide or insect repellent for Helicoverpa armigera.

Inhibiting the proteasome reduces molecular and biological impacts of the natural product insecticide, spinosad.

Insecticide targets are often identified by mutations that confer resistance, but the intricacies of insecticide binding and downstream processes leading to insect death often remain obscure. Mutations in α6-like nicotinic acetylcholine receptor subunit genes have been associated with high levels of resistance to spinosad in many insect species, including Drosophila melanogaster. Here, we aimed to expand our understanding of the effects of the natural product insecticide spinosad on its protein target, the α6 subunit, using genetic tools available in D. melanogaster. Functional, fluorescently tagged Dα6 subunits (Dα6YFP ) were developed to allow observation of the protein in vivo. Larvae expressing Dα6YFP were exposed to a sub-lethal concentration of spinosyn A (0.025 ppm) for 6 days, leading to a 64% reduction in fluorescence relative to unexposed larvae. Direct application of high doses of spinosyn A to dissected larval brains resulted in a visible 38.25% decrease in Dα6YFP within 20 min, indicating that degradation of the Dα6 protein occurred in response to spinosyn A exposure. Chemical inhibition of the proteasome system using the multiple myeloma treatment drug, PS-341 reduced loss of Dα6YFP in response to spinosyn A at the 20-min time point to 6.35%. In addition, in vivo administration of PS-341 prior to spinosad exposure reduced the effect of spinosad on larval activity. Based on these data, we propose that exposure to spinosad leads to degradation of the α6-like target protein, a potentially novel element in the mode of action of spinosyns that may contribute to their toxicity towards insects. © 2021 Society of Chemical Industry.

Role of nicotinic acetylcholine receptor subunits in the mode of action of neonicotinoid, sulfoximine and spinosyn insecticides in Drosophila melanogaster

Insecticides remain valuable tools for the control of insect pests that significantly impact human health and agriculture. A deeper understanding of insecticide targets is important in maintaining this control over pests. Our study systematically investigates the nicotinic acetylcholine receptor (nAChR) gene family, in order to identify the receptor subunits critical to the insect response to insecticides from three distinct chemical classes (neonicotinoids, spinosyns and sulfoximines). Applying the CRISPR/Cas9 gene editing technology in D. melanogaster, we were able to generate and maintain homozygous mutants for eight nAChR subunit genes. A ninth gene (Dβ1) was investigated using somatic CRISPR in neural cells to overcome the low viability of the homozygous germline knockout mutant. These findings highlight the specificity of the spinosyn class insecticide, spinosad, to receptors containing the Dα6 subunit. By way of contrast, neonicotinoids are likely to target multiple receptor subtypes, beyond those receptor subunit combinations previously identified. Significant differences in the impacts of specific nAChR subunit deletions on the resistance level of flies to neonicotinoids imidacloprid and nitenpyram indicate that the receptor subtypes they target do not completely overlap. While an R81T mutation in β1 subunits has revealed residues co-ordinating binding of sulfoximines and neonicotinoids differ, the resistance profiles of a deletion of Dβ1 examined here provide new insights into the mode of action of sulfoxaflor (sulfoximine) and identify Dβ1 as a key component of nAChRs targeted by both these insecticide classes. A comparison of resistance phenotypes found in this study to resistance reported in insect pests reveals a strong conservation of subunit targets across many different insect species and that mutations have been identified in most of the receptor subunits that our findings would predict to have the potential to confer resistance.

Crystal Structures of Insect Ryanodine Receptor Domains

Diamide insecticides are among the top-selling pesticides, generating worldwide sales over 2 billion U.S. dollars annually. They target insect ryanodine receptors (RyRs) and cause misregulation of calcium signaling in insect muscles and neurons. However, due to heavy usage, several resistance mutations identified in insect RyRs have been reported to reduce the efficacy of the diamides, but the exact binding site of diamides and the mechanism of resistance mutations remain elusive. Two options are available to overcome the resistance: modify the diamide insecticide to target the same binding pocket in resistant RyRs or develop new compounds to target a different binding site in insect RyRs. The recent breakthrough in the structural studies of mammalian RyRs has deepened our understanding of the channel, but the structural information about insect RyRs is still scarce. Here we report four crystal structures of insect RyR domains, including N-terminal domain (NTD), SPRY2 domain, and repeat34 domain from diamondback moth (DBM), and NTD from the honeybee. Several regions in these domains showed distinguished conformations in DBM relative to honeybee and mammalian RyRs. Many of these pest-specific structural features are located in the domain-domain interfaces that would change conformation upon channel gating, making them good target for candidate insecticides. We identified several protein kinase a (PKA) phosphorylation sites clustering in a loop and a newly identified α-helix in DBM repeat34 domain, showing a pest-specific phosphorylation pattern. Interestingly, the phosphorylation of insect RyR is temperature-dependent, facilitated by the low thermal stability and dynamic structure of insect Repeat34 domain. The accurate structural information of insect RyRs would provide valuable templates for the design of insecticidal molecules.

