Interaction of Whitefly Effector G4 with Tomato Proteins Impacts Whitefly Performance.

The phloem-feeding insect Bemisia tabaci is an important pest, responsible for the transmission of several crop-threatening virus species. While feeding, the insect secretes a cocktail of effectors to modulate plant defense responses. Here, we present a set of proteins identified in an artificial diet on which B. tabaci was salivating. We subsequently studied whether these candidate effectors can play a role in plant immune suppression. Effector G4 was the most robust suppressor of an induced- reactive oxygen species (ROS) response in Nicotiana benthamiana. In addition, G4 was able to suppress ROS production in Solanum lycopersicum (tomato) and Capsicum annuum (pepper). G4 localized predominantly in the endoplasmic reticulum in N. benthamiana leaves and colocalized with two identified target proteins in tomato: REF-like stress related protein 1 (RSP1) and meloidogyne-induced giant cell protein DB141 (MIPDB141). Silencing of MIPDB141 in tomato reduced whitefly fecundity up to 40%, demonstrating that the protein is involved in susceptibility to B. tabaci. Together, our data demonstrate that effector G4 impairs tomato immunity to whiteflies by interfering with ROS production and via an interaction with tomato susceptibility protein MIPDB141. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Analysis of insecticide resistance and de novo transcriptome assembly of resistance associated genes in the European grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae).

The European grapevine moth Lobesia botrana (Denis & Shiffermüller 1776) is an economically important pest of the vine-growing areas worldwide. Chemical insecticides have been used for its control; however, its resistance status is largely unknown in many regions. We monitored the susceptibility of several L. botrana populations from Greece and Turkey. In addition, based on RNAseq transcriptome analysis, we identified and phylogenetically classify the cytochrome P450 genes of L. botrana, as well as analysed target site sequences and looked for the presence of known resistance mutations. Resistance against chlorantraniliprole, alpha-cypermethrin, spinetoram, etofenprox, and acetamiprid was very low (below 2.5-fold in all cases, compared to a reference strain from Greece) in all populations from Greece that were included in the study. However, resistance against indoxacarb (4-30-fold), spinosad (5-59-fold), and deltamethrin (18-30 fold) was detected in the L. botrana populations from Turkey, compared to a reference population from Turkey. De novo transcriptome assembly and manual annotation, and subsequent PCR-based analysis of insecticide target sequences (i.e. voltage-gated sodium channel - VGSC: target of pyrethroids and oxadiazines; nicotinic acetylcholine receptor subunit a6 - nAChR_α6: target of spinosad; ryanodine receptor - RyR: target of diamides; glutamate-gated chloride channel - GluCl: target of avermectins and; acetylcholinesterase - AChE: target of organophosphates) showed the absence of known resistance mutations in all specimens from both countries. Finally, the L. botrana CYPome (116 genes) was manually analysed and phylogenetically characterised, to provide resources for future studies that will aim the analysis of metabolic resistance.

A spatiotemporal atlas of the lepidopteran pest Helicoverpa armigera midgut provides insights into nutrient processing and pH regulation.

BACKGROUND: Caterpillars from the insect order Lepidoptera are some of the most widespread and destructive agricultural pests. Most of their impact is at the larval stage, where the midgut epithelium mediates the digestion and absorption of an astonishing amount of food. Although this tissue has been the subject of frequent investigation in Lepidoptera, a comprehensive expression atlas has yet to be generated. RESULTS: Here, we perform RNA-sequencing and proteomics on the gut of the polyphagous pest Helicoverpa armigera across, life stages, diet types, and compartments of the anterior-posterior axis. A striking relationship between the structural homology and expression pattern of a group of sugar transporters was observed in the early larval stages. Further comparisons were made among the spatial compartments of the midgut, which suggested a putative role for vATPases and SLC9 transporters in the generation of alkaline conditions in the H. armigera midgut. CONCLUSIONS: This comprehensive resource will aid the scientific community in understanding lepidopteran gut physiology in unprecedented resolution. It is hoped that this study advances the understanding of the lepidopteran midgut and also facilitates functional work in this field.

