Functional Response of Four Phytoseiid Mites to Eggs and First-Instar Larvae of Western Flower Thrips, Frankliniella occidentalis.

The predation capacity and functional responses of adult females of the phytoseiid mites Amblyseius largoensis (Muma), Proprioseiopsis lenis (Corpuz and Rimando), Paraphytoseius cracentis (Corpuz and Rimando), and Amblyseius swirskii (Athias-Henriot) were studied on eggs and first instars of the western flower thrips, Frankliniella occidentalis (Pergande), in the laboratory at 25 °C and 30 °C. At both temperatures, the functional response of all four phytoseiid mites was type II to first instars of the thrips. In contrast, when offered thrips eggs, the functional response was type III. At both temperatures tested, A. swirskii had the highest mean daily consumption of first-instar F. occidentalis, followed by A. largoensis, P. cracentis, and P. lenis. Amblyseius largoensis had the shortest handling time and the highest maximum attack rate when first-instar thrips were the prey. When fed on thrips eggs, A. largoensis had the highest mean daily consumption, followed by A. swirskii, P. cracentis, and P. lenis. On thrips eggs, A. swirskii showed the shortest handling time and highest maximum attack rate. Our findings indicate that all four phytoseiids had a better ability to prey on first-instar larvae of F. occidentalis compared to thrips eggs. At 25 and 30 °C, A. largoensis was the better predator on thrips larvae, whereas A. swirskii was superior in consuming eggs of F. occidentalis. Proprioseiopsis lenis was the inferior predator on both thrips larvae and eggs compared to the other phytoseiids tested.

Acaricide resistance mechanisms and host plant responses in the tomato specialist Aculops lycopersici.

BACKGROUND: The mite Aculops lycopersici is a major tomato pest with extremely reduced gene families involved in chemoreception and detoxification. How this limited detoxification toolbox affects the evolution of resistance to acaricides in tomato russet mite(s) (TRM) remains enigmatic. Moreover, although a tomato specialist, TRM has been observed on other Solanaceae and Convolvulaceae plant species, raising questions about transcriptional plasticity underlying host exchange. RESULTS: We identified a field strain with strongly decreased susceptibility to both abamectin and spiromesifen. We detected target-site resistance caused by mutations at conserved positions in two glutamate-gated chloride channels (GluCl), as well as four overexpressed detoxification genes. We then examined transcriptional responses after host shift from tomato to two Solanaceae (potato and black nightshade) and two Convolvulaceae (sweet potato and hedge bindweed) species, as more challenging host plants. Transcriptional responses varied significantly between host plant families, with key differentially expressed genes (DEGs) related to proteolytic, metabolic and detoxification processes. Last, we also identified DEGs encoding for secreted proteins potentially involved in TRM-host plant interactions. CONCLUSIONS: Despite a limited detoxification toolbox, A. lycopersici might quickly evolve target-site resistance, probably facilitated by strong selection pressure on the genetic variation associated with enormous population size in field settings. Responses to host plant changes include plasticity in genes related to digestion and detoxification, while most responsive genes are of unknown function and remain to be investigated. © 2024 Society of Chemical Industry.

The cytochrome P450 subfamilies CYP392A and CYP392D are key players in acaricide metabolism in Tetranychusurticae.