Flonicamid and knockdown of inward rectifier potassium channel gene CsKir2B adversely affect the feeding and development of Chilo suppressalis.

The selective insecticide flonicamid shows highly insecticidal activities against piercing-sucking insects and has been widely used for the control of Hemipteran insect pests, whereas its effects on Lepidopteran insect pests remain largely unknown. Recently, inward rectifier potassium (Kir) channel has been verified to be a target of flonicamid, however, functional characterization of Lepidopteran Kir genes is still lacking. Flonicamid shows no insecticidal toxicity against Chilo suppressalis larvae. However, the feeding and growth of larvae were reversibly inhibited by flonicamid (50-1200 mg L-1 ). Flonicamid treatment also remarkably reduced and delayed the pupation and eclosion of Chilo suppressalis. Additionally, five distinct Kir channel genes (CsKir1, CsKir2A, CsKir2B, CsKir3A and CsKir3B) were cloned from Chilo suppressalis. Expression profiles analysis revealed that CsKir2A was predominately expressed in the hindgut of larvae, whereas CsKir2B had high expressions in the Malpighian tubules and hindgut. RNA interference (RNAi)-mediated knockdown of CsKir2B significantly reduced the growth and increased the mortalities of larvae, whereas silencing of CsKir2A had no obvious effects on Chilo suppressalis. Flonicamid exhibits adverse effects on the growth and development of Chilo suppressalis. CsKir2B might be involved in the feeding behavior of Chilo suppressalis. These results provide valuable information on the effects of flonicamid on non-target insects as well as the function of insect Kir channels, and are helpful in developing new insecticide targeting insect Kir channels. © 2020 Society of Chemical Industry.

Modulation of membrane physical properties by natural insecticidal ketones

The insecticidal activity of Mentha oil and its main components has been tested and established for various insects/pests. Several mint ketones have demonstrated to act on GABAA receptors (GABAA-R), a transmembrane channel target of several important insecticides whose activity can be modulated by surface-active compounds and by changes in the physical properties of the lipid membrane. In the present work, we analyze the capacity of monoterpenic ketones most commonly found in Mentha species, pulegone and menthone, to interact with DPPC membranes by molecular dynamics (MD) simulations and Langmuir monolayers. The experimental results indicate that the presence of menthone and pulegone in the subphase modify the interfacial characteristics of DPPC isotherms. The changes were reflected as expansion of the isotherms and disappearance or bringing forward of DPPC phase transition. MD simulation corroborate these results and indicate that both ketones are located at the region of the carbonyl group, at the interface with the acyl chains. Ketone intercalation between lipid molecules would induce an increasing intermolecular interaction, diminishing the film elasticity and causing an ordering effect. Our results suggest that the insecticidal activity of both ketones could involve their interaction with lipid molecules causing disturbance of the cell membrane as postulated for several larvicide compounds, or at least modulating the receptor surrounding.

Identification of the Aedes aegypti nAChR gene family and molecular target of spinosad.

Spinosad is an insecticide with unique mode of action (MOA) of disrupting nicotinic acetylcholine receptor and is efficacious against many insect species. Mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit have been identified that are associated with levels of spinosad resistance, but the molecular characterization of the nAChR gene family and a causative association between nAChR α6 and resistance to spinosad in Aedes aegypti, a primary vector of many arboviruses, have not yet been reported. In this study, we identified 10 candidate nAChR subunits in Ae. Aegypti, nAChRα1-α9 and nAChRβ1, showing similarly orthologous relationships with Anopheles gambiae. With the application of the CRISPR/Cas9 genome editing system, we introduced a 32-bp deletion at the 5' end of the Aaeα6 (Ae. aegypti nAChR α6) gene in a homozygous mutant strain (Aaeα6-KO). The mutation produced two successive pre-mature stop codons, resulting in loss of function in the target receptor. The Aaeα6-KO mutant strain exhibited a 320-fold level of resistance to spinosad compared with wildtype. A recessive mode of inheritance for spinosad resistance was found in the Aaeα6-KO strain. CRISPR/Cas9 introduced truncated Aaeα6 receptor in Ae. aegypti resulted in an increased level of resistance to spinosad, suggesting that the conserved nAChR α6 subunit is the target for spinosad insecticide. © 2020 Society of Chemical Industry.

FastD: Fast detection of insecticide target-site mutations and overexpressed detoxification genes in insect populations from RNA-Seq data.

