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Host shifts can lead to ecological speciation and the emergence of new pests and pathogens. However, the mutational events that facilitate the exploitation of novel hosts are poorly understood. Here, we characterize an adaptive walk underpinning the host shift of the aphid Myzus persicae to tobacco, including evolution of mechanisms that overcame tobacco chemical defenses. A series of mutational events added as many as 1.5 million nucleotides to the genome of the tobacco-adapted subspecies, M. p. nicotianae, and yielded profound increases in expression of an enzyme that efficiently detoxifies nicotine, both in aphid gut tissue and in the bacteriocytes housing the obligate aphid symbiont Buchnera aphidicola. This dual evolutionary solution overcame the challenge of preserving fitness of a mutualistic symbiosis during adaptation to a toxic novel host. Our results reveal the intricate processes by which genetic novelty can arise and drive the evolution of key innovations required for ecological adaptation.

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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

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.

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.

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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.