The spider mite Tetranychus urticae is a major agricultural pest with a global distribution, extremely diverse host range and a remarkable ability to develop resistance to a wide variety of acaricides. P450 mono-oxygenases have been frequently associated with resistance development in this species. In particular enzymes of the CYP392A-subfamily were shown to metabolize a number of key acaricides, including abamectin, amitraz, fenpyroximate and the active metabolite of pyflubumide. However, transcriptomic studies comparing highly resistant and susceptible populations have often revealed high expression of members of the CYP392D-subfamily, but these have been only poorly studied. Here, we conducted a meta-analysis of gene expression data of 20 populations and identified two key enzymes of this family, CYP392D2 and CYP392D8, whose expression is associated with resistance. We subsequently functionally expressed these enzymes, together with CYP392A11 and CYP392A16 as known metabolizers, and compared their potential to accept a wide diversity of acaricides as substrate. This study overall confirms previous discovered substrates for CYP392A11 and CYP392A16, but also reveals unreported metabolic activity towards new acaricides. These include carbaryl, chlorpyrifos and etoxazole for CYP392A16 and carbaryl, chlorpyrifos and NNI-0711-NH pyflubumide for CYP392A11. For the newly studied CYP392D-family, we show that CYP392D2 metabolizes pyridaben, fenpyroximate, etoxazole and chlorpyrifos, while CYP392D8 metabolizes carbaryl, fenazaquin and tebufenpyrad. Last, we observed that both CYP392A- and CYP392D-subfamily enzymes activate chlorpyrifos to its corresponding oxon. Our study indicates that there is both overlap and specificity in the activity of A- and D-subfamily enzymes against acaricides and model substrates. With the recent advent of highly efficient CRISPR/Cas9 gene editing protocols in T. urticae, the way is now paved to conduct further genetic experiments revealing and quantifying the role of these enzymes in the resistance phenotype in field populations.

Identification and CRISPR-Cas9 validation of a novel ß-adrenergic-like octopamine receptor mutation associated with amitraz resistance in Varroa destructor.

Varroa destructor is widely recognized as a significant contributor to colony collapse disorder. Chemical acaricides, such as amitraz, have been extensively used for Varroa control due to their selectivity within beehives. However, the increasing number of cases of amitraz resistance across global V. destructor populations poses a significant challenge. In this study, we conducted a comprehensive molecular screening of the ß-adrenergic-like octopamine receptor (Octß2R), the target-site of amitraz, across 66 Turkish and 63 Belgian V. destructor populations. Although previously reported amitraz resistance mutations were not detected, the screening revealed a novel Y337F mutation located within transmembrane 7 (TM7) of Octß2R in Turkish Varroa populations. Notably, this mutation was identified in the last residue of the highly conserved NPxxY motif associated with the activation of G-protein coupled receptors (GPCR). Among the 66 Varroa samples from Türkiye, twenty harbored the Y337F mutation, with eight samples exhibiting fixation of the mutation. Subsequent bioassays revealed over 8-fold resistance to amitraz in populations that contain the Y337F mutation. Genotyping of mites after exposure to 10 mg a.i./l amitraz demonstrated that all surviving mites were homozygous for the Y337F mutation, whereas dead mites carried susceptible alleles, providing genetic linkage between mutation and phenotype. Further, we used CRISPR-Cas9 editing to introduce the Y337F mutation in the orthologous Octß2R of the model organism Tetranychus urticae. Crispants exhibited over threefold resistance to amitraz. In conclusion, this study identified and validated a novel amitraz resistance mutation. Additional research is required to further evaluate the phenotypic strength of Y337F in the context of operational resistance with current treatment strategies.

Unveiling the diet of two generalist stink bugs, Halyomorpha halys and Pentatoma rufipes (Hemiptera: Pentatomidae), through metabarcoding of the ITS2 region from gut content.

BACKGROUND: The use of DNA metabarcoding has become an increasingly popular technique to infer feeding relationships in polyphagous herbivores and predators. Understanding host plant preference of native and invasive herbivore insects can be helpful in establishing effective integrated pest management (IPM) strategies. The invasive Halyomorpha halys and native Pentatoma rufipes are piercing-sucking stink bug pests that are known to cause economic damage in commercial fruit orchards. RESULTS: In this study, we performed molecular gut content analysis (MGCA) on field-collected specimens of these two herbivorous pentatomids using next-generation amplicon sequencing (NGAS) of the internal transcribed spacer 2 (ITS2) barcode region. Additionally, a laboratory experiment was set up where H. halys was switched from a mixed diet to a monotypic diet, allowing us to determine the detectability of the initial diet in a time series of ≤3 days after the diet switch. We detected 68 unique plant species from 54 genera in the diet of two stink bug species, with fewer genera found per sample and a smaller diet breadth for P. rufipes than for H. halys. Both stink bug species generally prefer deciduous trees over gymnosperms and herbaceous plants. Landscape type significantly impacted the observed genera in the diet of both stink bug species, whereas season only had a significant effect on the diet of H. halys. CONCLUSION: This study provides further insights into the dietary composition of two polyphagous pentatomid pests and illustrates that metabarcoding can deliver a relevant species-level resolution of host plant preference. © 2024 Society of Chemical Industry.