Target‐site mutations and detoxification gene overexpression are two major mechanisms conferring insecticide resistance. Molecular assays applied to detect these resistance genetic markers are time‐consuming and with high false‐positive rates. RNA‐Seq data contains information on the variations within expressed genomic regions and expression of detoxification genes. However, there is no corresponding method to detect resistance markers at present. Here, we collected 66 reported resistance mutations of four insecticide targets (AChE, VGSC, RyR, and nAChR) from 82 insect species. Next, we obtained 403 sequences of the four target genes and 12,665 sequences of three kinds of detoxification genes including P450s, GSTs, and CCEs. Then, we developed a Perl program, FastD, to detect target‐site mutations and overexpressed detoxification genes from RNA‐Seq data and constructed a web server for FastD (http://www.insect-genome.com/fastd). The estimation of FastD on simulated RNA‐Seq data showed high sensitivity and specificity. We applied FastD to detect resistant markers in 15 populations of six insects, Plutella xylostella, Aphis gossypii, Anopheles arabiensis, Musca domestica, Leptinotarsa decemlineata and Apis mellifera. Results showed that 11 RyR mutations in P. xylostella, one nAChR mutation in A. gossypii, one VGSC mutation in A. arabiensis and five VGSC mutations in M. domestica were found to be with frequency difference >40% between resistant and susceptible populations including previously confirmed mutations G4946E in RyR, R81T in nAChR and L1014F in VGSC. And 49 detoxification genes were found to be overexpressed in resistant populations compared with susceptible populations including previously confirmed detoxification genes CYP6BG1, CYP6CY22, CYP6CY13, CYP6P3, CYP6M2, CYP6P4 and CYP4G16. The candidate target‐site mutations and detoxification genes were worth further validation. Resistance estimates according to confirmed markers were consistent with population phenotypes, confirming the reliability of this program in predicting population resistance at omics‐level.

Piper capitarianum essential oil: a promising insecticidal agent for the management of Aedes aegypti and Aedes albopictus.

Mosquitoes are responsible for serious public health problems worldwide, and as such, Aedes aegypti and Aedes albopictus are important vectors in the transmission of dengue, chikungunya, and Zika in Brazil and other countries of the world. Due to growing resistance to chemical insecticides among populations of vectors, environmentally friendly strategies for vector management are receiving ever more attention. Essential oils (EOs) extracted from plants have activities against insects with multiple mechanisms of action. These mechanisms hinder the development of resistance, and have the advantages of being less toxicity and biodegradable. Thus, the present study aimed to evaluate the chemical composition of the EOs obtained from Piper capitarianum Yunck, as well as evaluating their insecticidal potential against Aedes aegypti and A. albopictus, and their toxicity in relation to Artemia salina. The yields of the EOs extracted from the leaves, stems, and inflorescences of P. capitarianum were 1.2%, 0.9%, and 0.6%, respectively, and their main constituents were trans-caryophyllene (20.0%), α-humulene (10.2%), β-myrcene (10.5%), α-selinene (7.2%), and linalool (6.0%). The EO from the inflorescences was the most active against A. aegypti and A. albopictus, and exhibited the respective larvicidal (LC50 = 87.6 μg/mL and 76.1 μg/mL) and adulticide activities (LC50 = 126.2 μg/mL and 124.5 μg/mL). This EO was also the most active in the inhibition of AChE, since it presented an IC50 value of 14.2 μg/mL. Its larvicidal effect was observed under optical and scanning electron microscopy. Additionally, non-toxic effects against A. salina were observed. Docking modeling of trans-caryophyllene and α-humulene on sterol carrier protein-2 (SCP-2) suggests that both molecules have affinity with the active site of the enzyme, which indicates a possible mechanism of action. Therefore, the essential oil of P. capitarianum may be used in the development of new insecticide targets for the control of A. aegypti and A. albopictus in the Amazonian environment.

Fitness effects for Ace insecticide resistance mutations are determined by ambient temperature

BackgroundInsect pest control programs often use periods of insecticide treatment with intermittent breaks, to prevent fixing of mutations conferring insecticide resistance. Such mutations are typically costly in an insecticide-free environment, and their frequency is determined by the balance between insecticide treatment and cost of resistance. Ace, a key gene in neuronal signaling, is a prominent target of many insecticides and across several species, three amino acid replacements (I161V, G265A, and F330Y) provide resistance against several insecticides. Because temperature disturbs neuronal signaling homeostasis, we reasoned that the cost of insecticide resistance could be modulated by ambient temperature.ResultsExperimental evolution of a natural Drosophila simulans population at hot and cold temperature regimes uncovered a surprisingly strong effect of ambient temperature. In the cold temperature regime, the resistance mutations were strongly counter selected (s = − 0.055), but in a hot environment, the fitness costs of resistance mutations were reduced by almost 50% (s = − 0.031). We attribute this unexpected observation to the advantage of the reduced enzymatic activity of resistance mutations in hot environments.ConclusionWe show that fitness costs of insecticide resistance genes are temperature-dependent and suggest that the duration of insecticide-free periods need to be adjusted for different climatic regions to reflect these costs. We suggest that such environment-dependent fitness effects may be more common than previously assumed and pose a major challenge for modeling climate change.