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.

ß-Cyanoalanine synthase protects mites against Arabidopsis defenses.

Glucosinolates are antiherbivory chemical defense compounds in Arabidopsis (Arabidopsis thaliana). Specialist herbivores that feed on brassicaceous plants have evolved various mechanisms aimed at preventing the formation of toxic isothiocyanates. In contrast, generalist herbivores typically detoxify isothiocyanates through glutathione conjugation upon exposure. Here, we examined the response of an extreme generalist herbivore, the two-spotted spider mite Tetranychus urticae (Koch), to indole glucosinolates. Tetranychus urticae is a composite generalist whose individual populations have a restricted host range but have an ability to rapidly adapt to initially unfavorable plant hosts. Through comparative transcriptomic analysis of mite populations that have differential susceptibilities to Arabidopsis defenses, we identified ß-cyanoalanine synthase of T. urticae (TuCAS), which encodes an enzyme with dual cysteine and ß-cyanoalanine synthase activities. We combined Arabidopsis genetics, chemical complementation and mite reverse genetics to show that TuCAS is required for mite adaptation to Arabidopsis through its ß-cyanoalanine synthase activity. Consistent with the ß-cyanoalanine synthase role in detoxification of hydrogen cyanide (HCN), we discovered that upon mite herbivory, Arabidopsis plants release HCN. We further demonstrated that indole glucosinolates are sufficient for cyanide formation. Overall, our study uncovered Arabidopsis defenses that rely on indole glucosinolate-dependent cyanide for protection against mite herbivory. In response, Arabidopsis-adapted mites utilize the ß-cyanoalanine synthase activity of TuCAS to counter cyanide toxicity, highlighting the mite's ability to activate resistant traits that enable this extreme polyphagous herbivore to exploit cyanogenic host plants.

Rapid specialization of counter defenses enables two-spotted spider mite to adapt to novel plant hosts.

Genetic adaptation, occurring over a long evolutionary time, enables host-specialized herbivores to develop novel resistance traits and to efficiently counteract the defenses of a narrow range of host plants. In contrast, physiological acclimation, leading to the suppression and/or detoxification of host defenses, is hypothesized to enable broad generalists to shift between plant hosts. However, the host adaptation mechanisms used by generalists composed of host-adapted populations are not known. Two-spotted spider mite (TSSM; Tetranychus urticae) is an extreme generalist herbivore whose individual populations perform well only on a subset of potential hosts. We combined experimental evolution, Arabidopsis thaliana genetics, mite reverse genetics, and pharmacological approaches to examine mite host adaptation upon the shift of a bean (Phaseolus vulgaris)-adapted population to Arabidopsis. We showed that cytochrome P450 monooxygenases are required for mite adaptation to Arabidopsis. We identified activities of two tiers of P450s: general xenobiotic-responsive P450s that have a limited contribution to mite adaptation to Arabidopsis and adaptation-associated P450s that efficiently counteract Arabidopsis defenses. In approximately 25 generations of mite selection on Arabidopsis plants, mites evolved highly efficient detoxification-based adaptation, characteristic of specialist herbivores. This demonstrates that specialization to plant resistance traits can occur within the ecological timescale, enabling the TSSM to shift to novel plant hosts.

Multiple indole glucosinolates and myrosinases defend Arabidopsis against Tetranychus urticae herbivory.

Arabidopsis (Arabidopsis thaliana) defenses against herbivores are regulated by the jasmonate (JA) hormonal signaling pathway, which leads to the production of a plethora of defense compounds. Arabidopsis defense compounds include tryptophan-derived metabolites, which limit Arabidopsis infestation by the generalist herbivore two-spotted spider mite, Tetranychus urticae. However, the phytochemicals responsible for Arabidopsis protection against T. urticae are unknown. Here, we used Arabidopsis mutants disrupted in the synthesis of tryptophan-derived secondary metabolites to identify phytochemicals involved in the defense against T. urticae. We show that of the three tryptophan-dependent pathways found in Arabidopsis, the indole glucosinolate (IG) pathway is necessary and sufficient to assure tryptophan-mediated defense against T. urticae. We demonstrate that all three IGs can limit T. urticae herbivory, but that they must be processed by myrosinases to hinder T. urticae oviposition. Putative IG breakdown products were detected in mite-infested leaves, suggesting in planta processing by myrosinases. Finally, we demonstrate that besides IGs, there are additional JA-regulated defenses that control T. urticae herbivory. Together, our results reveal the complexity of Arabidopsis defenses against T. urticae that rely on multiple IGs, specific myrosinases, and additional JA-dependent defenses.