Life table parameters of Amblyseius largoensis, Amblyseius swirskii and Proprioseiopsis lenis (Acari: Phytoseiidae) fed on eggs and larvae of Frankliniella occidentalis.

The immature development and reproduction of the predatory mites Amblyseius largoensis (Muma), Proprioseiopsis lenis (Corpuz and Rimando), and Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) were investigated using both thrips eggs and first instars of the western flower thrips, Frankliniella occidentalis Pergande, as prey in a controlled laboratory environment at 25 °C and 60% relative humidity. When provided with thrips eggs as food, A. largoensis exhibited a notably shorter immature development period for both males (7.05 days) and females (6.51 days) as compared with A. swirskii (8.05 and 7.19 days, respectively) and P. lenis (8.10 days and 7.05 days, respectively). Amblyseius largoensis also displayed a higher oviposition rate (2.19 eggs/female/day) than A. swirskii and P. lenis (1.79 and 1.78 eggs/female/day, respectively). Moreover, it exhibited the highest fecundity (25.34 eggs/female), followed by P. lenis (24.23 eggs/female) and A. swirskii (22.86 eggs/female). These variations led to A. largoensis having the highest intrinsic rate of increase (rm) at 0.209, followed by A. swirskii at 0.188, and P. lenis at 0.165. However, when the predatory mites were provided with first instars of F. occidentalis, A. swirskii demonstrated a faster immature development period for both males (7.67 days) and females (7.59 days) as compared with P. lenis (9.00 days and 7.86 days, respectively) and A. largoensis (8.47 days and 8.61 days, respectively). While the oviposition rates of P. lenis (1.92 eggs/female/day) and A. swirskii (1.90 eggs/female/day) were similar when feeding on this prey, A. largoensis produced fewer eggs (1.83 eggs/female/day). Further, A. swirskii exhibited the highest fecundity (31.93 eggs/female), followed by A. largoensis (25.71 eggs/female) and P. lenis (23 eggs/female). Consequently, the intrinsic rate of increase (rm) on thrips first instars was highest in A. swirskii (0.190), followed by A. largoensis (0.186), and P. lenis (0.176). In summary, our findings indicate that in terms of life history parameters A. largoensis performs optimally when feeding on thrips eggs, whereas A. swirskii performs best when preying on the mobile first instars of the thrips. These insights into the dietary preferences and reproductive capabilities of the studied predatory mite species have important implications for their potential use as biological control agents against F. occidentalis in agricultural settings.

A novel target-site mutation (H146Q) outside the ubiquinone binding site of succinate dehydrogenase confers high levels of resistance to cyflumetofen and pyflubumide in Tetranychus urticae.

Mitochondrial electron transfer inhibitors at complex II (METI-II), also referred to as succinate dehydrogenase inhibitors (SDHI), represent a recently developed class of acaricides encompassing cyflumetofen, cyenopyrafen, pyflubumide and cyetpyrafen. Despite their novelty, resistance has already developed in the target pest, Tetranychus urticae. In this study a new mutation, H146Q in a highly conserved region of subunit B of complex II, was identified in a T. urticae population resistant to all METI-IIs. In contrast to previously described mutations, H146Q is located outside the ubiquinone binding site of complex II. Marker-assisted backcrossing of this mutation in a susceptible genetic background validated its association with resistance to cyflumetofen and pyflubumide, but not cyenopyrafen or cyetpyrafen. Biochemical assays and the construction of inhibition curves with isolated mitochondria corroborated this selectivity. In addition, phenotypic effects of H146Q, together with the previously described H258L, were further examined via CRISPR/Cas9 gene editing. Although both mutations were successfully introduced into a susceptible T. urticae population, the H146Q gene editing event was only recovered in individuals already harboring the I260V mutation, known to confer resistance towards cyflumetofen. The combination of H146Q + I260V conferred high resistance levels to all METI-II acaricides with LC50 values over 5000 mg a.i./L for cyflumetofen and pyflubumide. Similarly, the introduction of H258L via gene editing resulted in high resistance levels to all tested acaricides, with extreme LC50 values (>5000 mg a.i./L) for cyenopyrafen and cyetpyrafen, but lower resistance levels for pyflubumide and cyflumetofen. Together, these findings indicate that different mutations result in a different cross-resistance spectrum, probably also reflecting subtle differences in the binding mode of complex II acaricides.

