Molecular monitoring of insecticide resistance in major disease vectors in Armenia.

BACKGROUND: Armenia is considered particularly vulnerable to life-threatening vector-borne diseases (VBDs) including malaria, West Nile virus disease and leishmaniasis. However, information relevant for the control of the vectors of these diseases, such as their insecticide resistance profile, is scarce. The present study was conducted to provide the first evidence on insecticide resistance mechanisms circulating in major mosquito and sand fly populations in Armenia. METHODS: Sampling sites were targeted based mainly on previous historical records of VBD occurrences in humans and vertebrate hosts. Initially, molecular species identification on the collected vector samples was performed. Subsequently, molecular diagnostic assays [polymerase chain reaction (PCR), Sanger sequencing, PCR-restriction fragment length polymorphism (RFLP), quantitative PCR (qPCR)] were performed to profile for major insecticide resistance mechanisms, i.e. target site insensitivity in voltage-gated sodium channel (vgsc) associated with pyrethroid resistance, acetylcholinesterase (ace-1) target site mutations linked to organophosphate (OP) and carbamate (CRB) resistance, chitin synthase (chs-1) target site mutations associated with diflubenzuron (DFB) resistance and gene amplification of carboxylesterases (CCEs) associated with resistance to the OP temephos. RESULTS: Anopheles mosquitoes were principally represented by Anopheles sacharovi, a well-known malaria vector in Armenia, which showed no signs of resistance mechanisms. Contrarily, the knockdown resistance (kdr) mutations V1016G and L1014F/C in the vgsc gene were detected in the arboviral mosquito vectors Aedes albopictus and Culex pipiens, respectively. The kdr mutation L1014S was also detected in the sand fly, vectors of leishmaniasis, Phlebotomus papatasi and P. tobbi, whereas no mutations were found in the remaining collected sand fly species, P. sergenti, P. perfiliewi and P. caucasicus. CONCLUSIONS: This is the first study to report on molecular mechanisms of insecticide resistance circulating in major mosquito and sand fly disease vectors in Armenia and highlights the need for the establishment of systematic resistance monitoring practices for the implementation of evidence-based control applications.

Development, application and evaluation of three novel TaqMan qPCR assays for phosphine resistance monitoring in major stored product pests Tribolium castaneum and Rhyzopertha dominica.

BACKROUND: Stored product protection from insect pests relies heavily on the use of phosphine. The most serious drawback of phosphine is the development of resistance in major stored product insects worldwide, including the red flour beetle, Tribolium castaneum (Herbst) and the lesser grain borer, Rhyzopertha dominica (F.). Two genetic loci are responsible for phosphine resistance: the rph1 (S349G mutation in the cyt-b5-r homolog) in T. castaneum and the rph2 (P45/49S mutation in the dihydrolipoamide dehydrogenase (dld) gene) in T. castaneum and R. dominica. RESULTS: In this study, we have developed and applied high-throughput, practical and specific molecular diagnostics (TaqMan qPCR) for monitoring mutations S349G, P45S and P49S. In our pilot monitoring application, we have included phosphine-resistant and susceptible populations from different parts of the world (USA, Australia, Brazil) and European strains from Greece and Serbia. Our results for the resistant T. castaneum showed a P45S mutant allele frequency (MAF) of 100% and 75.0% in the populations from Serbia and Brazil, respectively. Regarding the susceptible T. castaneum, P45S was detected in Greece (MAF = 62.5%) and was absent in Australia (MAF = 0.0%). Additionally, the S349G mutation was found to be fixed in all resistant populations, while it was also detected in susceptible ones (frequencies: 65.0% and 100.0%). The only case where both mutations were fixed (100%) was a resistant population from Serbia. In R. dominica, the P49S mutation was found only in the two resistant R. dominica populations from Serbia and Greece (50.0% and 100%) and was absent from the susceptible one from Greece; thus, P49S seems to be a satisfactory indicator for monitoring phosphine resistance. CONCLUSIONS: Our P49S detection assay in R. dominica seems to be a viable option in this direction, yet its utilization needs additional large-scale confirmatory work. The identification of additional resistance markers also should be prioritized. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

DDT Resistance in Anopheles pharoensis from Northern Cameroon Associated with High Cuticular Hydrocarbon Production.

Despite the contribution of secondary vectors to malaria transmission, there is still not enough information on their susceptibility status to insecticides. The present study assesses the resistance profile of Anopheles pharoensis to DDT. WHO tube tests were used to screen mosquito populations collected from the far-north region of Cameroon for susceptibility to 4% DDT. High DDT resistance in An. pharoensis populations from Maga, Simatou and Yangah with mortality rates ranging from 62.79% to 80% was recorded. Direct sequencing (Sanger) of the VGSC gene was undertaken to search for kdr L1014F/S mutations. However, no kdr allele was detected in the resistant samples. We then looked for cuticle alterations and CHC identification and quantitation were undertaken using GC-MS and GC-FID. High production of cuticular hydrocarbon was recorded in the populations of Yangah and Simatou, with 2420.9 ± 265 and 2372.5 ± 225 ng CHCs/mg dry weight, respectively. The present findings are the first ever describing the development of cuticle resistance in An. pharoensis. The data suggest the need to expand surveillance activities on other vector species.

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.