Plant-Herbivore Interactions: A Case of an Extreme Generalist, the Two-Spotted Spider Mite Tetranychus urticae.

Plant-herbivore interactions evolved over long periods of time, resulting in an elaborate arms race between interacting species. While specialist herbivores evolved specific strategies to cope with the defenses of a limited number of hosts, our understanding of how generalist herbivores deal with the defenses of a plethora of diverse host plants is largely unknown. Understanding the interaction between a plant host and a generalist herbivore requires an understanding of the plant's mechanisms aimed at defending itself and the herbivore's mechanisms intended to counteract diverse defenses. In this review, we use the two-spotted spider mite (TSSM), Tetranychus urticae (Koch) as an example of a generalist herbivore, as this chelicerate pest has a staggering number of plant hosts. We first establish that the ability of TSSM to adapt to marginal hosts underlies its polyphagy and agricultural pest status. We then highlight our understanding of direct plant defenses against spider mite herbivory and review recent advances in uncovering mechanisms of spider mite adaptations to them. Finally, we discuss the adaptation process itself, as it allows TSSM to overcome initially effective plant defenses. A high-quality genome sequence and developing genetic tools, coupled with an ease of mite experimental selection to new hosts, make TSSM an outstanding system to study the evolution of host range, mechanisms of pest xenobiotic resistance and plant-herbivore interactions. In addition, knowledge of plant defense mechanisms that affect mite fitness are of practical importance, as it can lead to development of new control strategies against this important agricultural pest. In parallel, understanding mechanisms of mite counter adaptations to these defenses is required to maintain the efficacy of these control strategies in agricultural practices.

Adaptation of a polyphagous herbivore to a novel host plant extensively shapes the transcriptome of herbivore and host.

Generalist arthropod herbivores rapidly adapt to a broad range of host plants. However, the extent of transcriptional reprogramming in the herbivore and its hosts associated with adaptation remains poorly understood. Using the spider mite Tetranychus urticae and tomato as models with available genomic resources, we investigated the reciprocal genomewide transcriptional changes in both spider mite and tomato as a consequence of mite's adaptation to tomato. We transferred a genetically diverse mite population from bean to tomato where triplicated populations were allowed to propagate for 30 generations. Evolving populations greatly increased their reproductive performance on tomato relative to their progenitors when reared under identical conditions, indicative of genetic adaptation. Analysis of transcriptional changes associated with mite adaptation to tomato revealed two main components. First, adaptation resulted in a set of mite genes that were constitutively downregulated, independently of the host. These genes were mostly of an unknown function. Second, adapted mites mounted an altered transcriptional response that had greater amplitude of changes when re-exposed to tomato, relative to nonadapted mites. This gene set was enriched in genes encoding detoxifying enzymes and xenobiotic transporters. Besides the direct effects on mite gene expression, adaptation also indirectly affected the tomato transcriptional responses, which were attenuated upon feeding of adapted mites, relative to the induced responses by nonadapted mite feeding. Thus, constitutive downregulation and increased transcriptional plasticity of genes in a herbivore may play a central role in adaptation to host plants, leading to both a higher detoxification potential and reduced production of plant defence compounds.

Reciprocal responses in the interaction between Arabidopsis and the cell-content-feeding chelicerate herbivore spider mite.

Most molecular-genetic studies of plant defense responses to arthropod herbivores have focused on insects. However, plant-feeding mites are also pests of diverse plants, and mites induce different patterns of damage to plant tissues than do well-studied insects (e.g. lepidopteran larvae or aphids). The two-spotted spider mite (Tetranychus urticae) is among the most significant mite pests in agriculture, feeding on a staggering number of plant hosts. To understand the interactions between spider mite and a plant at the molecular level, we examined reciprocal genome-wide responses of mites and its host Arabidopsis (Arabidopsis thaliana). Despite differences in feeding guilds, we found that transcriptional responses of Arabidopsis to mite herbivory resembled those observed for lepidopteran herbivores. Mutant analysis of induced plant defense pathways showed functionally that only a subset of induced programs, including jasmonic acid signaling and biosynthesis of indole glucosinolates, are central to Arabidopsis's defense to mite herbivory. On the herbivore side, indole glucosinolates dramatically increased mite mortality and development times. We identified an indole glucosinolate dose-dependent increase in the number of differentially expressed mite genes belonging to pathways associated with detoxification of xenobiotics. This demonstrates that spider mite is sensitive to Arabidopsis defenses that have also been associated with the deterrence of insect herbivores that are very distantly related to chelicerates. Our findings provide molecular insights into the nature of, and response to, herbivory for a representative of a major class of arthropod herbivores.