Fungicide resistance frequencies of Botrytis cinerea greenhouse isolates and molecular detection of a novel SDHI resistance mutation.

Sensitivity of B. cinerea to commonly used fungicides against Gray mold with emphasis to the newer quinone outside inhibitor (QoIs), and succinate dehydrogenase inhibitors (SDHIs) was assessed during a monitoring survey from vegetable greenhouses in four representative regions of Crete. 42% from a total of 168 isolates were simultaneously resistant to boscalid, fluopyram, pyraclostrobin and fenhexamid but not to fludioxonil making this phenylpyrrole fungicide an excellent anti-resistance antifungal agent. Isolates with double resistance to SDHIs and QoIs were found in very high frequencies indicating a selection towards double resistance due to the use of pyraclostrobin-boscalid mixtures. A number of sdhB resistance mutations (H272R, N230I and P225F/H) were found in isolates also carrying the G143A cytb resistance mutation in the above isolates. A novel sdhB point mutation (I274V) was identified for the first time in B. cinerea isolates collected from greenhouses with a fluopyram spray history with specific resistance to SDHIs. A PCR-RFLP diagnostic assay was developed for the detection of this mutation in the sdhB gene. Mutations P225F/H and I274V were found to be associated with fitness penalties in terms of mycelial growth, sporulation or pathogenicity. Results suggest that, in order to retain effective control of gray mold in Crete, appropriate anti-resistance strategies should be implemented taking into account the high double SDHI and QoI resistance frequencies. Additional studies for monitoring the already known and the new SDHI-resistance mutations, are necessary in order to hinder the further spread and establishment of single or double resistant isolates of B. cinerea detected in greenhouses in Crete.

A transcriptomic and proteomic atlas of expression in the Nezara viridula (Heteroptera: Pentatomidae) midgut suggests the compartmentalization of xenobiotic metabolism and nutrient digestion.

BACKGROUND: Stink bugs are an emerging threat to crop security in many parts of the globe, but there are few genetic resources available to study their physiology at a molecular level. This is especially true for tissues such as the midgut, which forms the barrier between ingested material and the inside of the body. RESULTS: Here, we focus on the midgut of the southern green stink bug Nezara viridula and use both transcriptomic and proteomic approaches to create an atlas of expression along the four compartments of the anterior-posterior axis. Estimates of the transcriptome completeness were high, which led us to compare our predicted gene set to other related stink bugs and Hemiptera, finding a high number of species-specific genes in N. viridula. To understand midgut function, gene ontology and gene family enrichment analyses were performed for the most highly expressed and specific genes in each midgut compartment. These data suggested a role for the anterior midgut (regions M1-M3) in digestion and xenobiotic metabolism, while the most posterior compartment (M4) was enriched in transmembrane proteins. A more detailed characterization of these findings was undertaken by identifying individual members of the cytochrome P450 superfamily and nutrient transporters thought to absorb amino acids or sugars. CONCLUSIONS: These findings represent an initial step to understand the compartmentalization and physiology of the N. viridula midgut at a genetic level. Future studies will be able to build on this work and explore the molecular physiology of the stink bug midgut.

Molecular diagnostics for detecting pyrethroid and abamectin resistance mutations in Tetranychus urticae.

Avermectin and pyrethroid resistance mutations (the G314D and the G326E in the glutamate gated chloride channels, and the F1538I in the voltage gated sodium channel) have been reported in the spider mite Tetranychus urticae, one of the most devastating pests of protected and open field crops worldwide. We developed three TaqMan molecular diagnostic assays for monitoring the presence and frequency of these mutations in T. urticae field populations. The TaqMan assays were validated against known genotypes and subsequently used to monitor the frequency of the resistance mutations in eleven T. urticae populations from Greece and Cyprus, with variable history of avermectin and pyrethroids applications. The frequency of the F1538I pyrethroid resistance mutation largely varied among samples, with highest frequencies (75%-97%) detected in four populations derived from protected and open field crops from Crete and Peloponnesus, low frequencies in three populations (2.5%-11%) from Attiki, Cyprus and Crete and not detected in four populations from Crete, Peloponnesus and Cyprus. The frequency of the abamectin resistance mutations G314D and G326E also varied across populations (from 0 to 100%), showing fixation in two populations (>97.5% for the G314D and 100% for the G326E), originating from rose greenhouses from Greece, low frequencies in three populations (5%-12.5%) also originating from rose greenhouses (Crete, Peloponnesus and Cyprus) and not detected in six populations from protected and open field vegetable crops. The TaqMan diagnostics showed higher resolution in detecting specific alleles in low frequency, compared to massive quantitative sequencing approaches previously employed. They can be used, together with classical bioassays, to support evidence - based insecticide resistance management strategies.