The role of ATP-binding cassette transporters in arthropod pesticide toxicity and resistance.

Pesticide resistance in arthropods threatens agricultural productivity and the control of vector-borne diseases. The ATP-binding cassette (ABC) transporters have emerged as important factors in the toxicity of synthetic pesticides, as well as for Bacillus thuringiensis insecticidal Cry protein binding. Depending on the localization of expression, both higher and lower expression of ABCs have been linked with pesticide resistance. The recent development of genetic-based approaches such as RNAi and CRISPR/Cas9 gene editing in nonmodel species, has greatly contributed to unveil their functional importance in pesticide toxicity and resistance. Using these tools, we are now poised to further unravel the molecular genetic mechanisms of gene regulation uncovering more elusive regulatory resistance genes.

SYNCAS: Efficient CRISPR/Cas9 gene-editing in difficult to transform arthropods.

The genome editing technique CRISPR/Cas9 has led to major advancements in many research fields and this state-of-the-art tool has proven its use in genetic studies for various arthropods. However, most transformation protocols rely on microinjection of CRISPR/Cas9 components into embryos, a method which is challenging for many species. Alternatively, injections can be performed on adult females, but transformation efficiencies can be very low as was shown for the two-spotted spider mite, Tetranychus urticae, a minute but important chelicerate pest on many crops. In this study, we explored different CRISPR/Cas9 formulations to optimize a maternal injection protocol for T. urticae. We observed a strong synergy between branched amphipathic peptide capsules and saponins, resulting in a significant increase of CRISPR/Cas9 knock-out efficiency, exceeding 20%. This CRISPR/Cas9 formulation, termed SYNCAS, was used to knock-out different T. urticae genes - phytoene desaturase, CYP384A1 and Antennapedia - but also allowed to develop a co-CRISPR strategy and facilitated the generation of T. urticae knock-in mutants. In addition, SYNCAS was successfully applied to knock-out white and white-like genes in the western flower thrips, Frankliniella occidentalis. The SYNCAS method allows routine genome editing in these species and can be a game changer for genetic research in other hard to transform arthropods.

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.

Contrasting roles of cytochrome P450s in amitraz and chlorfenapyr resistance in the crop pest Tetranychus urticae.

The molecular mechanisms of amitraz and chlorfenapyr resistance remain only poorly understood for major agricultural pests and vectors of human diseases. This study focusses on a multi-resistant field strain of the crop pest Tetranychus urticae, which could be readily selected in the laboratory to high levels of amitraz and chlorfenapyr resistance. Toxicity experiments using tralopyril, the active toxophore of chlorfenapyr, suggested decreased activation as a likely mechanism underlying resistance. Starting from the same parental strain, transcriptome profiling revealed that a cluster of detoxifying genes was upregulated after amitraz selection, but unexpectedly downregulated after chlorfenapyr selection. Further functional validation associated the upregulation of CYP392A16 with amitraz metabolism and the downregulation of CYP392D8 with reduced activation of chlorfenapyr to tralopyril. Genetic mapping (QTL analysis by BSA) was conducted in an attempt to unravel the genetic mechanisms of expression variation and resistance. This revealed that chlorfenapyr resistance was associated with a single QTL, while 3 QTLs were uncovered for amitraz resistance. Together with the observed contrasting gene expression patterns, we argue that transcriptional regulators most likely underly the distinct expression profiles associated with resistance, but these await further functional validation.

Adaptation of an arthropod predator to a challenging environment is associated with a loss of a genome-wide plastic transcriptional response.

