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.

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.

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.

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.

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.

Intradiol ring cleavage dioxygenases from herbivorous spider mites as a new detoxification enzyme family in animals.

BACKGROUND: Generalist herbivores such as the two-spotted spider mite Tetranychus urticae thrive on a wide variety of plants and can rapidly adapt to novel hosts. What traits enable polyphagous herbivores to cope with the diversity of secondary metabolites in their variable plant diet is unclear. Genome sequencing of T. urticae revealed the presence of 17 genes that code for secreted proteins with strong homology to "intradiol ring cleavage dioxygenases (DOGs)" from bacteria and fungi, and phylogenetic analyses show that they have been acquired by horizontal gene transfer from fungi. In bacteria and fungi, DOGs have been well characterized and cleave aromatic rings in catecholic compounds between adjacent hydroxyl groups. Such compounds are found in high amounts in solanaceous plants like tomato, where they protect against herbivory. To better understand the role of this gene family in spider mites, we used a multi-disciplinary approach to functionally characterize the various T. urticae DOG genes. RESULTS: We confirmed that DOG genes were present in the T. urticae genome and performed a phylogenetic reconstruction using transcriptomic and genomic data to advance our understanding of the evolutionary history of spider mite DOG genes. We found that DOG expression differed between mites from different plant hosts and was induced in response to jasmonic acid defense signaling. In consonance with a presumed role in detoxification, expression was localized in the mite's gut region. Silencing selected DOGs expression by dsRNA injection reduced the mites' survival rate on tomato, further supporting a role in mitigating the plant defense response. Recombinant purified DOGs displayed a broad substrate promiscuity, cleaving a surprisingly wide array of aromatic plant metabolites, greatly exceeding the metabolic capacity of previously characterized microbial DOGs. CONCLUSION: Our findings suggest that the laterally acquired spider mite DOGs function as detoxification enzymes in the gut, disarming plant metabolites before they reach toxic levels. We provide experimental evidence to support the hypothesis that this proliferated gene family in T. urticae is causally linked to its ability to feed on an extremely wide range of host plants.