Transcription analysis of neonicotinoid resistance in Mediterranean (MED) populations of B. tabaci reveal novel cytochrome P450s, but no nAChR mutations associated with the phenotype.

BACKGROUND: Bemisia tabaci is one of the most damaging agricultural pests world-wide. Although its control is based on insecticides, B. tabaci has developed resistance against almost all classes of insecticides, including neonicotinoids. RESULTS: We employed an RNA-seq approach to generate genome wide expression data and identify genes associated with neonicotinoid resistance in Mediterranean (MED) B. tabaci (Q1 biotype). Twelve libraries from insecticide resistant and susceptible whitefly populations were sequenced on an Illumina Next-generation sequencing platform, and genomic sequence information of approximately 73 Gbp was generated. A reference transcriptome was built by de novo assembly and functionally annotated. A total of 146 P450s, 18 GSTs and 23 CCEs enzymes (unigenes) potentially involved in the detoxification of xenobiotics were identified, along with 78 contigs encoding putative target proteins of six different insecticide classes. Ten unigenes encoding nicotinic Acetylcholine Receptors (nAChR), the target of neoinicotinoids, were identified and phylogenetically classified. No nAChR polymorphism potentially related with the resistant phenotypes, was observed among the studied strains. DE analysis revealed that among the 550 differentially (logFC > 1) over-transcribed unigenes, 52 detoxification enzymes were over expressed including unigenes with orthologues in P450s, GSTs, CCE and UDP-glucuronosyltransferases. Eight P450 unigenes belonging to clades CYP2, CYP3 and CYP4 were highly up-regulated (logFC > 2) including CYP6CM1, a gene already known to confer imidacloprid resistance in B. tabaci. Using quantitative qPCRs, a larger screening of field MED B. tabaci from Crete with known neonicotinoid phenotype was performed to associate expression levels of P450s with resistance levels. Expression levels of five P450s, including CYP6CM1, were found associated with neonicotinoid resistance. However, a significant correlation was found only in CYP303 and CYP6CX3, with imidacloprid and acetamiprid respectively. CONCLUSION: Our work has generated new toxicological data and genomic resources which will significantly enrich the available dataset and substantially facilitate the molecular studies in MED B. tabaci. No evidence of target site neonicotinoid resistance has been found. Eight P450 unigenes, including CYP6CM1, were found significantly over-expressed in resistant B. tabaci. This study suggests at least two novel P450s (CYP303 and CYP6CX3) as candidates for their functional characterization as detoxification mechanisms of neonicotinoid resistance in B. tabaci.

Molecular diagnostics for monitoring insecticide resistance in the western flower thrips Frankliniella occidentalis.