BACKGROUND: Structural and chemical plant defence traits may reduce the efficacy of biological control agents in integrated pest management. Breeding programmes have shown arthropod predators' potential to acclimate to challenging host plants. However, whether and how these predators adapt to novel plant environments remain unclear. Using the predatory mite Phytoseiulus persimilis - herbivorous mite Tetranychus urticae system in an experimental evolution setup, we studied the adaptation mechanisms to tomato and cucumber, plants that possess a distinct repertoire of defensive traits. RESULTS: Experimental evolution experiments on whole plants revealed that allowing P. persimilis to adapt to tomatoes led to an ~100% larger population size. Independent feeding assays showed that tomato- and cucumber-adapted prey reduced predator fecundity. The deleterious effect of ingesting low-quality prey persisted after adaptation of the predator to both cucumber and tomato. We demonstrated that jasmonic acid (JA)-dependent defences reduce prey quality by evaluating predator performance on prey fed on JA defence-deficient tomato plants. Transcriptomic profiling of the replicated P. persimilis lines showed that long-term propagation on tomato and cucumber plants produces distinctive gene-expression levels. Predator adaptation to tomatoes results in the loss of a large transcriptional response, in which predicted cuticle-building rather than detoxification pathways are affected. CONCLUSION: We showed that the adaptation of predatory arthropods to a novel, challenging plant does not necessarily occur via the prey, but rather through the physical environment of the plant. We provided first insights into the underlying molecular mechanisms. © 2023 Society of Chemical Industry.

Structural and functional studies reveal the molecular basis of substrate promiscuity of a glycosyltransferase originating from a major agricultural pest.

The two-spotted spider mite, Tetranychus urticae, is a major cosmopolitan pest that feeds on more than 1100 plant species. Its genome contains an unprecedentedly large number of genes involved in detoxifying and transporting xenobiotics, including 80 genes that code for UDP glycosyltransferases (UGTs). These enzymes were acquired via horizontal gene transfer from bacteria after loss in the Chelicerata lineage. UGTs are well-known for their role in phase II metabolism; however, their contribution to host adaptation and acaricide resistance in arthropods, such as T. urticae, is not yet resolved. TuUGT202A2 (Tetur22g00270) has been linked to the ability of this pest to adapt to tomato plants. Moreover, it was shown that this enzyme can glycosylate a wide range of flavonoids. To understand this relationship at the molecular level, structural, functional, and computational studies were performed. Structural studies provided specific snapshots of the enzyme in different catalytically relevant stages. The crystal structure of TuUGT202A2 in complex with UDP-glucose was obtained and site-directed mutagenesis paired with molecular dynamic simulations revealed a novel lid-like mechanism involved in the binding of the activated sugar donor. Two additional TuUGT202A2 crystal complexes, UDP-(S)-naringenin and UDP-naringin, demonstrated that this enzyme has a highly plastic and open-ended acceptor-binding site. Overall, this work reveals the molecular basis of substrate promiscuity of TuUGT202A2 and provides novel insights into the structural mechanism of UGTs catalysis.

The host plant strongly modulates acaricide resistance levels to mitochondrial complex II inhibitors in a multi-resistant field population of Tetranychus urticae.

The two-spotted spider mite Tetranychus urticae is a polyphagous pest with an extraordinary ability to develop acaricide resistance. Here, we characterize the resistance mechanisms in a T. urticae population (VR-BE) collected from a Belgian tomato greenhouse, where the grower was unsuccessful in chemically controlling the mite population resulting in crop loss. Upon arrival in the laboratory, the VR-BE population was established both on bean and tomato plants as hosts. Toxicity bioassays on both populations confirmed that the population was highly multi-resistant, recording resistance to 12 out of 13 compounds tested from various mode of action groups. DNA sequencing revealed the presence of multiple target-site resistance mutations, but these could not explain resistance to all compounds. In addition, striking differences in toxicity for six acaricides were observed between the populations on bean and tomato. The highest difference was recorded for the complex II inhibitors cyenopyrafen and cyflumetofen, which were 4.4 and 3.3-fold less toxic for VR-BE mites on tomato versus bean. PBO synergism bioassays suggested increased P450 based detoxification contribute to the host-dependent toxicity. Given the involvement of increased detoxification, we subsequently determined genome-wide gene expression levels of VR-BE on both hosts, in comparison to a reference susceptible population, revealing overexpression of a large set of detoxification genes in VR-BE on both hosts compared to the reference. In addition, a number of mainly detoxification genes with higher expression in VR-BE on tomato compared to bean was identified, including several cytochrome P450s. Together, our work suggests that multi-resistant field populations can accumulate a striking number of target-site resistance mutations. We also show that the host plant can have a profound effect on the P450-associated resistance levels to cyenopyrafen and cyflumetofen.