Over-expression in cis of the midgut P450 CYP392A16 contributes to abamectin resistance in Tetranychus urticae.

Cytochrome P450 mediated metabolism is a well-known mechanism of insecticide resistance. However, to what extent qualitative or quantitative changes are responsible for increased metabolism, is not well understood. Increased expression of P450 genes is most often reported, but the underlying regulatory mechanisms remain widely unclear. In this study, we investigate CYP392A16, a P450 from the polyphagous and major agricultural pest Tetranychus urticae. High expression levels of CYP392A16 and in vitro metabolism assays have previously associated this P450 with abamectin resistance. Here, we show that CYP392A16 is primarily localized in the midgut epithelial cells, as indicated by immunofluorescence analysis, a finding also supported by a comparison between feeding and contact toxicity bioassays. Silencing via RNAi of CYP392A16 in a highly resistant T. urticae population reduced insecticide resistance levels from 3400- to 1900- fold, compared to the susceptible reference strain. Marker-assisted backcrossing, using a single nucleotide polymorphism (SNP) found in the CYP392A16 allele from the resistant population, was subsequently performed to create congenic lines bearing this gene in a susceptible genetic background. Toxicity assays indicated that the allele derived from the resistant strain confers 3.6-fold abamectin resistance compared to the lines with susceptible genetic background. CYP392A16 is over-expressed at the same levels in these lines, pointing to cis-regulation of gene expression. In support of that, functional analysis of the putative promoter region from the resistant and susceptible parental strains revealed a higher reporter gene expression, confirming the presence of cis-acting regulatory mechanisms.

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.

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.

Untangling a Gordian knot: the role of a GluCl3 I321T mutation in abamectin resistance in Tetranychus urticae.

BACKGROUND: The cys-loop ligand-gated ion channels, including the glutamate-gated chloride channel (GluCl) and GABA-gated chloride channel (Rdl) are important targets for drugs and pesticides. The macrocyclic lactone abamectin primarily targets GluCl and is commonly used to control the spider mite Tetranychus urticae, an economically important crop pest. However, abamectin resistance has been reported for multiple T. urticae populations worldwide, and in several cases was associated with the mutations G314D in GluCl1 and G326E in GluCl3. Recently, an additional I321T mutation in GluCl3 was identified in several abamectin resistant T. urticae field populations. Here, we aim to functionally validate this mutation and determine its phenotypic strength. RESULTS: The GluCl3 I321T mutation was introgressed into a T. urticae susceptible background by marker-assisted backcrossing, revealing contrasting results in phenotypic strength, ranging from almost none to 50-fold. Next, we used CRISPR-Cas9 to introduce I321T, G314D and G326E in the orthologous Drosophila GluCl. Genome modified flies expressing GluCl I321T were threefold less susceptible to abamectin, while CRISPRed GluCl G314D and G326E flies were lethal. Last, functional analysis in Xenopus oocytes revealed that the I321T mutation might reduce GluCl3 sensitivity to abamectin, but also suggested that all three T. urticae Rdls are affected by abamectin. CONCLUSION: Three different techniques were used to characterize the role of I321T in GluCl3 in abamectin resistance and, combining all results, our analysis suggests that the I321T mutation has a complex role in abamectin resistance. Given the reported subtle effect, additional synergistic factors in resistance warrant more investigation. © 2020 Society of Chemical Industry.

Identification and functional characterization of a novel acetyl-CoA carboxylase mutation associated with ketoenol resistance in Bemisia tabaci.

Insecticides of the tetronic/tetramic acid family (cyclic ketoenols) are widely used to control sucking pests such as whiteflies, aphids and mites. They act as inhibitors of acetyl-CoA carboxylase (ACC), a key enzyme for lipid biosynthesis across taxa. While it is well documented that plant ACCs targeted by herbicides have developed resistance associated with mutations at the carboxyltransferase (CT) domain, resistance to ketoenols in invertebrate pests has been previously associated either with metabolic resistance or with non-validated candidate mutations in different ACC domains. A recent study revealed high levels of spiromesifen and spirotetramat resistance in Spanish field populations of the whitefly Bemisia tabaci that was not thought to be associated with metabolic resistance. We confirm the presence of high resistance levels (up to >640-fold) against ketoenol insecticides in both Spanish and Australian B. tabaci strains of the MED and MEAM1 species, respectively. RNAseq analysis revealed the presence of an ACC variant bearing a mutation that results in an amino acid substitution, A2083V, in a highly conserved region of the CT domain. F1 progeny resulting from reciprocal crosses between susceptible and resistant lines are almost fully resistant, suggesting an autosomal dominant mode of inheritance. In order to functionally investigate the contribution of this mutation and other candidate mutations previously reported in resistance phenotypes, we used CRISPR/Cas9 to generate genome modified Drosophila lines. Toxicity bioassays using multiple transgenic fly lines confirmed that A2083V causes high levels of resistance to commercial ketoenols. We therefore developed a pyrosequencing-based diagnostic assay to map the spread of the resistance alleles in field-collected samples from Spain. Our screening confirmed the presence of target-site resistance in numerous field-populations collected in Sevilla, Murcia and Almeria. This emphasizes the importance of implementing appropriate resistance management strategies to prevent or slow the spread of resistance through global whitefly populations.

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.