BACKGROUND: Insecticide resistance has emerged in various western flower thrips (WFT) populations across the world, threatening the efficiency of chemical control applications. Elucidation of insecticide resistance mechanisms at the molecular level provides markers for the development of diagnostics to monitor the trait and support evidence-based resistance management. RESULTS: TaqMan and Droplet Digital polymerase chain reaction (ddPCR) diagnostics were developed and validated, against Sanger sequencing, in individual and pooled WFT samples respectively, for the G275E mutation (nicotinic acetylcholine receptor α6 gene, nAChR α6) associated with resistance to nAChR allosteric modulators, site I (spinosyns); L1014F, T929I, T929C and T292V mutations (voltage-gated sodium channel gene, vgsc) linked with pyrethroid resistance; and I1017M (chitin synthase 1 gene, chs1) conferring resistance to growth inhibitors affecting CHS1 (benzoylureas). The detection limits of ddPCR assays for mutant allelic frequencies (MAF) were in the range of 0.1%-0.2%. The assays were applied in nine WFT field populations from Crete, Greece. The G275E (MAF = 29.66%-100.0%), T929I and T929V (combined MAF = 100%), L1014F (MAF = 11.01%-37.29%), and I1017M (MAF = 17.74%-51.07%) mutations were present in all populations. CONCLUSION: The molecular diagnostics panel that was developed in this study can facilitate the quick and sensitive resistance monitoring of WFT populations at the molecular level, to support evidence-based insecticide resistance management strategies. © 2022 Society of Chemical Industry.

Multiple TaqMan qPCR and droplet digital PCR (ddPCR) diagnostics for pesticide resistance monitoring and management, in the major agricultural pest Tetranychus urticae.

BACKGROUND: Decisions on which pesticide to use in agriculture are expected to become more difficult, as the number of available chemicals is decreasing. For Tetranychus urticae (T. urticae), a major pest for which a number of candidate markers for pesticide resistance are in place, molecular diagnostics could support decision-making for the rational use of acaricides. RESULTS: A suite of 12 TaqMan qPCR assays [G314D (GluCl1), G326E, I321T (GluCl3), G119S, F331W (Ace-1), H92R (PSST), L1024V, F1538I (VGSC), I1017F (CHS1), G126S, S141F, P262T (cytb)], were validated against Sanger-sequencing, and subsequently adapted for use with the ddPCR technology. The concordance correlation coefficient between the actual and ddPCR measured mutant allelic frequencies was 0.995 (95% CI = 0.991-0.998), and no systematic, proportional, or random differences were detected. The achieved Limit of Detection (LoD) was 0.1% (detection of one mutant in a background of 999 wild type mites). The ddPCR assay panel was then assessed in terms of agreement with phenotypic resistance, through a pilot application in field populations from Crete, with strong correlation and thus predictive and diagnostic value of the molecular assays in some cases (e.g., etoxazole and abamectin resistance). Molecular diagnostics were able to capture incipient resistance that was otherwise missed by phenotypic bioassays. The molecular and phenotypic resistance screening of T. urticae field populations from Crete, revealed both multi-resistant and susceptible populations. CONCLUSION: The highly sensitive T. urticae molecular diagnostic platforms developed in this study could prove a valuable tool for pesticide resistance management. © 2021 Society of Chemical Industry.

Next-generation molecular diagnostics (TaqMan qPCR and ddPCR) for monitoring insecticide resistance in Bemisia tabaci.

BACKGROUND: Insecticide resistance has developed in several populations of the whitefly Bemisia tabaci worldwide and threatens to compromise the efficacy of chemical control. The molecular mechanisms underpinning resistance have been characterized and markers associated with the trait have been identified, allowing the development of diagnostics for individual insects. RESULTS: TaqMan and Droplet Digital PCR (ddPCR) assays were developed and validated, in individual and pooled whitefly samples, respectively, for the following target-site mutations: the acetylcholinesterase (ace1) F331W mutation conferring organophosphate-resistance; the voltage-gated sodium channel (vgsc) mutations L925I and T929V conferring pyrethroid-resistance; and the acetyl-CoA carboxylase (acc) A2083V mutation conferring ketoenol-resistance. The ddPCR's limit of detection (LoD) was <0.2% (i.e. detection of one heterozygote whitefly in a pool of 249 wild-type individuals). The assays were applied in 11 B. tabaci field populations from four locations in Crete, Greece. The F331W mutation was detected to be fixed or close to fixation in eight of 11 B. tabaci populations, and at lower frequency in the remaining ones. The pyrethroid-resistance mutations were detected at very high frequencies. The A2083V spiromesifen resistance mutation was detected in eight of 11 populations (frequencies = 6.16-89.56%). Spiromesifen phenotypic resistance monitoring showed that the populations tested had variable levels of resistance, ranging from full susceptibility to high resistance. A strong spiromesifen-resistance phenotype-genotype (A2083V) correlation (rs  = -0.839, P = 0.002) was observed confirming the ddPCR diagnostic value. CONCLUSION: The ddPCR diagnostics developed in this study are a valuable tool to support evidence-based rational use of insecticides and resistance management strategies. © 2022 Society of Chemical Industry.