Localized efficacy of environmental RNAi in Tetranychus urticae.

Environmental RNAi has been developed as a tool for reverse genetics studies and is an emerging pest control strategy. The ability of environmental RNAi to efficiently down-regulate the expression of endogenous gene targets assumes efficient uptake of dsRNA and its processing. In addition, its efficiency can be augmented by the systemic spread of RNAi signals. Environmental RNAi is now a well-established tool for the manipulation of gene expression in the chelicerate acari, including the two-spotted spider mite, Tetranychus urticae. Here, we focused on eight single and ubiquitously-expressed genes encoding proteins with essential cellular functions. Application of dsRNAs that specifically target these genes led to whole mite body phenotypes-dark or spotless. These phenotypes were associated with a significant reduction of target gene expression, ranging from 20 to 50%, when assessed at the whole mite level. Histological analysis of mites treated with orally-delivered dsRNAs was used to investigate the spatial range of the effectiveness of environmental RNAi. Although macroscopic changes led to two groups of body phenotypes, silencing of target genes was associated with the distinct cellular phenotypes. We show that regardless of the target gene tested, cells that displayed histological changes were those that are in direct contact with the dsRNA-containing gut lumen, suggesting that the greatest efficiency of the orally-delivered dsRNAs is localized to gut tissues in T. urticae.

Environmental RNA interference in two-spotted spider mite, Tetranychus urticae, reveals dsRNA processing requirements for efficient RNAi response.

Comprehensive understanding of pleiotropic roles of RNAi machinery highlighted the conserved chromosomal functions of RNA interference. The consequences of the evolutionary variation in the core RNAi pathway genes are mostly unknown, but may lead to the species-specific functions associated with gene silencing. The two-spotted spider mite, Tetranychus urticae, is a major polyphagous chelicerate pest capable of feeding on over 1100 plant species and developing resistance to pesticides used for its control. A well annotated genome, susceptibility to RNAi and economic importance, make T. urticae an excellent candidate for development of an RNAi protocol that enables high-throughput genetic screens and RNAi-based pest control. Here, we show that the length of the exogenous dsRNA critically determines its processivity and ability to induce RNAi in vivo. A combination of the long dsRNAs and the use of dye to trace the ingestion of dsRNA enabled the identification of genes involved in membrane transport and 26S proteasome degradation as sensitive RNAi targets. Our data demonstrate that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the components of the RNAi machinery make T. urticae a potent experimental system to study the evolution of RNAi pathways.

Application of two-spotted spider mite Tetranychus urticae for plant-pest interaction studies.

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with more than 150 of economic value. It is a major pest of greenhouse crops, especially in Solanaceae and Cucurbitaceae (e.g., tomatoes, eggplants, peppers, cucumbers, zucchini) and greenhouse ornamentals (e.g., roses, chrysanthemum, carnations), annual field crops (such as maize, cotton, soybean, and sugar beet), and in perennial cultures (alfalfa, strawberries, grapes, citruses, and plums)1,2. In addition to the extreme polyphagy that makes it an important agricultural pest, T. urticae has a tendency to develop resistance to a wide array of insecticides and acaricides that are used for its control3-7. T. urticae is an excellent experimental organism, as it has a rapid life cycle (7 days at 27 °C) and can be easily maintained at high density in the laboratory. Methods to assay gene expression (including in situ hybridization and antibody staining) and to inactivate expression of spider mite endogenous genes using RNA interference have been developed8-10. Recently, the whole genome sequence of T. urticae has been reported, creating an opportunity to develop this pest herbivore as a model organism with equivalent genomic resources that already exist in some of its host plants (Arabidopsis thaliana and the tomato Solanum lycopersicum)11. Together, these model organisms could provide insights into molecular bases of plant-pest interactions. Here, an efficient method for quick and easy collection of a large number of adult female mites, their application on an experimental plant host, and the assessment of the plant damage due to spider mite feeding are described. The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage (eggs, larvae, nymphs, adult males, and females) that can be used for subsequent experimental application.