A glutamate-gated chloride channel as the mite-specific target-site of dicofol and other diphenylcarbinol acaricides.

Dicofol has been widely used to control phytophagous mites. Although dicofol is chemically related to DDT, its mode of action has remained elusive. Here, we mapped dicofol resistance in the spider mite Tetranychus urticae to two genomic regions. Each region harbored a glutamate-gated chloride channel (GluCl) gene that contained a mutation-G314D or G326E-known to confer resistance against the unrelated acaricide abamectin. Using electrophysiology assays we showed that dicofol and other diphenylcarbinol acaricides-bromopropylate and chlorobenzilate-induce persistent currents in Xenopus oocytes expressing wild-type T. urticae GluCl3 receptors and potentiate glutamate responses. In contrast, the G326E substitution abolished the agonistic activity of all three compounds. Assays with the wild-type Drosophila GluClα revealed that this receptor was unresponsive to dicofol. Homology modeling combined with ligand-docking confirmed the specificity of electrophysiology assays. Altogether, this work elucidates the mode of action of diphenylcarbinols as mite-specific agonists of GluCl.

A nuclear receptor HR96-related gene underlies large trans-driven differences in detoxification gene expression in a generalist herbivore.

The role, magnitude, and molecular nature of trans-driven expression variation underlying the upregulation of detoxification genes in pesticide resistant arthropod populations has remained enigmatic. In this study, we performed expression quantitative trait locus (eQTL) mapping (n = 458) between a pesticide resistant and a susceptible strain of the generalist herbivore and crop pest Tetranychus urticae. We found that a single trans eQTL hotspot controlled large differences in the expression of a subset of genes in different detoxification gene families, as well as other genes associated with host plant use. As established by additional genetic approaches including RNAi gene knockdown, a duplicated gene with a nuclear hormone receptor HR96-related ligand-binding domain was identified as causal for the expression differences between strains. The presence of a large family of HR96-related genes in T. urticae may enable modular control of detoxification and host plant use genes, facilitating this species' known and rapid evolution to diverse pesticides and host plants.

Host adaptation and specialization in Tetranychidae mites.

Composite generalist herbivores are comprised of host-adapted populations that retain the ability to shift hosts. The degree and overlap of mechanisms used by host-adapted generalist and specialist herbivores to overcome the same host plant defenses are largely unknown. Tetranychidae mites are exceptionally suited to address the relationship between host adaptation and specialization in herbivores as this group harbors closely related species with remarkably different host ranges-an extreme generalist the two-spotted spider mite (Tetranychus urticae Koch [Tu]) and the Solanaceous specialist Tetranychus evansi (Te). Here, we used tomato-adapted two-spotted spider mite (Tu-A) and Te populations to compare mechanisms underlying their host adaptation and specialization. We show that both mites attenuate induced tomato defenses, including protease inhibitors (PIs) that target mite cathepsin L digestive proteases. While Te solely relies on transcriptional attenuation of PI induction, Tu and Tu-A have elevated constitutive activity of cathepsin L proteases, making them less susceptible to plant anti-digestive proteins. Tu-A and Te also rely on detoxification of tomato constitutive defenses. Te uses esterase and P450 activities, while Tu-A depends on the activity of all major detoxification enzymatic classes to disarm tomato defensive compounds to a lesser extent. Thus, even though both Tu-A and Te use similar mechanisms to counteract tomato defenses, Te can better cope with them. This finding is congruent with the ecological and evolutionary times required to establish mite adaptation and specialization states, respectively.