Flupyradifurone resistance in Myzus persicae populations from peach and tobacco in Greece.

BACKGROUND: Myzus persicae has evolved resistance to various insecticides in Greece. Here we examine the effectiveness of the insecticide flupyradifurone against aphid clones collected from tobacco and peach in Greece during 2017-2020. Furthermore, we monitored the frequency of the neonicotinoid resistance mutation R81T in the sampled clones, and the association between the responses to flupyradifurone and acetamiprid. RESULTS: Of 43 clones tested with flupyradifurone, 6.977%, 60.465% and 32.558% showed low (10-14), moderate (19-89) and high (104-1914) resistance factor (RF) values, respectively. Resistance was higher in clones from peach than from tobacco with 42.308% and 17.647% of clones (respectively) failing into the high RF category (median RF values 67.5 and 36.4 for clones from peach and tobacco, respectively). Acetamiprid resistance was detected in clones collected in 2019-2020, in line with our previous study in Greece. The analysis of the whole dataset (54 clones collected during 2017-2020) revealed that all tobacco clones had RF < 7.5, whereas 55.263%, 18.421% and 26.316% of the peach clones exhibited low (<12), moderate (20-48) and high (100-145) RF values, respectively. A significant but moderate association between flupyradifurone and acetamiprid responses was detected (r = 0.513, P < 0.001). The R81T mutation was detected in aphids from peach (5.6% and 32.6% as homozygotes and heterozygotes, respectively) and in one aphid specimen (heterozygote) from tobacco. R81T was partially associated with the resistance to both insecticides, but many highly resistant clones did not possess the mutation, indicating the possible operation of one or more alternative underlying resistance mechanisms. CONCLUSIONS: The use of flupyradifurone and acetamiprid in IPM/IRM should be based on further ongoing susceptibility monitoring. © 2021 Society of Chemical Industry.

Identification and characterization of striking multiple-insecticide resistance in a Tetranychus urticae field population from Greece.

BACKGROUND: Tetranychus urticae is a notorious crop pest with a worldwide distribution that has developed resistance to a wide range of acaricides. Here, we investigated the resistance levels of a T. urticae population collected from an ornamental greenhouse in Peloponnese, Greece, and analyzed its resistance mechanisms at the molecular level. RESULTS: Toxicological assays showed resistance against compounds with different modes of action, with resistance ratios of: 89-fold for abamectin; > 1000-fold for clofentezine; > 5000-fold for etoxazole; 27-fold for fenpyroximate and pyridaben; 20- and 36-fold for spirodiclofen and spirotetramat, respectively; and 116- and > 500-fold for cyenopyrafen and cyflumetofen, respectively. Bioassays with synergists indicated the involvement of detoxification enzymes in resistance to abamectin, but not to cyflumetofen and spirodiclofen. RNA sequencing (RNA-seq) analysis showed significant over-expression of several genes encoding detoxification enzymes such as cytochrome P450 monooxygenases and UDP-glycosyltransferases, which have been previously associated with acaricide resistance. Known target-site resistance mutations were identified in acetyl-choline esterase, chitin synthase 1 and NDUFS7/psst, but putative novel resistance mutations were also discovered in targets such as glutamate-gated chloride channel subunit 3. Interestingly, target-site resistance mutations against pyrethroids or bifenazate were not identified, possibly indicating a recent reduced selection pressure in Greece, as well as a possible opportunity to rotate these chemistries. CONCLUSION: We identified and characterized a striking case of multiple acaricide resistance in a field population of T. urticae. Exceptionally strong resistance phenotypes, with accumulation of multiple resistance mutations and over-expression of P450s and other detoxification genes in the same field population are reported.