The complex II resistance mutation H258Y in succinate dehydrogenase subunit B causes fitness penalties associated with mitochondrial respiratory deficiency.

BACKGROUND: The acaricides cyflumetofen, cyenopyrafen and pyflubumide inhibit the mitochondrial electron transport chain at complex II [succinate dehydrogenase (SDH) complex]. A target site mutation H258Y was recently discovered in a resistant strain of the spider mite pest Tetranychus urticae. H258Y causes strong cross-resistance between cyenopyrafen and pyflubumide, but not cyflumetofen. In fungal pests, fitness costs associated with substitutions at the corresponding H258 position that confer resistance to fungicidal SDH inhibitors have not been uncovered. Here, we used H258 and Y258 near-isogenic lines of T. urticae to quantify potential pleiotropic fitness effects on mite physiology. RESULTS: The H258Y mutation was not associated with consistent significant changes of single generation life history traits and fertility life table parameters. In contrast, proportional Sanger sequencing and droplet digital polymerase chain reaction showed that the frequency of the resistant Y258 allele decreased when replicated 50:50 Y258:H258 experimentally evolving populations were maintained in an acaricide-free environment for approximately 12 generations. Using in vitro assays with mitochondrial extracts from resistant (Y258) and susceptible (H258) lines, we identified a significantly reduced SDH activity (48% lower activity) and a slightly enhanced combined complex I and III activity (18% higher activity) in the Y258 lines. CONCLUSION: Our findings suggest that the H258Y mutation is associated with a high fitness cost in the spider mite T. urticae. Importantly, while it is the most common approach, it is clear that only comparing life history traits and life table fecundity does not allow to reliably estimate fitness costs of target site mutations in natural pest populations. © 2023 Society of Chemical Industry.

A review of the molecular mechanisms of acaricide resistance in mites and ticks.

The Arachnida subclass of Acari comprises many harmful pests that threaten agriculture as well as animal health, including herbivorous spider mites, the bee parasite Varroa, the poultry mite Dermanyssus and several species of ticks. Especially in agriculture, acaricides are often used intensively to minimize the damage they inflict, promoting the development of resistance. Beneficial predatory mites used in biological control are also subjected to acaricide selection in the field. The development and use of new genetic and genomic tools such as genome and transcriptome sequencing, bulked segregant analysis (QTL mapping), and reverse genetics via RNAi or CRISPR/Cas9, have greatly increased our understanding of the molecular genetic mechanisms of resistance in Acari, especially in the spider mite Tetranychus urticae which emerged as a model species. These new techniques allowed to uncover and validate new resistance mutations in a larger range of species. In addition, they provided an impetus to start elucidating more challenging questions on mechanisms of gene regulation of detoxification associated with resistance.

Intraspecific diversity in the mechanisms underlying abamectin resistance in a cosmopolitan pest.

Pesticide resistance relies on a myriad of mechanisms, ranging from single mutations to a complex and polygenic architecture, and it involves mechanisms such as target-site insensitivity, metabolic detoxification, or a combination of these, with either additive or synergistic effects. Several resistance mechanisms against abamectin, a macrocyclic lactone widely used in crop protection, have been reported in the cosmopolitan pest Tetranychus urticae. However, it has been shown that a single mechanism cannot account for the high levels of abamectin resistance found across different mite populations. Here, we used experimental evolution combined with bulked segregant analyses to map quantitative trait loci (QTL) associated with abamectin resistance in two genetically unrelated populations of T. urticae. In these two independent QTL mapping experiments, three and four QTLs were identified, of which three were shared between experiments. Shared QTLs contained genes encoding subunits of the glutamate-gated chloride channel (GluCl) and harboured previously reported mutations, including G314D in GluCl1 and G326E in GluCl3, but also novel resistance candidate loci, including DNA helicases and chemosensory receptors. Surprisingly, the fourth QTL, present only in only one of the experiments and thus unique for one resistant parental line, revealed a non-functional variant of GluCl2, suggesting gene knock-out as resistance mechanism. Our study uncovers the complex basis of abamectin resistance, and it highlights the intraspecific diversity of genetic mechanisms underlying resistance in a cosmopolitan pest.