Co-Expression of a Homologous Cytochrome P450 Reductase Is Required for In Vivo Validation of the Tetranychus urticae CYP392A16-Based Abamectin Resistance in Drosophila.

Overexpression of the cytochrome P450 monooxygenase CYP392A16 has been previously associated with abamectin resistance using transcriptional analysis in the two-spotted spider mite Tetranychus urticae, an important pest species worldwide; however, this association has not been functionally validated in vivo despite the demonstrated ability of CYP392A16 to metabolize abamectin in vitro. We expressed CYP392A16 in vivo via a Gal4 transcription activator protein/Upstream Activating Sequence (GAL4/UAS) system in Drosophila melanogaster flies, driving expression with detoxification tissue-specific drivers. We demonstrated that CYP392A16 expression confers statistically significant abamectin resistance in toxicity bioassays in Drosophila only when its homologous redox partner, cytochrome P450 reductase (TuCPR), is co-expressed in transgenic flies. Our study shows that the Drosophila model can be further improved, to facilitate the functional analysis of insecticide resistance mechanisms acting alone or in combination.

Identification and geographical distribution of pyrethroid resistance mutations in the poultry red mite Dermanyssus gallinae.

BACKGROUND: The poultry red mite (PRM) Dermanyssus gallinae is the most common ectoparasite on poultry and causes high economic losses in poultry farming worldwide. Pyrethroid acaricides have been widely used for its control and, consequently, pyrethroid resistance has arisen. In this study we aim to investigate the occurrence of resistance and study the geographical distribution of pyrethroid resistance mutations across PRM populations in Europe. RESULTS: Full dose-response contact bioassays revealed very high levels of resistance against several pyrethroids (α-cypermethrin, fluvalinate, and cyfluthrin) in two PRM populations from Greece, compared to a susceptible reference strain. Resistance was associated with mutations in the gene encoding the target site of pyrethroids, the voltage-gated sodium channel (VGSC). Mutations, M918L and L925V in domain IIS4-S5 and F1534L in domain IIIS6, were found at positions known to play a role in pyrethroid resistance in other arthropod species. Subsequent screening by sequencing VGSC gene fragments IIS4-S5 and IIIS6 revealed the presence and distribution of these mutations in many European populations. In some populations, we identified additional or different mutations including M918V/T, L925M, T929I, I936F, and F1538L. The latter mutation is a possible alternative for F1538I that has been previously associated with pyrethroid resistance in other Acari species. CONCLUSION: We report very high levels of pyrethroid resistance in PRM populations from Greece, as well as the identification and geographical distribution of 10 pyrethroid resistance mutations in PRM populations across Europe. Our results draw attention to the need for an evidence-based implementation of PRM control, taking acaricide resistance management into consideration. © 2019 Society of Chemical Industry.

The evolution of multiple-insecticide resistance in UK populations of tomato leafminer, Tuta absoluta.

BACKGROUND: The tomato leafminer, Tuta absoluta, is an economically important pest of tomatoes in Europe, Africa, Asia and South America. In the UK this species is controlled using an integrated pest management (IPM) programme which incorporates the insecticides spinosad and chlorantraniliprole. In response to UK grower concerns of loss of efficacy of these compounds at certain sites, insecticide bioassays were performed on five populations collected from four commercial glasshouses and potential mechanisms of resistance investigated. RESULTS: We observed high levels of resistance to spinosad in four of the strains, and in two of these tolerance to chlorantraniliprole. Selection of one of these strains with chlorantraniliprole rapidly resulted in a line exhibiting potent resistance to this compound. Sequencing of messenger RNA encoding the nicotinic acetylcholine receptor (nAChR) α6 subunit, target of spinosad, revealed Taα6 transcripts in the spinosad-resistant strains that lack exon 4 and encode a highly truncated protein, or contain a triplet deletion in the predicted first transmembrane domain resulting in the loss of a highly conserved amino acid. Sequencing of the ryanodine receptor gene, encoding the target of diamide insecticides, of the chlorantraniliprole-selected line revealed an amino acid substitution (G4903V) that has been previously linked to diamide resistance in populations of T. absoluta in the Mediterranean and South America. CONCLUSION: Taken together our results reveal emerging resistance in UK populations of T. absoluta to two of the most important insecticides used as part of IPM, with significant implications for the control of this species in the UK. © 2019 Society of Chemical Industry.

Insecticide resistance in Trialeurodes vaporariorum populations and novel diagnostics for kdr mutations.

BACKGROUND: Neonicotinoids, pyrethroids and ketoenols are currently used for the control of Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). In this study, insecticide resistance status and mechanisms were investigated using classical bioassays and molecular techniques. RESULTS: Dose-response bioassays were performed on 19 Greek populations, among the 35 different whitefly populations used for the whole analysis. Resistance factors scaled up to 207-, 4657- and 59-fold for imidacloprid, bifenthrin and spiromesifen, respectively. Molecular assays were used to investigate the frequency of known resistance mutations. A simple polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed for detecting the pyrethroid-resistant alleles r1 (mutation L925I) and r2 (mutation T929I) of the para-type voltage-gated sodium channel gene (VGSC). Both alleles were present at high frequencies (on average 65% and 33%, respectively) in 14 populations from Greece. The M918 L pyrethroid resistance mutation was not detected in any of the Greek populations. Sequencing and a Taqman allelic discrimination were used to monitor the frequency of the mutation E645K of the acetyl-coenzyme A carboxylase gene (ACC) recently linked to spiromesifen resistance. This mutation was detected in 20 of the 24 populations examined in ∼38% frequency among the 433 individuals tested. However, its association with the spiromesifen resistance phenotype was not confirmed in the Greek populations. Finally, two homologues of the CYP6CM1 Bemisia tabaci P450, the known neonicotinoid metabolizer, were found upregulated in two T. vaporariorum neonicotinoid-resistant populations; they were both functionally expressed in Escherichia coli, but the recombinant proteins encoded did not metabolize those neonicotinoid insecticides tested. CONCLUSION: The development of simple diagnostics and their use alongside classical and molecular techniques for the early detection of resistant populations are of great importance for pest management strategies. The practical implications of our results are discussed in light of whitefly control. © 2017 Society of Chemical Industry.

Investigation of the contribution of RyR target-site mutations in diamide resistance by CRISPR/Cas9 genome modification in Drosophila.

Diamide insecticides are used widely against lepidopteran pests, acting as potent activators of insect Ryanodine Receptors (RyRs) and thus inducing muscle contraction and eventually death. However, resistant phenotypes have recently evolved in the field, associated with the emergence of target site resistance mutations (G4946E/V and I4790M). We investigated the frequency of the mutations found in a resistant population of Tuta absoluta from Greece (G4946V ~79% and I4790M ~21%) and the associated diamide resistance profile: there are very high levels of resistance against chlorantraniliprole (9329-fold) and flubendiamide (4969-fold), but moderate levels against cyantraniliprole (191-fold). To further investigate functionally the contribution of each mutation in the resistant phenotype, we used CRISPR/Cas9 to generate genome modified Drosophila carrying alternative allele combinations, and performed toxicity bioassays against all three diamides. Genome modified flies bearing the G4946V mutation exhibited high resistance ratios to flubendiamide (91.3-fold) and chlorantraniliprole (194.7-fold) when compared to cyantraniliprole (5.4-fold). Flies naturally wildtype for the I4790M mutation were moderately resistant to flubendiamide (15.3-fold) but significantly less resistant to chlorantraniliprole (7.5-fold), and cyantraniliprole (2.3-fold). These findings provide in vivo functional genetic confirmation for the role and relative contribution of RyR mutations in diamide resistance and suggest that the mutations confer subtle differences on the relative binding affinities of the three diamides at an overlapping binding site on the RyR protein.

Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae).

Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance management strategies is strongly advised to delay the further spread of resistance.